Method for detecting thrombin activity in biological product by using functionalized electrode

Abstract

The invention discloses a method for detecting thrombin activity in a biological product by using a functionalized electrode, and belongs to the technical field of electrochemical detection. Firstly, thrombin specific substrate polypeptide is fixed on the surface of a silk-screen printing gold electrode through an Au-S bond, and the N end of the polypeptide is modified with ferrocene (Fc) to serve as an electroactive signal molecule; then the surface of the electrode is coated with a cellulose acetate membrane (CA) to obtain a functionalized electrode, and the CA membrane can reduce non-specific adsorption and prevent electrode pollution and signal attenuation; dropwise adding a solution containing thrombin onto the functionalized electrode for reaction; and washing the reacted electrode, testing by using a square wave voltammetry, and obtaining the thrombin activity according to the change of the peak current. The method is convenient to operate, high in specificity, short in consumed time and long in storage time of the functionalized electrode, and is an effective means for detecting the thrombin activity in the biological product.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological detection, and particularly relates to a method for detecting the activity of thrombin in biological products using a functional electrode. BACKGROUND

[0002] Thrombin is a serine protease that plays a crucial role in the coagulation cascade, converting soluble fibrinogen into insoluble fibrin, thereby promoting the formation of stable blood clots. In addition to its coagulation function, thrombin can also initiate cell signaling through protease-activated receptors (PARs), promoting angiogenesis and tissue repair. In recent years, as a rapid local hemostatic drug, thrombin has been widely used in surgical operations. The efficacy and safety of thrombin are closely related to its activity: insufficient enzyme activity can weaken the hemostatic effect, while excessive activity may induce thrombosis or exacerbate inflammation. Moreover, due to the inherent instability of proteins, the activity of thrombin is easily affected by external environmental factors. Especially in emergency clinical situations, when thrombin needs to be used quickly to control bleeding, there is usually not enough time for time-consuming laboratory tests.

[0003] The detection methods of thrombin activity mainly fall into two categories: one is based on the determination of clotting time, which evaluates enzyme activity by measuring the clotting time of fibrinogen and thrombin reaction; the other is ultraviolet-visible spectrophotometry (UV-Vis), which detects the change in absorbance before and after the hydrolysis of the chromogenic substrate. However, the sensitivity of clotting time determination is limited by the naked eye endpoint judgment, which is prone to differences due to the proficiency of the operator; the UV-Vis method may be interfered by the substrate or sample matrix, introducing errors. There are also reports of various detection strategies such as colorimetric method, fluorescence spectroscopy, surface-enhanced Raman spectroscopy (SERS), electrochemiluminescence (ECL), and photoelectrochemical method (PEC), but most of them only focus on the quantitative evaluation of thrombin concentration, and the detection of functional activity is relatively insufficient. In addition, optical detection methods are easily affected by environmental factors, and the stability of the results is easily affected.

[0004] In contrast, electrochemical detection methods have become a powerful tool for on-site rapid detection due to their rapid response, ease of miniaturization, and ability to handle multiple samples simultaneously, showing high application value in point-of-care testing (POCT) and drug therapy monitoring. However, in existing research using electrochemical methods to detect thrombin, the main purpose is to detect a small amount of thrombin in complex samples, focusing on sensitivity and specificity, and additional signal amplification methods are used, resulting in complex operation and long time consumption. How to quickly detect the activity of high-content thrombin in biological products has not been studied yet. SUMMARY

[0005] 1. Objectives

[0006] The application aims to provide a method for detecting the activity of thrombin in biological products by using a functional electrode.

[0007] 2. Technical solution

[0008] In order to achieve the above-mentioned application purposes, the technical solution adopted by the application is as follows:

[0009] The application provides a method for detecting the activity of thrombin in biological products by using a functional electrode, comprising the following steps:

[0010] S1: fixing a substrate polypeptide on the surface of a silk screen printed gold electrode;

[0011] S2: coating a cellulose acetate membrane (CA) on the surface of the electrode to obtain a functional electrode;

[0012] S3: adding a solution containing thrombin to the functional electrode for reaction;

[0013] S4: after the reaction, the electrode is washed and tested by square wave voltammetry, and the activity of thrombin is obtained according to the change of peak current.

[0014] Further, the substrate polypeptide is fixed on the surface of the silk screen printed gold electrode through an Au-S bond.

[0015] Further, the substrate polypeptide is further modified with ferrocene (Fc) at the N terminal as an electroactive signal molecule.

[0016] Further, the sequence of the substrate polypeptide is GLVPRGSC (SEQ ID NO. 1).

[0017] Further, before fixing the substrate polypeptide, the silk screen printed gold electrode is washed with ultrapure water.

[0018] Further, the fixing of the substrate polypeptide on the surface of the silk screen printed gold electrode is that the cleaned silk screen printed gold electrode is incubated with a polypeptide (Pe-Fc, Fc-GLVPRGSC) solution modified with ferrocene at the N terminal at a concentration of 1150-1300 μM at 4℃ overnight, and then washed with PBS buffer.

[0019] Through multiple optimization experiments, it is found that the functional electrode obtained by using a Pe-Fc solution of 1200 μM has the best detection effect.

[0020] Further, the Pe-Fc solution is prepared by using 0.01 M PBS with pH 7.40.

[0021] Further, the cellulose acetate film (CA) is coated on the surface of the screen-printed gold electrode by dropping 3-6 μL of the CA solution (volume ratio of acetone (AC) and N,N-dimethylformamide (DMF) is 2:8-4:6) with a concentration of 0.4-0.6 wt% on the surface of the screen-printed gold electrode.

[0022] Further, the concentration of the CA solution is 0.5 wt%, and it is proved by multiple optimization experiments that the functionalized electrode has the best detection effect at this concentration.

[0023] Further, the solvent in the CA solution is acetone and N,N-dimethylformamide with a volume ratio of 3:7.

[0024] Further, the reaction in S3 is carried out at 37±0.5℃ for 8-12 minutes.

[0025] Further, the potential window range of the square wave voltammetry in S4 is 0-0.8 V, the sensitivity is 10 -5 A / V, and the frequency is 10 Hz.

[0026] The application also provides a functionalized electrode for detecting the activity of thrombin in biological products, wherein the thrombin-specific substrate polypeptide (sequence: GLVPRGSC) is modified with ferrocene (Fc) at the N terminus and is fixed on the surface of the screen-printed gold electrode through an Au-S bond, and the electrode surface is coated with a cellulose acetate film.

[0027] 3. Beneficial effects

[0028] Compared with the prior art, the application has the following beneficial effects:

[0029] (1) The method for detecting the activity of thrombin in biological products using the functionalized electrode provided by the application realizes precise control of the molecular direction by fixing the substrate polypeptide through an end Au-S bond, realizes sensitive signal transmission by introducing the ferrocene (Fc) redox signal molecule, and effectively prevents electrode pollution by using the cellulose acetate (CA) protective film.

[0030] (2) The method for detecting the activity of thrombin in biological products using the functionalized electrode provided by the application uses the screen-printed electrode (SPE), has the advantages of small size, easy storage, and convenient carrying, and does not need to be equipped with an electrolytic cell or a large amount of electrolyte. The activity is quantified by using the thrombin-specific substrate peptide (Pe-Fc), which ensures the specificity, accuracy, and reliability of the detection.

[0031] (3) The method for detecting the activity of thrombin in biological products using the functionalized electrode provided by the application can realize convenient detection of the activity of thrombin within 15 minutes, which is extremely short in time consumption.

[0032] (4) The functionalized electrode for detecting thrombin activity in biological products provided by the application can not lose signal after long-term storage, and has good storage stability.

[0033] (5) The application provides a method for detecting thrombin activity in biological products using a functionalized electrode, which can rapidly determine the functional activity of thrombin on site without complex sample pretreatment, and provides an efficient solution for quality control of biological pharmaceutical production and safety monitoring of clinical treatment based on thrombin.

[0034] (6) The application provides a method for detecting thrombin activity in biological products using a functionalized electrode, which can simply and accurately evaluate the activity of thrombin biological products on site, and has important significance for rapid screening of final products in the production process, stability monitoring in the storage and flow process, balancing efficacy and risk in instant medical care, and ensuring patient safety. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 is the principle diagram of electrochemical detection of thrombin activity.

[0036] Figure 2 is the scanning electron microscope image of the bare electrode (A), SPGE / Pe-Fc (B) and SPGE / Pe-Fc / CA (C).

[0037] Figure 3 is the EDS spectrum of the bare electrode (A), SPGE / Pe-Fc (B) and SPGE / Pe-Fc / CA (C).

[0038] Figure 4 is the CV (A) and EIS (B) result diagram of the bare electrode, SPGE / Pe-Fc and SPGE / Pe-Fc / CA.

[0039] Figure 5 is (A) the SWV curve of different thrombin activity (200-1500 U / mL); (B) the linear relationship diagram of thrombin activity and current drop value (n=3, i.e. 3 groups of parallel experiments).

[0040] Figure 6 is the analysis result diagram of inter-electrode repeatability (A) and inter-day repeatability for five consecutive days (n=3) (B).

[0041] Figure 7 is the peak current corresponding to different interferents and 1000 U / mL thrombin (n=3).

[0042] Figure 8 is the storage stability diagram at 4℃ (A) and -20℃ (B) (n=3). DETAILED DESCRIPTION

[0043] The present application is further described in conjunction with the specific examples below.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the term "and / or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0045] Unless otherwise indicated, conventional methods or those described in the literature were employed in the examples. Unless otherwise indicated, reagents or instruments were commercially available and were used according to the manufacturer's instructions.

[0046] As used herein, the term "about" is used to provide flexibility to a numerical range endpoint by providing support for a somewhat greater or lesser inclusion of the numerical value as literally used in the endpoint. The skilled person will be able to determine in light of the skill in the art, the flexibility to be applied to a numerically used value.

[0047] As used herein, the term "at least one of" is intended to mean one or more of the listed items. For example, "at least one of A, B and C" is intended to mean: A alone; B alone; C alone; A and B together; A and C together; B and C together; or A, B, and C together.

[0048] Concentrations, amounts, and other numerical data can be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be construed as having been followed to following the principles of the application. Therefore, unless otherwise specified, this description is to be construed in a manner consistent with the assignment of meaning to numerals within the ranges. Therefore, for example, a concentration range of "from about 1 to about 4.5" should be interpreted to include not only the explicitly recited limits of about 1 to about 4.5, but also to include individual values and sub ranges within the indicated range. The application embraces that subset of this generally described interdependent disclosure which relies on the explicit recitation of those numerical limits in order to support the recited aspect. The application also embraces that subset of this generally described interdependent disclosure which relies on the implied recitation of those numerical limits in order to support the recited aspect. Subsumed in this disclosure is that that subset of the interdependent disclosure which consists of subranges established by some of the recited numerical limits. In this subsumed subset, the numerical limits used to establish the subranges are implicitly recited. For example, a disclosure of a range of "about 1 to about 4.5" subsumes the disclosure of about 1 to about 4, about 1 to about 3, about 1 to about 2, about 1 to about 4.5, about 2 to about 4.5, about 3 to about 4.5, about 4 to about 4.5, about 3.5 to about 4.5, about 3.9 to about 4.5, and about 4.1 to about 4.5. This subsumed subset also subsumes the disclosure of individual values and sub ranges within the indicated ranges. For example, the disclosure of a range of "about 1 to about 4.5" subsumes the disclosure of "about 1, about 2, about 3, about 4, about 4.5, about 1.5, about 2.5, about 3.5, and about 4.5."

[0049] Thrombin for condition optimization (5000 U) was purchased from Nanjing Ley Biological Technology Co., Ltd. (Nanjing, China).

[0050] Thrombin standard reference material (CRM) and fibrinogen standard reference material (CRM) were provided by National Institute for Food and Drug Control (NIFDC, Beijing, China).

[0051] Potassium dihydrogen phosphate (KH2PO4), dipotassium hydrogen phosphate (K2HPO4), cellulose acetate (CA, acetyl content: 39.8 wt%; hydroxyl content: 3.5 wt%), L-cysteine (L-Cys), and chymotrypsin (Chy) were provided by Shanghai Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China).

[0052] Potassium chloride (KCl), sodium chloride (NaCl), urea, L-alanine (L-Ala), and hemoglobin (Hb) were provided by Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China).

[0053] PNGase F and O-glycosidase were from New England Biolabs (Ipswich, MA, USA).

[0054] Human urinary kallikrein (HUK) was provided by Jiangsu Aikang Pharmaceutical Co., Ltd. (Yangzhou, China).

[0055] Tris-HCl buffer (1 M, pH 7.40) was provided by Shanghai Biyun Tian Biotechnology Co., Ltd. (Shanghai, China).

[0056] 0.01M PBS buffer (containing 0.01M KCl, pH 7.40) was prepared by mixing 0.01M K2HPO4 solution (containing 0.01M KCl) and KH2PO4 solution (containing 0.01M KCl) at a ratio of 2:1, and adjusting the pH to 7.40.

[0057] Example 1

[0058] This example provides a method for preparing a functionalized electrode for detecting thrombin activity.

[0059] The ferrocene-modified substrate peptide (Pe-Fc, sequence: Fc-GLVPRGSC) was provided by Shangon Biotech Co., Ltd. (Shanghai, China); the screen-printed gold electrode (SPGE, model: DRP-C220AT-U75, Swiss Vantec China Co., Ltd., Shanghai, China).

[0060] First, the screen-printed gold electrode was rinsed with ultrapure water; the cleaned SPGE was incubated with 10 μL of Pe-Fc solution (1200 μM in 0.01M PBS) at a concentration of 1200 μM overnight at 4°C; then, the unbound substrate polypeptide was removed by thoroughly rinsing with 0.01M PBS buffer; 4 μL of CA solution (AC:DMF = 3:7, v:v) at a concentration of 0.5 wt% was added to the SPGE / Pe-Fc surface to form a CA film; after drying, a functionalized electrode was obtained, named SPGE / Pe-Fc / CA. Stored in a refrigerator at 4°C for subsequent use.

[0061] Example 2

[0062] The functionalized electrode prepared in Example 1 was used to detect the activity of thrombin standard reference material to obtain a standard curve.

[0063] SPGE / Pe-Fc / CA was incubated with 10 μL of thrombin standard reference material (prepared with 0.05 M Tris-HCl, pH 7.40) at different activity concentrations (200, 500, 750, 1000, 1250, 1500 U / mL) at 37 °C for 10 min. After washing with 0.05 M Tris-HCl, square wave voltammetry (SWV) tests were performed in 0.01 M PBS buffer, with a potential window range of 0 to 0.8 V and a sensitivity of 10 -5 A / V, and a frequency of 10 Hz. 0.05 M Tris-HCl without thrombin was used as a blank control group.

[0064] The working principle of the thrombin activity electrochemical detection platform is shown in Figure 1 The specific substrate polypeptide of thrombin is fixed on the gold electrode by Au-S bond through the C-terminal cysteine residue, and the N-terminal modified ferrocene (Fc) serves as an electrochemical signal molecule to generate a quantifiable current signal. To reduce electrode interface pollution, a layer of cellulose acetate (CA) membrane with a nanopore size of 50-200 nm, Figure 2 is coated on the electrode surface. After the introduction of thrombin, specific hydrolysis of the substrate polypeptide occurs, and the Fc-labeled fragment is released from the electrode surface into the solution, causing a decrease in Faraday current, and the signal drop is positively correlated with the activity level of thrombin.

[0065] As shown in Figure 5 A, there is a clear correlation between the peak current size of the SWV curve and the activity of thrombin. When there is no thrombin, the peak current of SWV is the highest, and within the activity range of 200-1500 U / mL, a dependent decrease in peak current is observed with increasing thrombin activity. This relationship can be attributed to the cleavage of the Fc-labeled substrate specific to thrombin, where an increase in enzyme activity leads to more substrate cleavage, resulting in a gradual decrease in peak current. The quantitative relationship between current change and thrombin activity is shown in Figure 5 B. The decrease in peak current is represented by Δip, and Δip has a clear linear relationship with the activity of thrombin (C Thrombin ) (R 2 = 0.9941), and the linear curve is Δip = 1.750C Thrombin + 1.311, where the slope represents the sensitivity of the method. The detection limit (LOD) is 81.1 U / mL, determined by the 3σ standard (where σ represents the standard deviation of the blank measurement).

[0066] Example 3

[0067] To study the surface morphology of the electrode, scanning electron microscopy (SEM) was used for observation. Compared with the bare electrode ( Figure 2 Compared to A), the surface morphology of SPGE / Pe-Fc did not show significant changes. Figure 2 B) indicates that peptide modification has little effect on the electrode surface morphology. This may be because peptide molecules are small and difficult to distinguish clearly under SEM. Conversely, when a cellulose acetate film is coated on the electrode surface ( Figure 2 C) The surface morphology of the electrode has changed significantly.

[0068] To further verify the modification of the peptide on the electrode surface, energy-dispersive X-ray spectroscopy (EDS) was used for elemental analysis of the electrode surface. Figure 3 As shown in Figure A, in addition to gold, metallic elements such as aluminum and sodium, as well as non-metallic elements such as silicon, carbon, and oxygen, were also detected on the surface of the bare electrode. These elements may originate from the ceramic substrate of the screen-printed gold electrode. After modifying the electrode surface with a substrate peptide ( Figure 3 B) In the EDS spectrum, in addition to the elements already present on the bare electrode, sulfur and iron were also observed, indicating that the peptide was successfully modified onto the electrode surface. This result is in good agreement with the presence of sulfur and iron in the Pe-Fc elemental composition. It is noteworthy that the potassium element appearing in the spectrum may originate from potassium ions in the PBS buffer used during peptide dissolution. After coating the electrode surface with a cellulose acetate film, only gold, carbon, and oxygen were detected. Figure 3 C). This is because the cellulose acetate membrane shields signals from other elements, indicating successful modification of the cellulose acetate membrane.

[0069] Example 4

[0070] The preparation process of SPGE / Pe-Fc / CA was further characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). EIS was performed at open-circuit potential in the presence of 5.0 mM [Fe(CN)6]. 3- / 4- The experiment was conducted in 0.1M KCl solution, with a frequency range of 10. 6 The Hz to 0.1Hz frequency range was maintained with an amplitude of 5.0mV. CV values ​​were acquired in the same electrolyte at a scan rate of 0.05V / s within a potential window of -0.4 to 0.9V.

[0071] CV and EIS results are as follows Figure 4 A and Figure 4 As shown in Figure B, the highest redox current and the lowest charge transfer resistance (RB) were observed for the bare electrode. ct After modifying the substrate peptide, the redox current decreased slightly, and R... ctThere was a slight increase. This phenomenon can be attributed to the steric hindrance caused by the substrate peptide, which hindered electron transfer at the electrode surface. Due to the non-conductivity of the polymer, the modification of the CA film led to a significant decrease in the redox current, thereby resulting in R... ct Significantly increased. The change in redox current in the CV plot and the change in R in the EIS plot. ct The consistent changes fully validated the successful preparation of the electrode.

[0072] Example 5

[0073] Given the non-reusable nature of screen-printed electrodes, this study evaluated the reproducibility between different electrodes prepared within the same batch. The current drop of ten electrodes treated with the same active thrombin (1000 U / mL) was measured, and the experiments were completed on the same day. The method followed Example 2, and the relative standard deviation (RSD) of the results was 6.0%. Figure 6 A) indicates good repeatability between different electrodes. Furthermore, the consistency of detection performance between different batches of electrodes prepared over five consecutive days was evaluated. Figure 6 B), the obtained RSD is 3.1%, indicating that the method has good day-to-day repeatability.

[0074] Example 6

[0075] To systematically evaluate the specificity of the developed method, we investigated a variety of potential interfering substances, including sodium chloride (NaCl), urea, L-alanine (L-Ala), L-cysteine ​​(L-Cys), hemoglobin (Hb), human urokinase (HUK), O-glucosidase, PNGase F, and chymotrypsin (Chy), at concentrations ten times that of thrombin. Figure 7 As shown, the peak current generated after the addition of interfering substances is very close to the background signal (blank measurement), indicating that they do not hydrolyze Pe-Fc and have no significant interference with thrombin assay. In contrast, thrombin acting on the target substrate causes a significant decrease in peak current. This result confirms that this method has high specificity for thrombin and can be used for the accurate determination of thrombin activity.

[0076] Example 7

[0077] The storage stability of the prepared electrode (SPGE / Pe-Fc / CA) was assessed by monitoring the current changes before and after the enzyme reaction at different storage durations. Figure 8As shown in Figure 4, the current change remained at the same level as the freshly prepared electrode during the 14-day storage at 4°C. This means that these electrodes have comparable performance with freshly prepared electrodes, indicating that they can be stably stored for at least 14 days under refrigerated conditions. In addition, after being stored at -20°C for different days (up to 28 days), the electrodes can also maintain a similar current change level to the non-frozen control group Figure 8 B).

[0078] Example 8

[0079] This example compares the method used in the present application with the methods for detecting thrombin activity in the existing literature. The references involved are as follows:

[0080] [1] D. Xu, Y. Wang, J. Zhang, X. Tan, D. Guo, S. Wang, X. Hou, Simple and low-cost thrombin activity assay using screen-printed electrodes with portable electrochemiluminescence device, Microchemical Journal 207 (2024) 112147. https: / / doi.org / 10.1016 / j.microc.2024.112147.

[0081] [2] Z. Deng, X. Tan, D. Guo, J. Zhang, D. Xu, X. Hou, S. Wang, J. Zhang, F. Wei, D. Zhang, MXene-sensitized electrochemiluminescence sensor for thrombin activity detection and inhibitor screening, Microchimica Acta 190 (8) (2023) 328. https: / / doi.org / 10.1007 / s00604-023-05906-9.

[0082] [3] Q. Hu, Y. Bao, S. Gan, Y. Zhang, D. Han, L. Niu, Amplified electrochemical biosensing of thrombin activity by RAFT polymerization, Analytical Chemistry 92 (4) (2020) 3470-3476. https: / / doi.org / 10.1021 / acs.analchem.9b05647.

[0083] [4] X. Gao, B. Hu, Z. Zhao, T. Niu, Y. Liu, K. Xu, B. Tang, Synthesis of AuSe bonded nanoprobe for specific detection of thrombin in lung cancer cells, Sensors and Actuators B: Chemical 352 (2022) 130999. https: / / doi.org / 10.1016 / j.snb.2021.130999.

[0084] [5] J. Zhang, D. Xu, Z. Deng, X. Tan, D. Guo, Y. Qiao, Y. Li, X. Hou, S. Wang, J. Zhang, Using tungsten oxide quantum-dot enhanced electrochemiluminescence to measure thrombin activity and screen its inhibitors, Talanta 267 (2024) 125267. https: / / doi.org / 10.1016 / j.talanta.2023.125267.

[0085] The comparison results of the time used in the thrombin activity detection method reported in the above literatures are shown in Table 1. The detection time of the present method can be shortened by more than 50% on the basis of ensuring the analysis performance, greatly improving the detection efficiency, and being suitable for rapid detection in clinic.

[0086] Table 1 Comparison of detection time of the present method and the method reported in the literature

[0087] Method Detection time References ECL 60 min 1 ECL 60 min 2 SWV 4.5h 3 Fluorimetry 35 min 4 SWV 15 min Invention

[0088] The storage stability results were compared with the results reported in the literature (Table 2). The electrode prepared by the proposed method did not show significant signal loss after storage for a certain period of time, indicating that the electrode prepared by the proposed method has good storage stability and can meet the preservation requirements in practical applications.

[0089] Table 2 Comparison of storage stability of the established method and the method reported in the literature

[0090]

[0091] Example 9

[0092] To verify the feasibility of the developed method for monitoring the activity of thrombin in biological products, we used electrochemical methods and the standard method for determining the potency of thrombin lyophilized powder specified in the Chinese Pharmacopoeia (2020 edition) Part II to analyze different specifications of commercially available thrombin preparations (reagent: 1000 U / branch; lyophilized powder drug: 500 U / branch) in parallel. The results are shown in Table 3 below.

[0093] Table 3 Comparison of thrombin potency results determined by electrochemical method and Chinese Pharmacopoeia method (n = 3)

[0094]

[0095]

[0096] The electrochemical method showed comparable accuracy to the Chinese Pharmacopoeia method, and all test samples showed similar activity levels between the two analysis methods. To statistically verify these observations, a paired sample t-test analysis was performed on the activity results of the 1000 U / branch thrombin sample. Statistical analysis showed no significant difference between the two measurement methods (p = 0.592 > 0.05), further verifying the reliability of the electrochemical method in assessing thrombin activity. Compared to the multi-step sample preparation process involved in the Chinese Pharmacopoeia method, the electrochemical method only requires simple dissolution and dilution steps, while eliminating the need for thrombin process endpoint determination, reducing the dependence of the determination results on the proficiency of the operator. The analysis time of the method is less than 15 minutes, and it is expected to become a reliable solution for rapid evaluation of the activity of thrombin biological products.

Claims

1. A method for detecting thrombin activity in biological products using a functionalized electrode, characterized in that, Includes the following steps: S1: Immobilize the substrate peptide onto the surface of a screen-printed gold electrode; S2: Coat the surface of the electrode with a cellulose acetate film to obtain a functionalized electrode; S3: Add a solution containing thrombin to the functionalized electrode for reaction; S4: After washing the reacted electrode, the thrombin activity was measured using square wave voltammetry and obtained from the change in peak current.

2. The method for detecting thrombin activity in biological products using a functionalized electrode according to claim 1, characterized in that, The substrate polypeptide is immobilized on the surface of a screen-printed gold electrode via Au-S bonds.

3. The method for detecting thrombin activity in biological products using a functionalized electrode according to claim 1 or 2, characterized in that, The substrate polypeptide is also modified with ferrocene at its N-terminus.

4. The method for detecting thrombin activity in biological products using a functionalized electrode according to claim 3, characterized in that, The amino acid sequence of the substrate polypeptide is: GLVPRGSC.

5. The method for detecting thrombin activity in biological products using a functionalized electrode according to claim 4, characterized in that, Before immobilizing the substrate peptide, the screen-printed gold electrode was washed with ultrapure water.

6. The method for detecting thrombin activity in biological products using a functionalized electrode according to any one of claims 1-5, characterized in that, The process of immobilizing the substrate peptide on the surface of a screen-printed gold electrode involves incubating the cleaned screen-printed gold electrode overnight at 4°C with a substrate peptide solution containing 1150-1300 μM N-terminus modified with ferrocene, followed by thorough rinsing with PBS buffer.

7. The method for detecting thrombin activity in biological products using a functionalized electrode according to claim 1, characterized in that, Coating the surface of a screen-printed gold electrode with a cellulose acetate film involves dropping 3-6 μL of a 0.4-0.6 wt% CA solution onto the surface of the screen-printed gold electrode. The volume ratio of acetone to N,N-dimethylformamide in the CA solution is 2:8-4:

6.

8. The method for detecting thrombin activity in biological products using a functionalized electrode according to claim 1, characterized in that, The reaction described in S3 was carried out at 37±0.5℃ for 8-12 minutes.

9. The method for detecting thrombin activity in biological products using a functionalized electrode according to claim 1, characterized in that, The square wave voltammetry potential window range described in S4 is 0 to 0.8V, and the sensitivity is 10. -5 A / V, frequency 10Hz.

10. A functionalized electrode for detecting thrombin activity in biological products, characterized in that, A thrombin-specific substrate peptide is immobilized on the surface of a screen-printed gold electrode via Au-S bonds. The N-terminus of the substrate peptide is modified with ferrocene, and the electrode surface is coated with a cellulose acetate membrane. The amino acid sequence of the substrate peptide is: GLVPRGSC.

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