A micro electrochemiluminescent biosensor for measuring thrombin activity, preparation method and application thereof

Abstract

The present invention discloses a micro electrochemiluminescent biosensor for measuring thrombin activity, a preparation method and its application, which belong to the field of electrochemiluminescent biosensors. A micro electrochemiluminescent biosensor is constructed using a thrombin-specific peptide as an activity recognition unit and a terpyridine ruthenium-tri-n-propylamine-ferrocene system as a luminescence quenching system. Only 10 μL of whole blood or serum is needed to achieve rapid and accurate measurement of thrombin activity. Combining the micro electrochemiluminescent biosensor with a portable device for thrombin activity detection can greatly improve the convenience and timeliness of the detection, and is expected to achieve real-time monitoring at the bedside, providing a fast and accurate diagnostic basis for the clinic and optimizing patient treatment management.

Description

Technical Field

[0001] The present invention belongs to the technical field of electrochemiluminescence biosensors, and in particular relates to a micro electrochemiluminescence biosensor for measuring thrombin activity, a preparation method and an application thereof. Background Art

[0002] Thrombin activity testing is crucial for assessing a patient's coagulation function and diagnosing and monitoring blood disorders. It helps physicians diagnose various coagulation disorders, such as bleeding disorders, thrombosis, and coagulation factor deficiencies or abnormalities. Before surgery, physicians typically perform a thrombin activity test to ensure the patient's coagulation function is normal, which helps prevent the risk of intraoperative or postoperative bleeding. Because certain medications (such as anticoagulants and antiplatelet drugs) can affect thrombin activity, regular testing can ensure that the patient maintains adequate coagulation status during drug treatment. For patients with coagulation disorders, regular thrombin activity testing helps monitor disease progression and treatment effectiveness. In summary, thrombin activity testing plays an important role in clinical practice, helping to ensure that the patient's coagulation function is normal, thereby maintaining health and preventing complications. Currently, the four coagulation tests (prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), and fibrinogen ionization (FIB)) are commonly used in clinical practice to assess coagulation or bleeding status in vivo, but they do not directly reflect thrombin activity.

[0003] Many studies on thrombin determination methods have been reported in the literature, such as colorimetry, fluorescence analysis (FL), bioluminescence resonance energy transfer (BRET), surface-enhanced Raman scattering (SERS) technology, and electrochemical analysis (EC). Most of these determination techniques are based on aptamers and can only reflect the amount of thrombin but cannot identify the "activity" of thrombin. Therefore, they cannot be used as methods for determining thrombin activity.

[0004] The activity of thrombin needs to be based on the recognition of thrombin-specific peptides and reacts through the effect of cleaving substrate peptides. Electrochemiluminescence is an analytical method that combines electrochemistry and chemiluminescence. When thrombin acts on an electrochemiluminescent-labeled substrate, it causes a change in the luminescent signal, thereby realizing the quantitative detection of thrombin activity. Xu et al. constructed an electrochemiluminescence sensor for measuring thrombin activity, using bifunctional peptides and low-toxic quantum dots (as luminescent markers), and achieved sensitive detection of target molecules by the ratio of colorimetric and electrochemiluminescence signals (Xu Y, Wang Z, Ding C, Luo X. Ratiometric antifouling electrochemiluminescence biosensor based on bi-functional peptides and low toxic quantum dots. Sensors and Actuators B: Chemical. 2020; 322.). This method is based on an MPI-E electrochemiluminescence instrument, which is large in size, has a relatively complex construction process, is difficult to control stability, and is subject to certain limitations in clinical application. Summary of the Invention

[0005] In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide a micro electrochemiluminescence biosensor for measuring thrombin activity, a preparation method and its application, so as to solve the technical problems of existing detection instruments being large in size, unable to detect thrombin activity, having a complex detection process and difficult to control stability.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention discloses a micro electrochemiluminescent biosensor for measuring thrombin activity, comprising a SPE electrode on which terpyridine ruthenium, gold, and a thrombin-specific peptide are sequentially immobilized, and blank sites on the surface of the SPE electrode are all blocked;

[0008] The thrombin-specific peptide segment is a peptide segment connected with ferrocene, and the peptide bond of the peptide segment can be cleaved by thrombin.

[0009] Preferably, the thrombin-specific peptide is Mpr-GGRK(Fc), Mpr-VPRK(Fc), Mpr-D(OBZL)PRK(Fc) or Mpr-FPRK(Fc);

[0010] The synthesis method of the thrombin-specific peptide segment comprises: coupling Mpr with a resin, then sequentially adding corresponding amino acids to the Mpr fixed on the resin, and then converting ferrocenecarboxylic acid into the corresponding acyl chloride, which is then connected to the amino acid terminal to obtain the thrombin-specific peptide segment.

[0011] Further preferably, the thrombin-specific peptide is Mpr-GGRK(Fc).

[0012] Preferably, terpyridine ruthenium is immobilized on the SPE electrode via a perfluorinated resin membrane.

[0013] Preferably, the blank sites on the surface of the SPE electrode are all blocked with mercaptohexanol.

[0014] The second aspect of the present invention discloses a method for preparing the micro electrochemiluminescent biosensor for measuring thrombin activity, comprising the following steps:

[0015] 1) Mixing a perfluorinated resin, ethanol, water, and terpyridine ruthenium to prepare a terpyridine ruthenium@perfluorinated resin solution; drop-coating the terpyridine ruthenium@perfluorinated resin solution onto the surface of a SPE electrode to prepare a terpyridine ruthenium@perfluorinated resin / SPE sensor;

[0016] 2) dripping a tetrachloroauric acid solution onto the surface of the terpyridine ruthenium@perfluorinated resin / SPE sensor prepared in step 1), and plating the sensor surface with gold to prepare a gold / terpyridine ruthenium@perfluorinated resin / SPE sensor;

[0017] 3) The thrombin-specific peptide solution was drop-coated onto the surface of the gold / terpyridine ruthenium@perfluorinated resin / SPE sensor for reaction, and the unbound blank sites were blocked with mercaptohexanol to prepare the peptide / gold / terpyridine ruthenium@perfluorinated resin / SPE sensor.

[0018] Preferably, in step 1), the volume ratio of the perfluorinated resin, ethanol, water and terpyridine ruthenium is 1:1:1:16.

[0019] Preferably, in step 2), the sensor surface is plated with gold using an electrodeposition method.

[0020] Further preferably, during the electrodeposition process, the electrodeposition potential is -0.6 V and the gold plating time is 5 s.

[0021] Preferably, in step 3), the reaction condition is 25° C. for 6 h.

[0022] The third aspect of the present invention discloses the use of the above-mentioned micro electrochemiluminescence biosensor for measuring thrombin activity in detecting thrombin activity.

[0023] Preferably, the micro electrochemiluminescent biosensor for measuring thrombin activity is capable of detecting thrombin activity in serum, plasma and whole blood.

[0024] The fourth aspect of the present invention discloses a thrombin activity detection system, comprising a portable electrochemiluminescence detector and the above-mentioned micro electrochemiluminescence biosensor for measuring thrombin activity.

[0025] The fifth aspect of the present invention discloses a method for detecting thrombin activity, wherein the blood to be tested is dropped onto the surface of the micro electrochemiluminescence biosensor for measuring thrombin activity, and the thrombin activity value is obtained based on the change in the thrombin luminescence signal before and after the reaction.

[0026] Furthermore, by preparing thrombin standard solutions of different activities and using the above-mentioned micro-electrochemiluminescence biosensor for measuring thrombin activity to detect the change value of the thrombin luminescence signal before and after the reaction, a linear regression equation of thrombin activity and thrombin luminescence signal change value is established; then, the thrombin luminescence signal change value of the blood to be tested is substituted into the linear regression equation of thrombin activity and thrombin luminescence signal change value to obtain the thrombin activity value.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention provides a micro electrochemiluminescent biosensor for measuring thrombin activity, which 1) adopts an SPE chip electrode to ensure the portability of the micro electrochemiluminescent biosensor. 2) Trispyridine ruthenium is fixed on the SPE electrode and can be used as an electrochemiluminescent signal probe. 3) Gold is fixed on the surface of trispyridine ruthenium@perfluorinated resin / SPE electrode, which not only enhances the electron transfer rate, but also provides a binding site for thrombin-specific peptide segments, accurately and directly reflecting the value of thrombin activity in the blood. 4) The blank sites on the surface of the SPE electrode are all blocked to ensure the accuracy of the test results. 5) The thrombin-specific peptide segment connected to the sensor can be specifically recognized and cut by thrombin, and the change in the electrochemiluminescent signal is affected by the distance of Fc, thereby realizing activity detection. Compared with fluorescence technology, this micro electrochemiluminescence biosensor can detect thrombin activity without relying on excitation light sources; compared with existing chemiluminescence instruments, the construction process is simple, the device is 1 / 5 of the volume of conventional instruments, it is easy to carry, the detection results are accurate, stable, and specific, and it has a wide range of application scenarios, such as bedside testing, home testing, outdoor tourist site testing, clinical diagnosis and monitoring, surgery and trauma management, drug efficacy monitoring, guidance for the use of new oral anticoagulants, disease status assessment, etc., which can provide an important basis for coagulation or bleeding risk diagnosis and personalized drug delivery.

[0029] Furthermore, using specific peptides Mpr-GGRK(Fc), Mpr-VPRK(Fc), Mpr-D(OBZL)PRK(Fc) or Mpr-FPRK(Fc) as thrombin activity recognition units has better specificity. The Fc in the specific peptide is away from the SPE surface, which increases the electrochemiluminescence signal. The activity of thrombin is evaluated by measuring the change in the electrochemiluminescence signal intensity, which can accurately and directly reflect the value of thrombin activity in the blood.

[0030] The present invention provides a method for detecting thrombin activity, which combines a miniature portable electrochemiluminescence detector with a prepared miniature electrochemiluminescence biosensor for measuring thrombin activity, carries out an enzyme-catalyzed reaction on an SPE chip electrode fixed with terpyridine ruthenium, gold, and a thrombin-specific peptide segment, and obtains the thrombin activity value based on the change in the thrombin luminescence signal before and after the reaction. Bedside detection can be achieved only by measuring the change in the electrochemiluminescence signal, with high anti-interference ability, and can realize real-time analysis of thrombin activity in the blood. In addition, the detection method only requires a sample volume of 10 μL, and micro-blood sampling from the fingertips can meet the measurement requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of the preparation process of the micro electrochemiluminescence biosensor for measuring thrombin activity of the present invention;

[0032] Figure 2 The diagrams show the molecular docking results of different specific peptides of the present invention with thrombin; wherein, (a) is Mpr-GGRK(Fc), (b) is Mpr-VPRK(Fc), (c) is Mpr-D(OBZL)PRK(Fc), and (d) is Mpr-FPRK(Fc);

[0033] Figure 3 This is a working curve result diagram of the micro electrochemiluminescence biosensor for measuring thrombin activity of the present invention;

[0034] Figure 4 This figure shows the specificity evaluation results of the micro electrochemiluminescence biosensor for measuring thrombin activity according to the present invention. DETAILED DESCRIPTION

[0035] To facilitate understanding of the features and effects of the present invention by those skilled in the art, the following provides a general description and definition of the terms and expressions used in the specification and claims. Unless otherwise indicated, all technical and scientific terms used herein have the ordinary meanings as understood by those skilled in the art regarding the present invention. In the event of conflict, the definitions in this specification shall prevail.

[0036] The theories or mechanisms described and disclosed herein, whether correct or incorrect, should not limit the scope of the present invention in any way, that is, the present invention can be implemented without being limited by any specific theory or mechanism.

[0037] Herein, all features such as values, amounts, amounts, and concentrations defined in numerical ranges or percentage ranges are for brevity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to include and specifically disclose all possible subranges and individual values ​​within the range (including integers and fractions).

[0038] In this document, unless otherwise specified, “include,” “including,” “contains,” “has” or similar terms cover the meanings of “consisting of” and “mainly consisting of,” for example, “A includes a” covers the meanings of “A includes a and other” and “A only includes a.”

[0039] In this document, for the sake of brevity, not all possible combinations of the various technical features in each embodiment or example are described. Therefore, as long as there are no contradictions in the combination of these technical features, the various technical features in each embodiment or example can be combined in any way, and all possible combinations should be considered to be within the scope of this specification.

[0040] As used herein, the term "thrombin-specific peptide" refers to a peptide linked to ferrocene whose peptide bond can be cleaved by thrombin. The thrombin-specific peptide includes, but is not limited to, Mpr-GGRK(Fc), Mpr-VPRK(Fc), Mpr-D(OBZL)PRK(Fc), and Mpr-FPRK(Fc); wherein "Mpr" represents thioglycolic acid, "G" represents glycine, "R" represents arginine, "K" represents lysine, "V" represents valine, "P" represents proline, "D(OBZL)" represents benzyl-D-aspartic acid, "F" represents phenylalanine, and "Fc" represents ferrocene.

[0041] Here, the synthesis method of Mpr-GGRK(Fc) is as follows: first, Mpr is coupled to a resin via solid-phase synthesis, so that Mpr is fixed to the resin via the thiol group; second, glycine (G), glycine (G), arginine (R), and lysine (K) are sequentially added to the resin-immobilized Mpr using standard solid-phase peptide synthesis (SPPS) methods to obtain Mpr-GGRK; finally, ferrocenecarboxylic acid is converted into the corresponding acyl chloride, which then reacts with the K at the end of Mpr-GGRK to obtain Mpr-GGRK(Fc).

[0042] Herein, the synthesis methods of Mpr-VPRK(Fc), Mpr-D(OBZL)PRK(Fc), and Mpr-FPRK(Fc) refer to the synthesis method of pr-GGRK(Fc), the difference being the different linked amino acids.

[0043] The present invention provides a method for preparing a micro electrochemiluminescent biosensor for measuring thrombin activity, such as Figure 1 As shown, the following steps are included:

[0044] 1. Terpyridine ruthenium@perfluorinated resin modified electrode

[0045] A Ru@perfluorinated resin solution was prepared by mixing perfluorinated resin, ethanol, water, and terpyridine (RtP) in a volume ratio of 1:1:1:16. This RtP@perfluorinated resin solution was then drop-coated onto the surface of a screen-printed electrode (SPE) to create a RtP@perfluorinated resin / SPE sensor.

[0046] In this step, ruthenium terpyridine was immobilized on the SPE electrode through a perfluorinated resin membrane as an electrochemiluminescence signal probe.

[0047] 2. Gold / terpyridine ruthenium@perfluorinated resin modified electrode

[0048] A tetrachloroauric acid solution was drop-coated onto the surface of a terpyridine ruthenium@perfluorinated resin / SPE sensor, and the sensor surface was gold-plated by electrodeposition. During the electrodeposition process, the electrodeposition potential was -0.6 V, and the gold plating time was 5 s. Thus, a gold / terpyridine ruthenium@perfluorinated resin / SPE sensor was prepared.

[0049] In this step, gold was fixed on the surface of terpyridine ruthenium@perfluorinated resin / SPE electrode by electrodeposition, which not only enhanced the electron transfer rate but also provided binding sites for thrombin-specific peptides.

[0050] 3. Preparation of micro electrochemiluminescence biosensor

[0051] The thrombin-specific peptide was drop-coated onto the surface of the gold / terpyridine ruthenium@perfluorinated resin / SPE sensor and reacted at 25°C for 6-12 hours. The unbound blank sites were blocked with mercaptohexanol to prepare the peptide / gold / terpyridine ruthenium@perfluorinated resin / SPE sensor.

[0052] In this step, a thrombin-specific peptide was immobilized on the surface of a gold / terpyridine ruthenium@perfluorinated resin / SPE electrode via a gold-sulfur bond as a specific recognition probe for thrombin.

[0053] The present invention also provides a micro-electrochemiluminescent biosensor for measuring thrombin activity, prepared by the above method. The micro-electrochemiluminescent biosensor comprises a SPE electrode, on which ruthenium terpyridine, gold, and a thrombin-specific peptide are sequentially immobilized. Blank sites on the SPE electrode surface are blocked with mercaptohexanol. This micro-electrochemiluminescent biosensor utilizes a ruthenium terpyridine-tri-n-propylamine-ferrocene system as a luminescent system. In the presence of active thrombin, the micro-electrochemiluminescent biosensor specifically recognizes and cleaves the thrombin-specific peptide, displacing the Fc residue in the thrombin-specific peptide from the SPE surface and resulting in an increased electrochemiluminescent signal. Thrombin activity is assessed by measuring changes in the electrochemiluminescent signal intensity.

[0054] The present invention also provides a method for detecting thrombin activity using the micro electrochemiluminescence biosensor, comprising the following steps:

[0055] 1. Preparation of thrombin standard solution

[0056] Take Na + Thrombin standard solutions with different activities were prepared by using 140 mmol / L phosphate buffered saline as diluent using thrombin freeze-dried powder.

[0057] 2. Electrochemiluminescence signal detection (before thrombin reaction)

[0058] Tripropylamine (TPA) was added to the surface of the peptide / gold / terpyridine ruthenium@perfluorinated resin / SPE biosensor, and then placed in a portable electrochemiluminescence instrument for electrochemiluminescence signal detection. The PMT was set to -800 V, the scan rate was set to 300 mV / s, and the potential range was set to -1 to 1.25 V to obtain the thrombin basal value.

[0059] 3. Electrochemiluminescence signal detection (after thrombin reaction)

[0060] Different active thrombin solutions were added dropwise onto the surface of the peptide / gold / terpyridine ruthenium@perfluorinated resin / SPE biosensor, reacted at 37°C for 30 to 60 minutes, and then placed in a portable electrochemiluminescence detector for electrochemiluminescence signal detection. The PMT was set to -800 V, the scan rate was set to 300 mV / s, and the potential range was set to -1 to 1.25 V to obtain the thrombin electrochemiluminescence signal value.

[0061] 4. Calculate the change in thrombin luminescence signal ΔECL

[0062] The thrombin luminescence signal change value ΔECL of each group was calculated according to the following formula:

[0063] ΔECL=│Thrombin electrochemiluminescence signal value - thrombin basal value│

[0064] 5. Establish a standard curve for thrombin activity detection

[0065] The logarithm of the activity of the thrombin standard solution was used as the abscissa x and ΔECL was used as the ordinate y. The linear regression equation of thrombin activity and ΔECL was obtained through linear regression analysis.

[0066] 6. Thrombin activity detection

[0067] The blood to be tested was dropped onto the surface of the peptide / gold / terpyridine ruthenium@perfluorinated resin / SPE biosensor and reacted at 37°C for 30 to 60 minutes. The peptide / gold / terpyridine ruthenium@perfluorinated resin / SPE biosensor with the blood to be tested was then placed in a portable electrochemiluminescence instrument for electrochemiluminescence signal detection. The PMT was set to -800 V, the scan rate was set to 300 mV / s, and the potential range was set to -1 to 1.25 V to obtain the electrochemiluminescence signal value of the thrombin to be tested.

[0068] The obtained electrochemiluminescence signal value of the thrombin to be tested is substituted into the linear regression equation of thrombin activity and ΔECL to obtain the thrombin activity value.

[0069] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0070] The following examples utilize conventional instruments and equipment in the art. Experimental methods in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or as recommended by the manufacturer. The various raw materials used in the following examples, unless otherwise specified, were conventional commercially available products, with specifications conventional in the art.

[0071] In the early stage of the experiment, the specific peptides Mpr-GGRK(Fc), Mpr-VPRK(Fc), Mpr-D(OBZL)PRK(Fc) and Mpr-FPRK(Fc) were synthesized by Shanghai Shenggong Biotechnology Co., Ltd. according to the above method, and the specific constant K cat / K m The affinity of each specific peptide to thrombin was tested, and the specific constants of each specific peptide were 209.4, 23.0, 43.1 and 1.5 respectively. The interaction between the specific peptide and thrombin was tested by molecular docking. The results are as follows Figure 2As shown in the figure, the docking effect was scored using the Surflex-dock algorithm, and Mpr-GGRK(Fc) had the highest total docking score of 13.35. These results indicate that the specific peptide Mpr-GGRK(Fc) has the highest affinity and specificity for thrombin.

[0072] Taking the specific peptide Mpr-GGRK (Fc) as an example, the micro electrochemiluminescence biosensor for thrombin activity and its preparation and detection methods are described below.

[0073] 1. Preparation of a micro-electrochemiluminescence biosensor for measuring thrombin activity

[0074] 1. Terpyridine ruthenium@perfluorinated resin modified electrode

[0075] A Ruthenium terpyridine@perfluorinated resin solution was prepared by mixing perfluorinated resin, ethanol, water, and 80 mmol / L Ruthenium terpyridine in a volume ratio of 1:1:1:16. A 2.5 μL droplet of this Ruthenium terpyridine@perfluorinated resin solution was applied to the surface of a SPE electrode and allowed to air-dry. The SPE electrode surface was then rinsed with 0.01 mol / L phosphate buffer solution and blotted dry with filter paper to prepare a Ruthenium terpyridine@perfluorinated resin / SPE sensor.

[0076] 2. Gold / terpyridine ruthenium@perfluorinated resin modified electrode

[0077] Add 20 μL of 0.1 mol / L tetrachloroauric acid solution to the surface of the terpyridine ruthenium@perfluorinated resin / SPE sensor prepared in Step 1. Perform potentiostatic deposition at -0.6 V for 5 seconds to plate gold on the surface of the terpyridine ruthenium@perfluorinated resin / SPE sensor. Carefully remove the remaining tetrachloroauric acid solution with filter paper, air-dry at room temperature in the dark, and rinse the unfixed gold on the electrode surface with 0.01 mol / L phosphate buffer solution. Blot dry with filter paper to complete the gold / terpyridine ruthenium@perfluorinated resin / SPE sensor.

[0078] 3. Preparation of micro electrochemiluminescence biosensor

[0079] First, 2.5 μL of 10 mmol / L -SH-modified peptide substrate Mpr-GGRK(Fc) (98% purity, synthesized by Shanghai Sangon Biotechnology Co., Ltd.) was added dropwise to the gold / terpyridine ruthenium@perfluorinated resin / SPE sensor surface prepared in step 2. The reaction was incubated at 25°C in the dark for 6 h. The -SH-modified Mpr-GGRK(Fc) was fixed to the modified electrode surface using a gold-sulfur bond to prepare a peptide / gold / terpyridine ruthenium@perfluorinated resin / SPE sensor. Then, 2.5 μL of 1 mmol / L mercaptohexanol was added to the surface of the peptide / gold / terpyridine ruthenium@perfluorinated resin / SPE sensor and placed in the dark at 25°C for 1 h to block the nonspecific binding sites on the electrode surface and prevent nonspecific adsorption. The sensor was washed several times with 0.01 mol / L phosphate buffer solution to remove excess peptide substrate and mercaptohexanol to obtain a peptide / gold / terpyridine ruthenium@perfluorinated resin / SPE biosensor.

[0080] 2. Determination of Thrombin Activity Using a Micro-electrochemiluminescence Biosensor

[0081] 1. Preparation of thrombin standard solution

[0082] Take Na + Thrombin standard solutions with activities of 0.1 m U / mL, 0.316 m U / mL, 1 m U / mL, 3.16 m U / mL, 10 m U / mL, 31.6 m U / mL, 0.1 U / mL, 0.316 U / mL, and 1.0 U / mL were prepared using 140 mmol / L phosphate buffered saline as the diluent.

[0083] 2. Electrochemiluminescence signal detection (before thrombin reaction)

[0084] 20 μL of 93.6 mmol / L TPA was added to the surface of the peptide / gold / terpyridine ruthenium@perfluorinated resin / SPE biosensor prepared in step 1, and then placed in a RPEL-B portable electrochemiluminescence instrument for electrochemiluminescence signal detection. The PMT was set to -800 V, the scan rate was set to 300 mV / s, and the potential range was set to -1 to 1.25 V to obtain the thrombin basal value.

[0085] After the test is completed, the sensor surface is cleaned multiple times with 0.01 mol / L phosphate buffer solution to remove residual TPA on the sensor surface to prevent it from affecting the subsequent thrombin incubation and test results.

[0086] 3. Electrochemiluminescence signal detection (after thrombin reaction)

[0087] The different active thrombin solutions prepared in step 1 were added dropwise to the surface of the peptide / gold / terpyridine ruthenium@perfluorinated resin / SPE biosensor prepared in step 1, reacted in a constant temperature incubator at 37°C for 60 minutes, and then placed in a RPEL-B portable electrochemiluminescence detector for electrochemiluminescence signal detection. The PMT was set to -800 V, the scan rate was set to 300 mV / s, and the potential range was set to -1 to 1.25 V to obtain the thrombin electrochemiluminescence signal value.

[0088] After the reaction was completed, the sensor surface was washed several times with 0.01 mol / L phosphate buffer solution to terminate the reaction.

[0089] 4. Calculate the change in thrombin luminescence signal ΔECL

[0090] The thrombin luminescence signal change value ΔECL of each group was calculated according to the following formula:

[0091] ΔECL=│Thrombin electrochemiluminescence signal value - thrombin basal value│

[0092] 5. Establish a standard curve for thrombin activity detection

[0093] like Figure 3 As shown, with the logarithm of thrombin activity as the abscissa x and ΔECL as the ordinate y, linear regression analysis was performed to obtain the linear regression equation of thrombin activity and ΔECL: y=526.36x+2519.88, and the correlation coefficient was r=0.997 (n=3).

[0094] In the linear regression equation, the linear correlation coefficient r is greater than 0.990, and the detection limit is 4.02×10 -5 U / mL. This result shows that the established method has good accuracy and high detection sensitivity.

[0095] 6. Thrombin activity detection

[0096] 2.5 μL of thrombin (purchased from Hunan Yige Pharmaceutical Co., Ltd.) was dropped onto the surface of the peptide / gold / terpyridine ruthenium@perfluorinated resin / SPE biosensor prepared in step 1 and reacted at 37°C for 1 hour. Then, the peptide / gold / terpyridine ruthenium@perfluorinated resin / SPE biosensor with thrombin was placed in a RPEL-B portable electrochemiluminescence instrument for electrochemiluminescence signal detection. The PMT was set to -800 V, the scan rate was set to 300 mV / s, and the potential range was set to -1 to 1.25 V to obtain the thrombin electrochemiluminescence signal detection value.

[0097] After the reaction was completed, the sensor surface was washed several times with 0.01 mol / L phosphate buffer solution to terminate the reaction.

[0098] The thrombin electrochemiluminescence signal detection value is substituted into the thrombin activity detection standard curve obtained in step 5 to obtain the thrombin activity value.

[0099] 3. Verification of the detection effect of micro electrochemiluminescence biosensor

[0100] 1. Sensor life evaluation experiment of micro electrochemiluminescence sensor

[0101] Twelve micro-electrochemical biosensors were prepared simultaneously according to the preparation method of step 1. Three sensors were taken out on the 1st day, 3rd day, 6th day and 9th day, and 1 U / mL thrombin solution was dripped on them. After reacting in a constant temperature incubator at 37°C for 60 minutes, they were placed in an RPEL-B portable electrochemiluminescence detector for detection. The PMT was set to -800 V, the scan rate was set to 300 mV / s, and the potential range was set to -1 to 1.25 V. The electrochemiluminescence response signal value of the sensor was recorded.

[0102] Table 1 Sensor life evaluation results of electrochemiluminescence sensors

[0103]

[0104] The test results are shown in Table 1, which show that the electrochemiluminescence response signal value measured by the micro-electrochemical biosensor can still be maintained at a relatively stable level during the 9-day storage period.

[0105] 2. Inter-sensor precision evaluation experiment of electrochemiluminescence sensor

[0106] Five micro electrochemiluminescence biosensors were prepared according to the preparation method of step 1. 100 μU / mL thrombin solution was drop-coated on each of them. After reacting in a constant temperature incubator at 37°C for 60 minutes, the sensors were placed in a RPEL-B portable electrochemiluminescence detector for detection. The PMT was set to -800 V, the scan rate was set to 300 mV / s, and the potential range was set to -1 to 1.25 V. The electrochemiluminescence response signal value of each electrode was recorded.

[0107] Table 2 Intermediate precision evaluation results of electrochemiluminescence sensor

[0108]

[0109] The test results are shown in Table 2, which show that the intermediate precision of the electrochemiluminescence response signal values ​​between sensors is less than 5%, indicating that the inter-batch differences of the electrochemiluminescence sensors are small.

[0110] 3. Specificity evaluation experiment of electrochemiluminescence sensor

[0111] Fifteen microelectrochemical biosensors were prepared simultaneously according to the preparation method in step 1. 10 U / mL human albumin (HSA), immunoglobulin G (IgG), hemoglobin (Hb), lysozyme (Lysozyme), and 1 U / mL thrombin (TB) were drop-coated three times per group. After incubation at 37°C for 60 minutes, the sensors were placed in a RPEL-B portable electrochemiluminescence detector. The PMT was set to -800 V, the scan rate was set to 300 mV / s, and the potential range was set to -1 to 1.25 V. The electrochemiluminescence response signal of each protein was recorded.

[0112] Test results see Figure 4 The results showed that the response signal of the micro-biosensor to thrombin was more than 8 times that of other proteins, indicating that the micro-biosensor had good selectivity.

[0113] 4. Accuracy evaluation experiment of electrochemiluminescence sensor

[0114] Thrombin activity in serum, plasma, and whole blood from healthy individuals was detected using the standard addition method. Microelectrochemical biosensors were prepared according to the preparation method in step 1. Serum, plasma, and whole blood sample solutions were drop-coated and incubated in a 37°C constant temperature incubator for 60 minutes. The sensors were then placed in an RPEL-B portable electrochemiluminescence detector (ECL) with the PMT set to -800 V, a scan rate set to 300 mV / s, and a potential range of -1 to 1.25 V. The ECL response signal from each electrode was recorded.

[0115] Table 3 Accuracy evaluation of electrochemiluminescence sensor in serum

[0116]

[0117] Table 4 Accuracy evaluation of electrochemiluminescence sensor in plasma

[0118]

[0119] Table 5 Accuracy evaluation of electrochemiluminescence sensor in whole blood

[0120]

[0121]

[0122] The test results are shown in Tables 3 to 5, which indicate that the biosensor has good accuracy in detecting thrombin activity in blood samples.

[0123] The above content is only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A micro electrochemiluminescence biosensor for measuring thrombin activity, characterized in that: The method comprises an SPE electrode, on which terpyridine ruthenium, gold and a thrombin-specific peptide are sequentially fixed, and blank sites on the surface of the SPE electrode are all blocked; The thrombin-specific peptide is a peptide connected to ferrocene, and the peptide bond can be cleaved by thrombin. The thrombin-specific peptide is Mpr-GGRK(Fc), Mpr-VPRK(Fc), Mpr-D(OBZL)PRK(Fc) or Mpr-FPRK(Fc); the synthesis method of the thrombin-specific peptide is: coupling Mpr with resin, and then sequentially adding the corresponding amino acids to the Mpr fixed on the resin, and then converting ferrocenecarboxylic acid into the corresponding acyl chloride, and then connecting it to the amino acid end to obtain the thrombin-specific peptide.

2. A micro electrochemiluminescence biosensor for measuring thrombin activity according to claim 1, characterized in that: Ruthenium terpyridine is immobilized on the SPE electrode via a perfluorinated resin membrane.

3. A micro electrochemiluminescence biosensor for measuring thrombin activity according to claim 1, characterized in that: The blank sites on the surface of the SPE electrode were blocked with mercaptohexanol.

4. The method for preparing the micro electrochemiluminescence biosensor for measuring thrombin activity according to any one of claims 1 to 3, characterized in that: The following steps are involved: 1) Mixing perfluorinated resin, ethanol, water, and terpyridine ruthenium to prepare a terpyridine ruthenium@perfluorinated resin solution; drop-coating the terpyridine ruthenium@perfluorinated resin solution onto the surface of a SPE electrode to prepare a terpyridine ruthenium@perfluorinated resin / SPE sensor; 2) dripping a tetrachloroauric acid solution onto the surface of the terpyridine ruthenium@perfluorinated resin / SPE sensor prepared in step 1), and then gold-plating the sensor surface to prepare a gold / terpyridine ruthenium@perfluorinated resin / SPE sensor; 3) The thrombin-specific peptide solution was drop-coated onto the surface of the gold / terpyridine ruthenium@perfluorinated resin / SPE sensor for reaction, and the unbound blank sites were blocked with mercaptohexanol to prepare the peptide / gold / terpyridine ruthenium@perfluorinated resin / SPE sensor.

5. The preparation method according to claim 4, characterized in that In step 1), the volume ratio of the perfluorinated resin, ethanol, water and terpyridine ruthenium is 1:1:1:

16.

6. Use of the micro electrochemiluminescence biosensor for measuring thrombin activity according to any one of claims 1 to 3 in detecting thrombin activity.

7. The use according to claim 6, characterized in that The micro electrochemiluminescence biosensor for measuring thrombin activity can detect thrombin activity in serum, plasma and whole blood.

8. A thrombin activity detection system, characterized in that: The invention comprises a portable electrochemiluminescence detector and the micro electrochemiluminescence biosensor for measuring thrombin activity according to any one of claims 1 to 3.

9. A method for detecting thrombin activity, characterized in that: The blood to be tested is dropped onto the surface of the micro electrochemiluminescence biosensor for measuring thrombin activity according to any one of claims 1 to 3 for reaction, and the thrombin activity value is obtained according to the change value of the thrombin luminescence signal before and after the reaction.

Images