Electrochemiluminescence biosensor based on antibacterial peptide as well as preparation method and application of electrochemical luminescence biosensor

Abstract

The invention discloses an electrochemical luminescence biosensor based on antibacterial peptide as well as a preparation method and application of the electrochemical luminescence biosensor, and belongs to the technical field of biological analysis and detection. The preparation method of the electrochemical luminescence biosensor based on the antibacterial peptide comprises the following steps: synthesizing Ru (at) SiO2 nanoparticles; modifying the Ru (at) SiO2 nano particles with streptavidin; the method comprises the following steps: incubating biotin-antibacterial peptide in streptavidin modified Ru (at) SiO2 nanoparticles, and taking the biotin-antibacterial peptide as an electrochemical luminescence signal probe; preparing a gold nanoparticle (AuNPs) suspension liquid; modifying the glassy carbon electrode; and adding the antibacterial peptide to the modified glassy carbon electrode, incubating the glassy carbon electrode, immersing the glassy carbon electrode in a lipopolysaccharide solution, and adding an electrochemical luminescence signal probe to obtain the electrochemical luminescence biosensor based on the antibacterial peptide. The method is simple in preparation process and can be used for detecting lipopolysaccharide in clinical common injection.

Description

Technical Field

[0001] The present invention relates to the technical field of biological analysis and detection, and more specifically to an electrochemiluminescent biosensor based on antimicrobial peptides and a preparation method and application thereof. Background Art

[0002] Lipopolysaccharides, abbreviated in English as LPS, also refer to bacterial endotoxins, are the main components of the cell wall of Gram-negative bacteria and are extremely toxic to the host. Due to their high lipoprotein content, there is a strong desire to monitor them quickly and sensitively. Bioactivity and toxicity, even at low picomoles per milliliter concentrations. They pose a serious threat to human health because they are naturally released from cells during their life cycle and are toxic in themselves. LPS are found in Gram-negative bacteria in the outer cell membrane and consist of a membrane-bound fatty acid region, called lipid A, bound to a core sugar region. Attached is a long polysaccharide region composed of repeating units of various sugars. The lipid A region of the LPS molecule is recognized by the immune system's TLR4 / MD2 receptors, where a cascade of immunogenic reactions is initiated. If not controlled, it can lead to septic shock, organ failure and ultimately death. Septic shock caused by LPS is a major problem in hospitals and other immediate care settings, such as battlefields.

[0003] In recent years, the detection technology of lipopolysaccharide has made significant progress. Colorimetric, fluorescent, electrochemical and electrochemiluminescent biosensors have been reported and applied to the detection of lipopolysaccharide. The lipopolysaccharide fluorescent probe is prepared by assembling tetramethylrhodamine-labeled lipopolysaccharide binding peptides on graphene oxide and fluorescent sensors based on the interaction between lipopolysaccharide and quaternary ammonium salts; some studies have shown that the colorimetric LPS sensor using functionalized polydiacetylene liposomes provides different responses to specific lipopolysaccharide strains. In addition, there are studies that use electrochemical impedance technology based on electronic tongue to detect and distinguish lipopolysaccharide in spiked culture medium containing potential interferents such as proteins, nucleic acids and phospholipids. However, the preparation of existing electrochemiluminescent biosensors usually requires the construction of a signal amplification system to sensitively detect lipopolysaccharide, but the signal amplification system is usually composed of nanomaterials, enzymes, aptamers, DNAzymes, CRISPR / Cas systems, etc. It needs to be constructed by selecting suitable nanomaterials and biomolecules, assembling biomolecules on the surface of nanomaterials to construct a signal amplification system, optimizing system parameters, verifying the performance of the signal amplification system, and integrating the signal amplification system into the electrochemiluminescent biosensor. The process is complicated. Summary of the invention

[0004] To solve the above problems, the present invention provides an electrochemiluminescent biosensor based on antimicrobial peptides and a preparation method and application thereof, which are used to solve the problem of complex preparation process of electrochemiluminescent biosensors.

[0005] The present invention is achieved through the following technical solutions:

[0006] A method for preparing an electrochemiluminescent biosensor based on an antimicrobial peptide is prepared by a method comprising the following steps:

[0007] Using polyethylene glycol octylphenyl ether, cyclohexane, n-hexanol, Ru(bpy)3 2+ Ru@SiO2 nanoparticles were synthesized by aqueous solution, tetraethyl orthosilicate and ammonia, wherein polyethylene glycol octylphenyl ether, cyclohexane, n-hexanol, Ru(bpy)3 2+ The volume ratio of aqueous solution, tetraethyl orthosilicate and ammonia water is 1.77-1.97:7.5-7.7:1.8-2.0:0.34-0.38:0.1-0.105:0.06-0.08.

[0008] The Ru@SiO2 nanoparticles were modified with a streptavidin solution to obtain a Ru@SiO2-streptavidin solution.

[0009] The biotin-antimicrobial peptide is placed in a Ru@SiO2-streptavidin solution and incubated to form a biotin-streptavidin complex as an electrochemiluminescent signal probe, wherein the volume ratio of the biotin-antimicrobial peptide to the Ru@SiO2-streptavidin solution is 1 to 1.2:1.

[0010] A gold nanoparticle AuNPs suspension is prepared using a tetrachloroauric acid solution and a sodium citrate solution, wherein the volume ratio of the sodium citrate solution to the tetrachloroauric acid solution is 25-26:1.

[0011] A glassy carbon electrode is modified by using a mixture of a gold nanoparticle AuNPs suspension and a perfluoro ion exchange resin solution, and then dried to obtain a modified glassy carbon electrode, wherein the volume ratio of the perfluoro ion exchange resin solution to the AuNPs suspension is 1:2-2.2.

[0012] The antimicrobial peptide is added to the modified glassy carbon electrode to cover the surface of the modified glassy carbon electrode, allowed to stand, washed, and incubated in a 6-mercapto-1-hexanol solution to obtain an incubated glassy carbon electrode. The incubated glassy carbon electrode is immersed in a lipopolysaccharide solution, and an electrochemiluminescent signal probe is added to obtain an electrochemiluminescent biosensor based on the antimicrobial peptide.

[0013] Preferably, the biotin-antimicrobial peptide is a biotin-bombesin 1 solution, and the purity of the biotin-bombesin 1 solution is 96.60%.

[0014] Preferably, the antimicrobial peptide is a bombesin 1 solution, and the concentration of the bombesin 1 solution is 1.0 μg·mL -1 ~4.0 μg mL -1 .

[0015] Preferably, when the streptavidin solution is modified with Ru@SiO2 nanoparticles, the concentration used is 0.8 mg·mL -1 ~1.2 mg mL -1 of streptavidin solution.

[0016] Preferably, the mass concentration of the tetrachloroauric acid solution is 0.01% to 0.015%, and the mass concentration of the sodium citrate solution is 0.8% to 1%.

[0017] Preferably, when the biotin-antimicrobial peptide is placed in the Ru@SiO2-streptavidin solution for incubation, the incubation temperature is 35°C to 40°C and the time is 1.5 to 2.5 hours.

[0018] Preferably, the molar concentration of the 6-mercapto-1-hexanol solution is 0.8 μmol / L to 1.3 μmol / L.

[0019] Preferably, when incubating in the 6-mercapto-1-hexanol solution, the incubation time is 1.5 hours to 2.5 hours.

[0020] The antimicrobial peptide-based electrochemiluminescent biosensor is used in detecting lipopolysaccharide.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention provides a method for preparing an electrochemiluminescent biosensor based on antimicrobial peptides, using polyethylene glycol octylphenyl ether, cyclohexane, n-hexanol, Ru(bpy)3 2+ Ru@SiO2 nanoparticles were synthesized by aqueous solution, tetraethyl orthosilicate and ammonia water; Ru@SiO2 nanoparticles were modified by streptavidin solution to obtain Ru@SiO2-streptavidin solution; biotin-antimicrobial peptide was placed in the Ru@SiO2-streptavidin solution for incubation to form a biotin-streptavidin bioaffinity complex as an electrochemiluminescent signal probe, and a gold nanoparticle AuNPs suspension was prepared by using tetrachloroauric acid solution and sodium citrate solution; a glassy carbon electrode was modified by using a mixture of the gold nanoparticle AuNPs suspension and a perfluoro ion exchange resin solution, and dried to obtain a modified glassy carbon electrode; antimicrobial peptide was added to the modified glassy carbon electrode to cover the surface of the modified glassy carbon electrode, allowed to stand, washed, and placed in a 6-mercapto-1-hexanol solution for incubation to obtain an incubated glassy carbon electrode, the incubated glassy carbon electrode was immersed in a lipopolysaccharide solution, and an electrochemiluminescent signal probe was added to obtain an electrochemiluminescent biosensor based on antimicrobial peptide. When the constructed sensor was combined with LPS, the sensor had a good electrochemiluminescence response to LPS at different concentrations. The electrochemiluminescence intensity was significantly correlated with the LPS concentration at 2.0×10-11 mol / L~3.0×10 -10 mol / L, and the detection limit is 1.0×10 -12 mol / L. The electrochemiluminescent biosensor based on antimicrobial peptide has a simple preparation process and high detection sensitivity. It can be used to detect lipopolysaccharide in common clinical injections and can selectively detect lipopolysaccharide of different strains and lipopolysaccharide on the membrane of living Escherichia coli. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 Schematic diagram of the electrochemiluminescence biosensor for detecting lipopolysaccharide provided in an embodiment of the present invention;

[0025] Figure 2 The antimicrobial peptide chain structure diagram provided by the embodiment of the present invention;

[0026] Figure 3 The detection of lipopolysaccharide at different concentrations provided by the embodiment of the present invention;

[0027] Figure 3 In the equation, a is 0 mol / L; b is 5.0×10 -11 mol / L; c is 3.0×10 -10 mol / L;

[0028] Figure 4 The relationship between the concentration of the antimicrobial peptide provided in the embodiment of the present invention and the change value of the electrochemical luminescence intensity, lipopolysaccharide concentration: 2.0×10 -10 mol / L;

[0029] Figure 5 The relationship between the fixed concentration of LPS and the electrochemiluminescent probe reaction time provided in the embodiment of the present invention, LPS concentration: 2.0×10 -10 mol / L;

[0030] Figure 6 The electrochemical luminescence intensity curve of different signal substances at the same LPS concentration provided in the embodiment of the present invention, LPS concentration: 2.0×10 -10 mol / L;

[0031] Figure 6 In the above, a is a ruthenium bipyridine derivative; b is Ru@SiO2;

[0032] Figure 7 A graph showing the relationship between the electrochemiluminescence intensity and the lipopolysaccharide concentration after the electrochemiluminescence sensor provided in an embodiment of the present invention reacts with lipopolysaccharides of different concentrations;

[0033] Figure 7 In the equation, a is 0 mol / L; b is 5.0×10 -12 mol / L; c is 2.0×10 -11 mol / L; d is 3.0×10 -11 mol / L; e is 5.0×10 -11 mol / L; f is 1.0×10 -10 mol / L; g is 2.0×10 -10 mol / L; h is 3.0×10 -10 mol / L. DETAILED DESCRIPTION

[0034] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively below, and preferred embodiments of the present invention are provided. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0036] In view of the fact that the preparation process of electrochemiluminescent biosensors in the prior art is complicated and needs to be solved urgently, the present invention provides an electrochemiluminescent biosensor based on antimicrobial peptides, and the preparation method includes:

[0037] Using polyethylene glycol octylphenyl ether, cyclohexane, n-hexanol, Ru(bpy)3 2+ Ru@SiO2 nanoparticles were synthesized by aqueous solution, tetraethyl orthosilicate and ammonia, wherein polyethylene glycol octylphenyl ether, cyclohexane, n-hexanol, Ru(bpy)3 2+ The volume ratio of the aqueous solution, ethyl orthosilicate and ammonia water is 1.77-1.97: 7.5-7.7: 1.8-2.0: 0.34-0.38: 0.1-0.15: 0.06-0.08.

[0038] The Ru@SiO2 nanoparticles were modified with a streptavidin solution to obtain a Ru@SiO2-streptavidin solution.

[0039] The biotin-antimicrobial peptide is placed in a Ru@SiO2-streptavidin solution and incubated to form a biotin-streptavidin complex as an electrochemiluminescent signal probe, wherein the volume ratio of the biotin-antimicrobial peptide to the Ru@SiO2-streptavidin solution is 1 to 1.2:1.

[0040] A gold nanoparticle AuNPs suspension is prepared using a tetrachloroauric acid solution and a sodium citrate solution, wherein the volume ratio of the sodium citrate solution to the tetrachloroauric acid solution is 25-26:1.

[0041] A glassy carbon electrode is modified by using a mixture of a gold nanoparticle AuNPs suspension and a perfluoro ion exchange resin solution, and then dried to obtain a modified glassy carbon electrode, wherein the volume ratio of the perfluoro ion exchange resin solution to the AuNPs suspension is 1:2-2.2.

[0042] The antimicrobial peptide is added to the modified glassy carbon electrode to cover the surface of the modified glassy carbon electrode, allowed to stand, washed, and incubated in a 6-mercapto-1-hexanol solution to obtain an incubated glassy carbon electrode. The incubated glassy carbon electrode is immersed in a lipopolysaccharide solution, and an electrochemiluminescent signal probe is added to obtain an electrochemiluminescent biosensor based on the antimicrobial peptide.

[0043] The principle of the electrochemiluminescent biosensor based on antimicrobial peptide for detecting lipopolysaccharide is as follows: Ru@SiO2 composite nanoparticles are used as signal substances, and Ru@SiO2 composite nanoparticles modified with streptavidin are specifically combined with biotin-antimicrobial peptide through streptavidin and biotin to construct an electrochemiluminescent probe. The antimicrobial peptide structure is as follows: Figure 2 The glassy carbon electrode is modified with a perfluorinated ion exchange resin solution doped with a gold nanoparticle AuNPs suspension, and the antimicrobial peptide is added to the modified glassy carbon electrode. The sulfur group of the antimicrobial peptide can combine with the gold nanoparticles to form a gold-sulfur bond, and then the blank site is blocked with a 6-mercapto-1-hexanol solution. This solves the problem of the complex preparation process of the electrochemiluminescent biosensor in the prior art.

[0044] Taking lipopolysaccharide as the detection object, the anions in the end groups of lipopolysaccharide and the antimicrobial peptides as well as the cationic amino acids in the peptides in the probe are combined together through electrostatic interaction, thereby detecting lipopolysaccharide, see Figure 1 .

[0045] The electrochemiluminescent biosensor based on antimicrobial peptides in the present invention has a simple preparation process, can be used for the detection of lipopolysaccharide in commonly used clinical injections, and can selectively detect lipopolysaccharide of different strains and lipopolysaccharide on the membrane of living Escherichia coli.

[0046] The technical solution of the present invention will be further described below in conjunction with specific embodiments.

[0047] The sources of the instruments and raw materials used in the present invention are as follows:

[0048] (1) Main reagents

[0049] The main reagents for the experiment are shown in Table 1.

[0050] Table 1 Main reagents for the experiment

[0051]

[0052] (2) Main instruments and equipment.

[0053] The experimental instruments and equipment are shown in Table 2.

[0054] Table 2 Main instruments and equipment for the experiment

[0055]

[0056] Example 1: A method for preparing an electrochemiluminescent biosensor based on antimicrobial peptides

[0057] (1) Synthesis of Ru@SiO2 nanoparticles

[0058] The ruthenium bipyridine derivative was mixed with water to obtain Ru(bpy)3 with a molar concentration of 40mmol / L 2+ aqueous solution, 0.34 mL of Ru(bpy)3 with a molar concentration of 40 mmol / L 2+ The aqueous solution, 1.77 mL of polyethylene glycol octylphenyl ether, 7.5 mL of cyclohexane and 1.8 mL of n-hexanol were mixed and stirred. After stirring for 30 minutes, 0.1 mL of tetraethyl orthosilicate was added to the mixture. Then, the polymerization reaction was initiated by adding 0.06 mL of 30 wt% ammonia water. The solution was stirred in the dark for 24 hours to obtain Ru@SiO2 nanoparticles, which were separated by acetone, centrifuged and washed several times with ethanol and ultrapure water to remove residual surfactant molecules and excess Ru(bpy)3 2+ , and obtain Ru@SiO2 nanoparticle solution.

[0059] (2) Streptavidin modified Ru@SiO2 nanoparticles

[0060] Use ethanol to mix 1.0 mL of 2.0 mg mL -1Ru@SiO2 nanoparticles were diluted to 5.0 mL with ethanol, and 400 μL of 3-aminopropyltriethoxysilane solution was added. Add to the solution. After stirring for 4 hours, centrifuge and wash with ethanol several times to remove excess 3-aminopropyltriethoxysilane to obtain Ru@SiO2 nanoparticles containing amino termini. Stir with 5.0 mL of 5% glutaraldehyde solution at 37 °C for 4 hours. Then, the mixture was centrifuged and washed with water 3 times to remove excess glutaraldehyde. The precipitate was dispersed in 2.0 mL of ultrapure water. Afterwards, 190 μL of 0.8 mg·mL -1 The streptavidin solution was added to the suspension, which was then stirred in a 37°C water bath for 4 h. The mixture was centrifuged and eluted with a pH of 7.4 and a molar concentration of 0.01 mol·L -1 After washing with a phosphate buffer solution, streptavidin-modified Ru@SiO2 nanoparticles were obtained. Finally, the streptavidin-modified Ru@SiO2 nanoparticles were redispersed in a phosphate buffer solution containing 1% bovine serum albumin per 1.0 mL, pH = 7.4, to obtain a Ru@SiO2-streptavidin solution, which was stored at 4°C for use.

[0061] (3) Synthesis of electrochemiluminescent signal probe

[0062] At 35 °C, 100 μL of biotin-antimicrobial peptide, i.e., biotin-bombesin 1 solution, with a purity of 96.60%, was incubated with 100 μL of Ru@SiO2-streptavidin solution for 1.5 h to form a biotin-streptavidin bioaffinity complex as an electrochemiluminescent signal probe, which was ultimately used to generate an electrochemiluminescent signal.

[0063] (4) Preparation of AuNPs suspension

[0064] 100 mL of 0.01% tetrachloroauric acid solution was refluxed, and then 4 mL of 0.8% sodium citrate solution was introduced while stirring, and then boiled for 30 minutes and cooled to room temperature to obtain a gold nanoparticle AuNPs suspension, wherein the diameter of the gold nanoparticle AuNPs was 12 nm.

[0065] (5) Modification of glassy carbon electrode

[0066] The glassy carbon electrode, i.e., GCE, was polished with alumina slurry having a diameter of 2.0 mm and then rinsed thoroughly with water. A 0.5% mass concentration of perfluoro ion exchange resin solution, the English name of which is Nafion, was mixed with the gold nanoparticle AuNPs suspension prepared above, the volume ratio of the perfluoro ion exchange resin solution to the gold nanoparticle AuNPs suspension being 1:2, and ultrasonically treated for 30 minutes to prepare a mixture of the gold nanoparticle AuNPs suspension and the perfluoro ion exchange resin solution. Then, a 10 μL mixture of the gold nanoparticle AuNPs suspension and the perfluoro ion exchange resin solution was used to modify the glassy carbon electrode with a microsyringe and dried at room temperature to obtain a modified glassy carbon electrode.

[0067] (6) Construction of electrochemiluminescent biosensor based on antimicrobial peptides

[0068] The molar concentration of 10 μL is 1.0 μg mL -1 The antimicrobial peptide, namely, bombesin 1 solution, was added to the modified glassy carbon electrode to cover the surface of the modified glassy carbon electrode to identify and capture the target lipopolysaccharide, and then left to stand for 12 hours, and then washed twice with a 0.1 mol / L phosphate buffer solution to remove the unbound recognition probe. The glassy carbon electrode was incubated in a 6-mercapto-1-hexanol solution with a molar concentration of 0.8 μmol / L for 1.5 hours to block the blank site and obtain the incubated electrode. The incubated electrode was immersed in a lipopolysaccharide solution, and an electrochemiluminescent signal probe was added to obtain an electrochemiluminescent biosensor based on antimicrobial peptides.

[0069] Example 2: A method for preparing an electrochemiluminescent biosensor based on antimicrobial peptides

[0070] (1) Synthesis of Ru@SiO2 nanoparticles

[0071] The ruthenium bipyridine derivative was mixed with water to obtain Ru(bpy)3 with a molar concentration of 40 mmol / L. 2+ aqueous solution, 0.38 mL of Ru(bpy)3 with a molar concentration of 40 mmol / L 2+ A mixture of aqueous solution, 1.97 mL of polyethylene glycol octylphenyl ether, 7.7 mL of cyclohexane and 2.0 mL of n-hexanol was stirred. After stirring for 30 minutes, 0.15 mL of tetraethyl orthosilicate was added to the mixture. Then, the polymerization reaction was initiated by adding 0.080 mL of 30 wt% ammonia water. The solution was stirred in the dark for 24 hours to obtain Ru@SiO2 nanoparticles, which were separated by acetone, then centrifuged and washed several times with ethanol and ultrapure water to remove residual surfactant molecules and excess Ru(bpy)3 2+ , and obtain Ru@SiO2 nanoparticle solution.

[0072] (2) Streptavidin modified Ru@SiO2 nanoparticles

[0073] Use ethanol to mix 1.0 mL of 2.0 mg mL -1 Ru@SiO2 nanoparticles were diluted to 5.0 mL with ethanol, and 400 μL of 3-aminopropyltriethoxysilane solution was added. Add to the solution. After stirring for 4 h, centrifuge and wash with ethanol several times to remove excess 3-aminopropyltriethoxysilane to obtain Ru@SiO2 nanoparticles containing amino termini. Stir with 5.0 mL of 5% glutaraldehyde solution at 37 °C for 4 h. Then, the mixture was centrifuged and washed with water 3 times to remove excess glutaraldehyde. The precipitate was dispersed in 2.0 mL of ultrapure water. Afterwards, 210 μL of 1.2 mg mL -1 The streptavidin solution was added to the suspension, which was then stirred in a 37°C water bath for 4 h. The mixture was centrifuged and eluted with a pH of 7.4 and a molar concentration of 0.01 mol·L -1 After washing with a phosphate buffer solution, streptavidin-modified Ru@SiO2 nanoparticles were obtained. Finally, the streptavidin-modified Ru@SiO2 nanoparticles were redispersed in a phosphate buffer solution containing 1% bovine serum albumin per 1.0 mL, pH = 7.4, to obtain a Ru@SiO2-streptavidin solution, which was stored at 4°C for use.

[0074] (3) Synthesis of electrochemiluminescent signal probe

[0075] At 40 °C, 120 μL of biotin-antimicrobial peptide, i.e., biotin-bombesin 1 solution, with a purity of 96.60%, was incubated with 100 μL of Ru@SiO2-streptavidin solution for 2.5 h to form a biotin-streptavidin bioaffinity complex as an electrochemiluminescent signal probe, which was ultimately used to generate an electrochemiluminescent signal.

[0076] (4) Preparation of AuNPs suspension

[0077] 104 mL of 0.015% tetrachloroauric acid solution was refluxed, and then 4 mL of 0.1% sodium citrate solution was introduced while stirring, and then boiled for 30 minutes and cooled to room temperature to obtain a gold nanoparticle AuNPs suspension, wherein the diameter of the gold nanoparticle AuNPs was 12 nm.

[0078] (5) Modification of glassy carbon electrode

[0079] The glassy carbon electrode, i.e., GCE, was polished with alumina slurry having a diameter of 2.0 mm and then rinsed thoroughly with water. A 0.5% mass concentration of perfluoro ion exchange resin solution, the English name of which is Nafion, was mixed with the gold nanoparticle AuNPs suspension prepared above, the volume ratio of the perfluoro ion exchange resin solution to the gold nanoparticle AuNPs suspension being 1:2.2, and ultrasonically treated for 30 minutes to prepare a mixture of the gold nanoparticle AuNPs suspension and the perfluoro ion exchange resin solution. Then, 10 μL of the mixture of the gold nanoparticle AuNPs suspension and the perfluoro ion exchange resin solution was modified with a microsyringe and dried at room temperature to obtain a modified glassy carbon electrode.

[0080] (6) Construction of electrochemiluminescent biosensor based on antimicrobial peptides

[0081] The molar concentration of 10 μL is 4.0 μg mL -1 The antimicrobial peptide, namely, bombesin 1 solution, was added to the modified glassy carbon electrode to cover the surface of the modified glassy carbon electrode to identify and capture the target lipopolysaccharide, and then left to stand for 12 hours, and then washed twice with a 0.1 mol / L phosphate buffer solution to remove the unbound recognition probe. The glassy carbon electrode was incubated in a 6-mercapto-1-hexanol solution with a molar concentration of 1.3 μmol / L for 2.5 hours to block the blank site and obtain the incubated electrode. The incubated glassy carbon electrode was immersed in a lipopolysaccharide solution, and an electrochemiluminescent signal probe was added to obtain an electrochemiluminescent biosensor based on antimicrobial peptides.

[0082] Example 3: A detection method based on an electrochemiluminescent biosensor for antimicrobial peptides

[0083] (1) The prepared antimicrobial peptide-based electrochemiluminescent biosensor was immersed in 100 μL of a lipopolysaccharide solution with a fixed concentration after incubation, and then rinsed with a rinse solution after reacting for 40 minutes.

[0084] (2) Then, 10 μL of 20 μmol / L electrochemiluminescent bioprobe was added to the rinsed glassy carbon electrode. After rinsing with the rinse solution, the electrode was placed in 3 mL of 100 mmol / L tripropylamine solution.

[0085] (3) When the potential is scanned from 0 to 1.3 V, the peak value of the electrochemiluminescence signal is recorded for analysis. The peak intensity of the electrochemiluminescence signal is proportional to the concentration of lipopolysaccharide within a certain concentration range, thereby enabling the detection of lipopolysaccharide.

[0086] Example 4: Feasibility of electrochemiluminescence analysis for detecting lipopolysaccharide

[0087] This example compares the electrochemiluminescence intensity of the electrochemiluminescence biosensor based on antimicrobial peptides under different concentrations of lipopolysaccharide.

[0088] Figure 3 The electrochemiluminescent biosensor based on antimicrobial peptides was shown to be -11 mol / L of lipopolysaccharide and 3.0×10 -10 The change of electrochemiluminescence intensity before and after the reaction of 1.5 mol / L lipopolysaccharide. Figure 3 As can be seen, the electrochemiluminescent biosensor based on antimicrobial peptides is comparable to 5.0×10 -11 The peak value of electrochemiluminescence intensity after the reaction of lipopolysaccharide with mol / L is 1510, as shown in curve b. -10 The peak value of the electrochemiluminescence intensity after the reaction with 1 mol / L lipopolysaccharide is 3124, as shown in curve c, and the electrochemiluminescence intensity is enhanced, which indicates that the constructed electrochemiluminescence biosensor based on antimicrobial peptides is feasible for detecting lipopolysaccharide.

[0089] Example 5: Detection of antimicrobial peptide-based electrochemiluminescent biosensor under different experimental conditions

[0090] This example detects the effects of different amounts of antimicrobial peptides and self-assembly time on the electrochemiluminescence intensity of the biosensor.

[0091] Figure 4 This is the relationship between the concentration of antimicrobial peptides and the change in electrochemiluminescence intensity. -1 ~8.0μg·mL -1 The results are shown in Figure 4 As shown, it shows that ΔI increases from 1.0 μg·mL -1 to 4.0 μg·mL -1 The concentration increased significantly when it reached 4.0 μg mL -1 This indicates that the antimicrobial peptides almost occupied all accessible active sites on the glassy carbon electrode. Therefore, 4.0 μg mL -1 concentration of antimicrobial peptides.

[0092] Figure 5 This is a graph showing the relationship between the reaction time of lipopolysaccharide and electrochemiluminescence probe and the change in electrochemiluminescence intensity. Figure 5As shown in the figure, when the reaction time of LPS and ECL probe increases from 5min to 20min, the ECL intensity change value increases sharply. From 20min to 40min, the ECL intensity change value increases slowly, reaches the maximum after 40min and remains stable. The results show that 40min is enough for the system to reach equilibrium. Therefore, the reaction time of LPS and ECL probe in this experiment is set to 40min.

[0093] Example 6: Differences between Ru@SiO2 and ruthenium bipyridine derivatives as signal substances

[0094] from Figure 6 It can be seen that the electrochemical luminescence intensity of Ru@SiO2 as a signal substance is significantly improved compared with that of ruthenium bipyridine derivatives as signal substances. This shows that Ru@SiO2 as a signal substance is of better quality than ruthenium bipyridine derivatives.

[0095] Example 7: Performance study of electrochemiluminescent biosensor based on antimicrobial peptides

[0096] This example quantitatively measures the electrochemiluminescence intensity of lipopolysaccharide at different concentrations.

[0097] Figure 7 The electrochemiluminescence intensity curves of the sensor reacting with different concentrations of lipopolysaccharide are shown. Figure 7 It can be seen that the peak of electrochemiluminescence intensity increases with the increase of LPS concentration. The increase of electrochemiluminescence peak intensity ΔI is related to LPS concentration. -11 mol / L~3.0×10 -10 The regression equation is ΔI = 1147 + 64C, where the unit of C is 10 -11 mol / L, correlation coefficient R=0.9970, detection limit is 1.0×10 -12 mol / / L.

[0098] Example 8. Reproducibility of electrochemiluminescent biosensor based on antimicrobial peptides

[0099] This example evaluates the reproducibility of an antimicrobial peptide-based electrochemiluminescent biosensor.

[0100] Five different antimicrobial peptide-based electrochemiluminescent biosensors were used to detect 2.0 × 10 -10 The relative standard deviation of lipopolysaccharide was 3.7% when the same electrochemiluminescent biosensor based on antimicrobial peptide was used for five repeated measurements, and the relative standard deviation was 3.2%, which showed that the electrochemiluminescent biosensor based on antimicrobial peptide was reproducible.

[0101] In summary, the antimicrobial peptides are self-assembled on the gold nanoparticle-modified glassy carbon electrode, and the synthesized streptavidin-labeled Ru@SiO2 and the biotin-labeled antimicrobial peptides are used to construct electrochemiluminescent signal probes, and electrochemiluminescent biosensors are constructed. When the lipopolysaccharide is used as the detection object, the electrochemiluminescence intensity increases when the detection object binds to the antimicrobial peptides, and a new method for detecting lipopolysaccharide is established. Compared with traditional biological detection methods, the designed electrochemiluminescent biosensor based on antimicrobial peptides has excellent performance, a complex preparation process, easy operation, high sensitivity, selectivity, a wide linear range and a low detection limit, and has broad application prospects.

Claims

1. A method for preparing an electrochemiluminescent biosensor based on antimicrobial peptides, characterized in that: The method is prepared by the following steps: Using polyethylene glycol octylphenyl ether, cyclohexane, n-hexanol, Ru(bpy)3 2+ Ru@SiO2 nanoparticles were synthesized by aqueous solution, tetraethyl orthosilicate and ammonia, wherein polyethylene glycol octylphenyl ether, cyclohexane, n-hexanol, Ru(bpy)3 2+ The volume ratio of aqueous solution, ethyl orthosilicate and ammonia water is 1.77-1.97:7.5-7.7:1.8-2.0:0.34-0.38:0.1-0.15:0.06-0.08; The Ru@SiO2 nanoparticles were modified with a streptavidin solution to obtain a Ru@SiO2-streptavidin solution; The biotin-antimicrobial peptide is placed in a Ru@SiO2-streptavidin solution and incubated to form a biotin-streptavidin complex as an electrochemiluminescent signal probe, wherein the volume ratio of the biotin-antimicrobial peptide to the Ru@SiO2-streptavidin solution is 1 to 1.2:1; A gold nanoparticle AuNPs suspension is prepared using a tetrachloroauric acid solution and a sodium citrate solution, wherein the volume ratio of the sodium citrate solution to the tetrachloroauric acid solution is 25-26:1; Modifying a glassy carbon electrode with a mixture of a gold nanoparticle AuNPs suspension and a perfluoro ion exchange resin solution, and drying to obtain a modified glassy carbon electrode, wherein the volume ratio of the perfluoro ion exchange resin solution to the AuNPs suspension is 1:2 to 2.2; The antimicrobial peptide is added to the modified glassy carbon electrode to cover the surface of the modified glassy carbon electrode, allowed to stand, washed, and incubated in a 6-mercapto-1-hexanol solution to obtain an incubated glassy carbon electrode. The incubated glassy carbon electrode is immersed in a lipopolysaccharide solution, and an electrochemiluminescent signal probe is added to obtain an electrochemiluminescent biosensor based on the antimicrobial peptide.

2. The method for preparing the electrochemiluminescent biosensor based on antimicrobial peptide according to claim 1, characterized in that: The biotin-antimicrobial peptide is a biotin-bombesin 1 solution, and the purity of the biotin-bombesin 1 solution is 96.60%.

3. The method for preparing the electrochemiluminescent biosensor based on antimicrobial peptide according to claim 1, characterized in that: The antimicrobial peptide is a bombesin 1 solution, and the concentration of the bombesin 1 solution is 1.0 μg·mL -1 ~4.0 μg mL -1 .

4. The method for preparing the electrochemiluminescent biosensor based on antimicrobial peptide according to claim 1, characterized in that: When streptavidin solution was used to modify Ru@SiO2 nanoparticles, the concentration was 0.8 mg mL -1 ~1.2 mg mL -1 of streptavidin solution.

5. The method for preparing the electrochemiluminescent biosensor based on antimicrobial peptide according to claim 1, characterized in that: The mass concentration of the tetrachloroauric acid solution is 0.01% to 0.015%, and the mass concentration of the sodium citrate solution is 0.8% to 1%.

6. The method for preparing the electrochemiluminescent biosensor based on antimicrobial peptide according to claim 1, characterized in that: When the biotin-antimicrobial peptide is placed in the Ru@SiO2-streptavidin solution for incubation, the incubation temperature is 35°C to 40°C and the time is 1.5 hours to 2.5 hours.

7. The method for preparing the electrochemiluminescent biosensor based on antimicrobial peptide according to claim 1, characterized in that: The molar concentration of the 6-mercapto-1-hexanol solution is 0.8 μmol / L to 1.3 μmol / L.

8. The method for preparing the electrochemiluminescent biosensor based on antimicrobial peptide according to claim 1, characterized in that: When the incubation is carried out in the 6-mercapto-1-hexanol solution, the incubation time is 1.5 hours to 2.5 hours.

9. An electrochemiluminescent biosensor based on antimicrobial peptides obtained according to the preparation method according to any one of claims 1 to 8.

10. Use of the electrochemiluminescent biosensor based on antimicrobial peptide according to claim 9 in detecting lipopolysaccharide.

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