Preparation method of electrochemical luminescence biosensor based on carbon material enhanced porphyrin hydrogel

Abstract

The invention belongs to the technical field of food-borne pathogenic bacterium detection, and discloses a preparation method of an electrochemical luminescence biosensor based on carbon material enhanced porphyrin hydrogel. The method comprises the following steps: synthesizing a sodium alginate-chitosan-TPPS (Thermoplastic Polystyrene) composite hydrogel; au NPs is prepared; modifying the electrode; and preparing the detection liquid. According to the invention, 5, 10, 15, 20-tetra (4-sulfophenyl) porphyrin is used as a functional material, the functional material is innovatively introduced into a sodium alginate-chitosan bi-crosslinking hydrogel system, the problems of poor stability and difficulty in modification of a water-soluble porphyrin material in sensing application are effectively overcome, and meanwhile, the conductivity and ECL signal strength of the porphyrin hydrogel are enhanced by adopting the carbon nanotubes, so that the sensing performance of the water-soluble porphyrin hydrogel is improved. Therefore, accurate detection of food-borne pathogenic bacteria represented by staphylococcus aureus is achieved, and the sensing electrode is easy and convenient to prepare, high in operability, rapid in response, high in sensitivity, high in controllability and suitable for on-site rapid detection and portable analysis requirements in the fields of food production and processing, circulation supervision, public health monitoring and the like.

Description

Technical Field

[0001] This invention relates to the field of foodborne pathogen detection technology, specifically to a method for preparing a carbon-reinforced porphyrin hydrogel electrochemiluminescence biosensor. Background Technology

[0002] Foodborne pathogens pose a significant threat. Some pathogens can evolve drug resistance, increasing the difficulty and cost of treatment. They also easily form biofilms that adhere to the surfaces of food contact materials, making them difficult to remove and causing continuous food contamination and iatrogenic infections. This leads to food recalls, strain on medical resources, and substantial economic losses. The World Health Organization has identified four major foodborne pathogens: Staphylococcus aureus, Escherichia coli, Salmonella, and Listeria monocytogenes. Among them, Staphylococcus aureus (S. aureus), a common Gram-positive bacterium, can infect humans through contaminated food, causing vomiting, diarrhea, sepsis, and even toxic shock syndrome. The mortality rate is high among vulnerable populations such as children and the elderly.

[0003] Detection of foodborne pathogens is crucial for ensuring food safety and public health. The National Food Safety Standard for Limits of Pathogenic Bacteria in Prepackaged Foods (GB29921-2021) clearly stipulates that the maximum limit for S. aureus in prepackaged foods is 1000 CFU / g (mL). Detection serves as the basis for compliant production by enterprises and for supervision and inspection by regulatory authorities. Through detection, food contamination can be detected promptly, disease transmission chains can be interrupted, and group infections can be avoided. It can also clarify the types of pathogens, providing support for precise clinical treatment and the formulation of food safety control strategies. Simultaneously, it encourages enterprises to improve their quality management levels, enhances consumer trust, and maintains the healthy development of the food industry. However, existing traditional detection methods, such as bacterial culture, are time-consuming; polymerase chain reaction (PCR) requires specialized technicians and expensive equipment, and has high requirements for the experimental environment. While enzyme-linked immunosorbent assay (ELISA) is simple, it is prone to false positives. Therefore, there is an urgent need to develop a simple, rapid, highly selective, and sensitive detection method to achieve accurate and rapid detection of S. aureus. In food safety analysis and public health monitoring, developing new, simple, and rapid technologies to achieve early and accurate detection of foodborne pathogens such as S. aureus is a core need in this field and a key measure to strengthen food safety defenses and enhance public health monitoring capabilities.

[0004] The basic principle of electrochemiluminescence (ECL) sensing technology lies in applying a specific potential to the electrode surface to generate highly reactive intermediates. These intermediates interact to form excited-state substances, which release photons when they return to the ground state. Qualitative or quantitative analysis of the target substance is achieved by detecting the photon signal. Due to its advantages such as low background, high sensitivity, and strong controllability, ECL sensing technology has been widely used in environmental monitoring, food safety inspection, and clinical diagnostics. Porphyrins and their derivatives possess tunable structures, excellent photophysical properties, strong light absorption / emission capabilities, and favorable electrochemical properties, making them highly attractive in the ECL field. However, porphyrins are prone to aggregation in aqueous phases, leading to luminescence quenching (ACQ). Water-soluble porphyrin materials are difficult to modify at the sensing interface, making it difficult to obtain stable ECL signals, which also poses a challenge to the ECL application of porphyrins.

[0005] To obtain stable ECL signals, reports have indicated the use of hydrogels to prepare gel-type electrochemiluminescence (ECL) active materials. However, the prepared hydrogel luminescent materials often involve metal components or organic solvents, resulting in poor biocompatibility and inconsistency with green chemistry principles. Furthermore, single-component hydrogels, such as sodium alginate hydrogels, exhibit weak mechanical stiffness and high brittleness, while also suffering from insufficient conductivity that severely hinders ECL signal emission, limiting their application effectiveness. Therefore, exploring environmentally friendly and readily available high-performance porphyrin gel luminescent materials to construct a highly sensitive Staphylococcus aureus ECL sensor is not only of significant scientific importance but also possesses considerable practical value. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention develops a method for preparing an electrochemiluminescence biosensor based on carbon-reinforced porphyrin hydrogel. The sensor, based on the synergistic effect of SCT hydrogel and carbon nanotubes, enables accurate detection of foodborne pathogens, represented by Staphylococcus aureus. Furthermore, the method is simple to prepare, highly operable, has a rapid response, high sensitivity, and strong controllability.

[0007] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: A method for fabricating a carbon-reinforced porphyrin hydrogel electrochemiluminescence biosensor, the sensing device comprising a working electrode and a detection solution, the method comprising the following steps: (1) Preparation of working electrode 1) Synthesis of SCT hydrogel Chitosan (CS) was dissolved in an aqueous acetic acid solution and stirred at room temperature to obtain CS hydrogel; sodium alginate (SA) was dissolved in NaCl solution and stirred at room temperature to obtain SA hydrogel; the prepared CS hydrogel was added to the SA hydrogel and mixed by magnetic stirring to obtain SA-CS hydrogel; 5,10,15,20-tetrakis(4-sulfonylphenyl)porphyrin (TPPS) was added to the prepared SA-CS hydrogel and stirred by magnetic stirring to obtain SCT hydrogel; 2) Preparation of Au NPs The tetrachloroauric acid (HAuCl4) solution was heated to a gentle boil; then, sodium citrate solution was rapidly added, and the mixture was refluxed at 90°C for 30 minutes; heating was stopped immediately when the solution color changed from colorless to red; the formation of gold nanoparticles (Au NPs) was confirmed by measuring its ultraviolet-visible absorption spectrum; the obtained Au NPs were stored below 4°C for later use. 3) Modified electrode Carbon nanotube solution was drop-coated onto the electrode surface, followed by SCT hydrogel prepared in step 1). After drying, Au NPs prepared in step 2) were added, and then Staphylococcus aureus antibody solution was added and incubated for 4-8 hours. BSA (bovine serum albumin) solution was then used to incubate at 4°C for 15-30 minutes to obtain the working electrode. (2) Preparation of detection solution Dissolve and dilute K2S2O8 with PBS buffer solution to prepare K2S2O8 detection solution.

[0008] Preferably, in step 1), 350-400 mg of chitosan (CS) is weighed and dissolved in 20 mL of acetic acid aqueous solution, and stirred at room temperature for 4 hours to obtain CS hydrogel; 790-805 mg of sodium alginate (SA) is dissolved in 40 mL of NaCl solution and stirred for 4 hours to obtain SA hydrogel; the prepared CS hydrogel and SA hydrogel are mixed at a volume ratio of 1:0.5-2 and mixed by magnetic stirring to obtain SA-CS hydrogel; 0.5-2 mg of 5,10,15,20-tetra(4-sulfophenyl)porphyrin (TPPS) is added to 1 mL of the prepared SA-CS hydrogel and stirred by magnetic stirring to form a homogeneous state, thus obtaining SCT hydrogel.

[0009] More preferably, in step 1), the mass fraction of the acetic acid aqueous solution is 0.5%, and the concentration of the NaCl solution is 0.5 mol / L.

[0010] Preferably, in step 2), 100-150 mL of tetrachloroauric acid (HAuCl4) solution is placed on a magnetic stirrer and stirred, and heated to a gentle boiling state; then, 3.0 mL of sodium citrate solution is quickly added, and the mixture is refluxed at 90°C for 30 minutes; when the solution color changes from colorless to red, heating is stopped immediately; the formation of gold nanoparticles (Au NPs) is confirmed by measuring its ultraviolet-visible absorption spectrum; the obtained Au NPs are stored below 4°C for later use.

[0011] More preferably, in step 2), the mass fraction of the tetrachloroauric acid (HAuCl4) solution is 0.01% and the mass fraction of the sodium citrate solution is 1%.

[0012] Preferably, in step 3), 5 μL of carbon nanotube solution is drop-coated onto the electrode surface, followed by 5 μL of SCT hydrogel prepared in step 1), and dried at 37°C; 5 μL of Au NPs prepared in step 2) is added, followed by 5 μL of Staphylococcus aureus antibody solution, and incubated at 4°C for 4–8 hours; 5 μL of BSA (bovine serum albumin) solution is used for incubation at 4°C for 15–30 minutes, and finally washed with PBS solution at pH 7.4, and excess water is absorbed with filter paper to obtain the working electrode.

[0013] The carbon nanotube solution is one of a multi-walled carbon nanotube solution, a carboxylated multi-walled carbon nanotube solution, a single-walled carbon nanotube solution, or a carboxylated single-walled carbon nanotube solution; the electrode is a glassy carbon electrode or a screen-printed electrode.

[0014] More preferably, in step 3), the concentration of the carbon nanotube solution is 1 mg / mL, the concentration of the Staphylococcus aureus antibody solution is 5 μg / mL, and the concentration of the BSA solution is 0.25 wt%.

[0015] Preferably, in step (2), 27-270 mg of K2S2O8 is weighed into a centrifuge tube, 10 mL of PBS solution with a pH of 7.4 is added, and the solution is sonicated to prepare a K2S2O8 detection solution of 10-100 mmol / L.

[0016] Preferably, the sensor is used as follows: the sample to be tested is drop-coated onto the working electrode and incubated at room temperature in the dark for 40–60 minutes. Then, it is gently rinsed with PBS solution at pH 7.4. The working electrode is then placed in a reaction cell containing the detection solution for ECL detection. ECL testing is performed on an electrochemiluminescence analyzer using a conventional three-electrode system: a modified glassy carbon electrode or screen-printed electrode as the working electrode, a platinum wire as the counter electrode, and Ag / AgCl as the reference electrode. The photomultiplier tube's operating voltage is set to 600–800 V, and the potential scan range is 0–-1.3 V.

[0017] The detection principle of the sensor of this invention is as follows: Figure 1 As shown, when Staphylococcus aureus is dropped onto the working electrode, specific sites on the Staphylococcus aureus bind to the antibody, hindering the transmission of photoelectric signals and causing a decrease in the ECL signal.

[0018] This invention selects 5,10,15,20-tetra(4-sulfonylphenyl)porphyrin (TPPS), which possesses electrochemiluminescence properties, photosensitivity, and good biocompatibility, as a functional material and innovatively introduces it into a sodium alginate-chitosan (SA-CS) double-crosslinked hydrogel system. In this system, chitosan acts as a second network, forming a stable three-dimensional porous network structure with sodium alginate through electrostatic crosslinking and hydrogen bonding. This effectively controls the pore size of the hydrogel, significantly enhances its mechanical strength, and inhibits the molecular aggregation of water-soluble phosphophores (TPPS) encapsulated within, thereby improving the luminescence performance of TPPS. The SA-CS hydrogel can effectively encapsulate water-soluble porphyrin phosphophores (such as TPPS), inhibiting molecular leakage, aggregation, and non-radiative transitions, thus overcoming the problems of poor stability and difficulty in modification of water-soluble porphyrin materials in sensing applications. Furthermore, carbon nanotubes are introduced, utilizing their excellent conductivity to compensate for the insufficient conductivity of the hydrogel matrix, thereby enhancing the intensity of the electrochemiluminescence signal. Furthermore, specific antibodies modified on the sensor surface are used to selectively capture Staphylococcus aureus. Based on the steric quenching effect of the electrochemiluminescence signal in the presence of bacteria, a linear relationship between the concentration of Staphylococcus aureus and the degree of quenching of the electrochemiluminescence signal is established, thereby achieving rapid and highly sensitive quantitative detection of Staphylococcus aureus.

[0019] In this sensor, porphyrin hydrogel provides a stable three-dimensional porous structure and ECL luminescence activity, while carbon nanotubes effectively enhance the conductivity and luminescence intensity of the porphyrin hydrogel. When Staphylococcus aureus (S. aureus) binds to a specific antibody (Ab) modified on the sensor surface, a significant change in the ECL signal occurs due to steric hindrance, thereby enabling rapid and sensitive quantitative detection of Staphylococcus aureus.

[0020] By replacing the water-soluble luminescent material with different dopants in the SA-CS hydrogel, this invention can be extended to prepare a variety of ECL hydrogel luminescent materials; by replacing the different specific antibodies, the sensor of this invention can also be extended to the detection of a variety of foodborne pathogens, which can meet the needs of rapid on-site detection and portable analysis, and has good versatility and potential for widespread application.

[0021] Compared with existing technologies, the beneficial effects of adopting the above technical solution are as follows: This invention innovatively introduces 5,10,15,20-tetra(4-sulfonylphenyl)porphyrin (TPPS) as a functional material into a sodium alginate-chitosan (SA-CS) double crosslinked hydrogel system, effectively overcoming the problems of poor stability and difficulty in modification of water-soluble porphyrin materials in sensing applications. At the same time, carbon nanotubes are used to enhance the conductivity and ECL signal intensity of the porphyrin hydrogel, thereby achieving accurate detection of foodborne pathogens such as Staphylococcus aureus. Furthermore, the sensing electrode is easy to prepare, highly operable, has a fast response, high sensitivity, and strong controllability, making it suitable for on-site rapid detection and portable analysis needs in fields such as food production and processing, circulation supervision, and public health monitoring. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of SCT hydrogel synthesis and sensor fabrication. Figure 2 Figures show the morphological characterization results of SA-CS hydrogel and SCT hydrogel. A, B: SEM results, CI: EDS results. Figure 3A Figure 1 shows the inverted vial experiment for the stability of porphyrin hydrogels. a: CS-TPPS hydrogel, b: SCT hydrogel, c: SA-TPPS hydrogel. Figure 3B The results show the stability of the ECL signal. a: CS-TPPS hydrogel, b: SCT hydrogel, c: SA-TPPS hydrogel, d: TPPS aqueous solution. Figure 3C Comparison of ECL properties of SCT hydrogels reinforced with different carbon nanotubes; a: SCT hydrogel, b: SCT hydrogel / MWCNTs-COOH / GCE, c: SCT hydrogel / SWCNTs-COOH / GCE, d: SCT hydrogel / SWCNTs / GCE, e: SCT hydrogel / MWCNTs / GCE. Figure 4A Linear detection results of the sensor for detecting Staphylococcus aureus are shown in the figure. The response curves of ECL intensity and Staphylococcus aureus concentration are: a: 5 CFU / mL, b: 10 CFU / mL, c: 102 CFU / mL, d: 103 CFU / mL, e: 104 CFU / mL, f: 105 CFU / mL, g: 106 CFU / mL, h: 107 CFU / mL, i: ​​108 CFU / mL, j: 2×108 CFU / mL, k: 5×108 CFU / mL. Figure 4B The curve showing the linear relationship between ECL intensity and the logarithm of Staphylococcus aureus concentration; Figure 5A This is a selective experimental plot; Figure 5B The graphs show the stability of the sensor for detecting Staphylococcus aureus at different concentrations: a: 10³ CFU / mL, b: 10⁶ CFU / mL, c: 10² CFU / mL. Detailed Implementation

[0023] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0024] In the following examples, ECL testing was performed on an electrochemiluminescence analyzer using a conventional three-electrode system: a modified glassy carbon electrode or a screen-printed electrode as the working electrode, a platinum wire as the counter electrode, and Ag / AgCl as the reference electrode. The photomultiplier tube's operating voltage was set to 600–800 V, and the potential scan range was 0–-1.3 V. Example

[0025] A method for preparing an electrochemiluminescence biosensor based on carbon-reinforced porphyrin hydrogel includes the following steps: (1) Preparation of working electrode 1) Synthesis of SCT hydrogel 400 mg of chitosan (CS) was dissolved in 20 mL of acetic acid aqueous solution and stirred at room temperature for 4 hours to obtain CS hydrogel; 800 mg of sodium alginate (SA) was dissolved in 40 mL of NaCl solution and stirred for 4 hours to obtain SA hydrogel; the prepared CS hydrogel and SA hydrogel were mixed at a volume ratio of 1:2 and stirred magnetically to obtain SA-CS hydrogel; 1 mg of 5,10,15,20-tetrakis(4-sulfonylphenyl)porphyrin (TPPS) was added to 1 mL of the prepared SA-CS hydrogel and stirred magnetically to form a homogeneous state, thus obtaining SCT hydrogel; the mass fraction of acetic acid aqueous solution was 0.5%, and the concentration of NaCl solution was 0.5 mol / L.

[0026] 2) Preparation of Au NPs 100 mL of tetrachloroauric acid (HAuCl4) solution was stirred on a magnetic stirrer and heated to a gentle boil. Then, 3.0 mL of sodium citrate solution was quickly added, and the mixture was refluxed at 90 °C for 30 minutes. Heating was stopped immediately when the solution color changed from colorless to red. The formation of gold nanoparticles (Au NPs) was confirmed by measuring its UV-Vis absorption spectrum. The obtained Au NPs were stored below 4 °C for later use. The mass fraction of the tetrachloroauric acid (HAuCl4) solution was 0.01%, and the mass fraction of the sodium citrate solution was 1%.

[0027] 3) Modified electrode The glassy carbon electrode surface was polished, and 5 μL of carboxylated multi-walled carbon nanotube solution was drop-coated onto the electrode surface. Then, 5 μL of SCT hydrogel prepared in step 1) was drop-coated and dried at 37 °C. 5 μL of Au NPs prepared in step 2) was added, followed by 5 μL of Staphylococcus aureus antibody solution, and the mixture was incubated at 4 °C for 4 hours. After rinsing with PBS solution at pH 7.4, the mixture was incubated with 5 μL of BSA solution at 4 °C for 15 minutes. Finally, it was rinsed with PBS solution at pH 7.4, and excess water was absorbed with filter paper to obtain the working electrode. The concentration of the carboxylated multi-walled carbon nanotube solution was 1 mg / mL, the concentration of the Staphylococcus aureus antibody solution was 5 μg / mL, and the concentration of the BSA solution was 0.25 wt%.

[0028] (2) Preparation of detection solution Weigh 27 mg of K2S2O8 into a centrifuge tube, add 10 mL of PBS solution with a pH of 7.4, and sonicate to dissolve and prepare a 10 mmol / L K2S2O8 detection solution.

[0029] The working electrode was used to detect Staphylococcus aureus. The detection method was as follows: the sample to be tested was dropped onto the working electrode and incubated at room temperature in the dark for 40 minutes. Then, it was gently rinsed with PBS solution with a pH of 7.4. The working electrode was then placed in a reaction cell containing the detection solution for ECL detection. Example

[0030] A method for preparing an electrochemiluminescence biosensor based on carbon-reinforced porphyrin hydrogel includes the following steps: (1) Preparation of working electrode 1) Synthesis of SCT hydrogel 385 mg of chitosan (CS) was dissolved in 20 mL of acetic acid aqueous solution and stirred at room temperature for 4 hours to obtain CS hydrogel; 805 mg of sodium alginate (SA) was dissolved in 40 mL of NaCl solution and stirred for 4 hours to obtain SA hydrogel; the prepared CS hydrogel and SA hydrogel were mixed at a volume ratio of 1:1.5 and stirred magnetically to obtain SA-CS hydrogel; 0.8 mg of 5,10,15,20-tetrakis(4-sulfonylphenyl)porphyrin (TPPS) was added to 1 mL of the prepared SA-CS hydrogel and stirred magnetically to form a homogeneous state, thus obtaining SCT hydrogel; the mass fraction of acetic acid aqueous solution was 0.5%, and the concentration of NaCl solution was 0.5 mol / L.

[0031] 2) Preparation of Au NPs 110 mL of tetrachloroauric acid (HAuCl4) solution was stirred on a magnetic stirrer and heated to a gentle boil. Then, 3.0 mL of sodium citrate solution was rapidly added, and the mixture was refluxed at 90 °C for 30 minutes. Heating was stopped immediately when the solution color changed from colorless to red. The formation of gold nanoparticles (Au NPs) was confirmed by measuring their UV-Vis absorption spectra. The obtained Au NPs were stored below 4 °C for later use. The mass fraction of the tetrachloroauric acid (HAuCl4) solution was 0.01%, and the mass fraction of the sodium citrate solution was 1%.

[0032] 3) Modified electrode The glassy carbon electrode surface was polished, and 5 μL of carboxylated single-walled carbon nanotube solution was drop-coated onto the electrode surface. Then, 5 μL of the SCT hydrogel prepared in step 1) was drop-coated and dried at 37 °C. 5 μL of Au NPs prepared in step 2) was added, followed by 5 μL of Staphylococcus aureus antibody solution, and the mixture was incubated at 4 °C for 5 hours. After rinsing with PBS solution at pH 7.4, the mixture was incubated with 5 μL of BSA solution at 4 °C for 20 minutes. Finally, it was rinsed with PBS solution at pH 7.4, and excess water was absorbed with filter paper to obtain the working electrode. The concentration of the carboxylated single-walled carbon nanotube solution was 1 mg / mL, the concentration of the Staphylococcus aureus antibody solution was 5 μg / mL, and the concentration of the BSA solution was 0.25 wt%.

[0033] (2) Preparation of detection solution Weigh 81 mg of K2S2O8 into a centrifuge tube, add 10 mL of PBS solution with a pH of 7.4, and sonicate to dissolve and prepare a 30 mmol / L K2S2O8 detection solution.

[0034] The working electrode was used to detect Staphylococcus aureus. The detection method was as follows: the sample to be tested was dropped onto the working electrode and incubated at room temperature in the dark for 50 minutes. Then, it was gently rinsed with PBS solution with a pH of 7.4. The working electrode was then placed in a reaction cell containing the detection solution for ECL detection. Example

[0035] A method for preparing an electrochemiluminescence biosensor based on carbon-reinforced porphyrin hydrogel includes the following steps: (1) Preparation of working electrode 1) Synthesis of SCT hydrogel 395 mg of chitosan (CS) was dissolved in 20 mL of acetic acid aqueous solution and stirred at room temperature for 4 hours to obtain CS hydrogel; 790 mg of sodium alginate (SA) was dissolved in 40 mL of NaCl solution and stirred for 4 hours to obtain SA hydrogel; the prepared CS hydrogel and SA hydrogel were mixed at a volume ratio of 1:1 and stirred magnetically to obtain SA-CS hydrogel; 1.5 mg of 5,10,15,20-tetrakis(4-sulfonylphenyl)porphyrin (TPPS) was added to 1 mL of the prepared SA-CS hydrogel and stirred magnetically to form a homogeneous state, thus obtaining SCT hydrogel; the mass fraction of acetic acid aqueous solution was 0.5%, and the concentration of NaCl solution was 0.5 mol / L.

[0036] 2) Preparation of Au NPs 130 mL of tetrachloroauric acid (HAuCl4) solution was stirred on a magnetic stirrer and heated to a gentle boil. Then, 3.0 mL of sodium citrate solution was rapidly added, and the mixture was refluxed at 90 °C for 30 minutes. Heating was stopped immediately when the solution color changed from colorless to red. The formation of gold nanoparticles (Au NPs) was confirmed by measuring their UV-Vis absorption spectra. The obtained Au NPs were stored below 4 °C for later use. The mass fraction of the tetrachloroauric acid (HAuCl4) solution was 0.01%, and the mass fraction of the sodium citrate solution was 1%.

[0037] 3) Modified electrode 5 μL of single-walled carbon nanotube solution was drop-coated onto the surface of a screen-printed electrode, followed by 5 μL of the SCT hydrogel prepared in step 1), and dried at 37 °C. 5 μL of Au NPs prepared in step 2) was added, followed by 5 μL of Staphylococcus aureus antibody solution, and the electrode was incubated at 4 °C for 7 hours. After rinsing with PBS solution at pH 7.4, the electrode was incubated with 5 μL of BSA solution at 4 °C for 25 minutes, and finally rinsed with PBS solution at pH 7.4. Excess water was absorbed with filter paper to obtain the working electrode. The concentration of the single-walled carbon nanotube solution was 1 mg / mL, the concentration of the Staphylococcus aureus antibody solution was 5 μg / mL, and the concentration of the BSA solution was 0.25 wt%.

[0038] (2) Preparation of detection solution Weigh 135 mg of K2S2O8 into a centrifuge tube, add 10 mL of PBS solution with a pH of 7.4, and sonicate to dissolve and prepare a 50 mmol / L K2S2O8 detection solution.

[0039] The working electrode was used to detect Staphylococcus aureus. The detection method was as follows: the sample to be tested was dropped onto the working electrode and incubated at room temperature in the dark for 40 minutes. Then, it was gently rinsed with PBS solution with a pH of 7.4. The working electrode was then placed in a reaction cell containing the detection solution for ECL detection. Example

[0040] A method for preparing an electrochemiluminescence biosensor based on carbon-reinforced porphyrin hydrogel includes the following steps: (1) Preparation of working electrode 1) Synthesis of SCT hydrogel 350 mg of chitosan (CS) was dissolved in 20 mL of acetic acid aqueous solution and stirred at room temperature for 4 hours to obtain CS hydrogel; 800 mg of sodium alginate (SA) was dissolved in 40 mL of NaCl solution and stirred for 4 hours to obtain SA hydrogel; the prepared CS hydrogel and SA hydrogel were mixed at a volume ratio of 1:0.5 and stirred magnetically to obtain SA-CS hydrogel; 0.5 mg of 5,10,15,20-tetrakis(4-sulfonylphenyl)porphyrin (TPPS) was added to 1 mL of the prepared SA-CS hydrogel and stirred magnetically to form a homogeneous state, thus obtaining SCT hydrogel; the mass fraction of acetic acid aqueous solution was 0.5%, and the concentration of NaCl solution was 0.5 mol / L.

[0041] 2) Preparation of Au NPs 150 mL of tetrachloroauric acid (HAuCl4) solution was stirred on a magnetic stirrer and heated to a gentle boil. Then, 3.0 mL of sodium citrate solution was rapidly added, and the mixture was refluxed at 90 °C for 30 minutes. Heating was stopped immediately when the solution color changed from colorless to red. The formation of gold nanoparticles (Au NPs) was confirmed by measuring their UV-Vis absorption spectra. The obtained Au NPs were stored below 4 °C for later use. The mass fraction of the tetrachloroauric acid (HAuCl4) solution was 0.01%, and the mass fraction of the sodium citrate solution was 1%.

[0042] 3) Modified electrode The glassy carbon electrode surface was polished, and 5 μL of multi-walled carbon nanotube solution was drop-coated onto the electrode surface. Then, 5 μL of SCT hydrogel prepared in step 1) was drop-coated and dried at 37 °C. 5 μL of Au NPs prepared in step 2) was added, followed by 5 μL of Staphylococcus aureus antibody solution. The electrode was incubated at 4 °C for 8 hours. After rinsing with PBS solution at pH 7.4, the electrode was incubated with 5 μL of BSA solution at 4 °C for 20 minutes. Finally, the electrode was rinsed with PBS solution at pH 7.4, and excess water was absorbed with filter paper to obtain the working electrode. The concentration of the multi-walled carbon nanotube solution was 1 mg / mL, the concentration of the Staphylococcus aureus antibody solution was 5 μg / mL, and the concentration of the BSA solution was 0.25 wt%.

[0043] (2) Preparation of detection solution Weigh 189 mg of K2S2O8 into a centrifuge tube, add 10 mL of PBS solution with a pH of 7.4, and sonicate to dissolve and prepare a 70 mmol / L K2S2O8 detection solution.

[0044] The working electrode was used to detect Staphylococcus aureus. The detection method was as follows: the sample to be tested was dropped onto the working electrode and incubated at room temperature in the dark for 60 minutes. Then, it was gently rinsed with PBS solution with a pH of 7.4. The working electrode was then placed in a reaction cell containing the detection solution for ECL detection. Example

[0045] A method for preparing an electrochemiluminescence biosensor based on carbon-reinforced porphyrin hydrogel includes the following steps: (1) Preparation of working electrode 1) Synthesis of SCT hydrogel 365 mg of chitosan (CS) was dissolved in 20 mL of acetic acid aqueous solution and stirred at room temperature for 4 hours to obtain CS hydrogel; 800 mg of sodium alginate (SA) was dissolved in 40 mL of NaCl solution and stirred for 4 hours to obtain SA hydrogel; the prepared CS hydrogel and SA hydrogel were mixed at a volume ratio of 1:2 and stirred magnetically to obtain SA-CS hydrogel; 2 mg of 5,10,15,20-tetrakis(4-sulfonylphenyl)porphyrin (TPPS) was added to 1 mL of the prepared SA-CS hydrogel and stirred magnetically to form a homogeneous state, thus obtaining SCT hydrogel; the mass fraction of acetic acid aqueous solution was 0.5%, and the concentration of NaCl solution was 0.5 mol / L.

[0046] 2) Preparation of Au NPs 120 mL of tetrachloroauric acid (HAuCl4) solution was stirred on a magnetic stirrer and heated to a gentle boil. Then, 3.0 mL of sodium citrate solution was rapidly added, and the mixture was refluxed at 90 °C for 30 minutes. Heating was stopped immediately when the solution color changed from colorless to red. The formation of gold nanoparticles (Au NPs) was confirmed by measuring their UV-Vis absorption spectra. The obtained Au NPs were stored below 4 °C for later use. The mass fraction of the tetrachloroauric acid (HAuCl4) solution was 0.01%, and the mass fraction of the sodium citrate solution was 1%.

[0047] 3) Modified electrode 5 μL of carboxylated multi-walled carbon nanotube solution was drop-coated onto the surface of a screen-printed electrode, followed by 5 μL of the SCT hydrogel prepared in step 1), and dried at 37 °C. 5 μL of Au NPs prepared in step 2) was added, followed by 5 μL of Staphylococcus aureus antibody solution, and the electrode was incubated at 4 °C for 6 hours. After rinsing with PBS solution at pH 7.4, the electrode was incubated with 5 μL of BSA solution at 4 °C for 25 minutes. Finally, the electrode was rinsed with PBS solution at pH 7.4, and excess water was absorbed with filter paper to obtain the working electrode. The concentration of the carboxylated multi-walled carbon nanotube solution was 1 mg / mL, the concentration of the Staphylococcus aureus antibody solution was 5 μg / mL, and the concentration of the BSA solution was 0.25 wt%.

[0048] (2) Preparation of detection solution Weigh 270 mg of K2S2O8 into a centrifuge tube, add 10 mL of PBS solution with a pH of 7.4, and sonicate to dissolve and prepare a 100 mmol / L K2S2O8 detection solution.

[0049] The working electrode was used to detect Staphylococcus aureus. The detection method was as follows: the sample to be tested was dropped onto the working electrode and incubated at room temperature in the dark for 55 minutes. Then, it was gently rinsed with PBS solution with a pH of 7.4. The working electrode was then placed in a reaction cell containing the detection solution for ECL detection.

[0050] The morphology and structure of the SCT hydrogel were characterized to comprehensively explore its properties, and the performance of the fabricated sensor was tested. The specific testing methods are as follows: Different concentrations of Staphylococcus aureus were sequentially dropped onto the prepared working electrode and allowed to stand at room temperature in the dark for 40 minutes. After reacting with Staphylococcus aureus, the working electrode was gently rinsed and placed in a reaction cell containing the detection solution to detect the ECL signal value.

[0051] 1) Characterization of SCT hydrogel properties SCT hydrogels were prepared according to the method described in Example 1, and the SCT hydrogels were tested using SEM and EDS. The test results are as follows. Figure 2As shown. Figure 2 Image A shows the SEM morphology of the SA-CS hydrogel, revealing its porous structure. Figure 2 As shown in Figures B and DI, the prepared SCT hydrogel has a three-dimensional porous structure. Figure 2 In the middle (B), the six elements C, N, O, S, Na, and Cl are uniformly distributed. Figure 2 (China DI).

[0052] 2) Investigation of gel stability For comparison, the same amount of TPPS (1 mg) as in Example 1 was introduced into 1 mL of SA hydrogel and CS hydrogel, respectively, and magnetically stirred to prepare the corresponding SA-TPPS and CS-TPPS hydrogels (TPPS concentration of 1 mg / mL). The stability of the obtained hydrogels was verified by inverted vial experiment.

[0053] like Figure 3A As shown, the inverted vial experiment results indicate that CS-TPPS hydrogel is inhomogeneous and has high flow resistance; while SA-TPPS hydrogel exhibits excessive fluidity and insufficient stability. The corresponding ECL signal stability (e.g.) Figure 3B As shown in the figure, the ECL signal of the pure TPPS solution fluctuates the most, with an RSD of 15.04%. The signals of CS-TPPS hydrogel and SA-TPPS hydrogel are improved, with RSDs of 7.94% and 4.39% respectively, but their signal values ​​are not high. Obviously, both CS and SA hydrogels inhibit the TPPS signal. The signal of SCT hydrogel (SA-CS-TPPS) is the most stable, with an RSD as low as 2.90% (as shown in the figure). Figure 3B (Curve b). The above results fully demonstrate that the SA-CS composite hydrogel matrix can effectively restrict the free movement of TPPS molecules and inhibit their self-aggregation behavior, thereby generating a highly stable ECL signal.

[0054] Figure 3CThe ECL signal intensities of four types of carbon nanotubes as SCT hydrogel substrates are shown. Compared to the ECL signal of the SCT hydrogel-modified electrode (curve a), all carbon nanotube modifications enhance the ECL intensity of the SCT hydrogel, resulting in a significant improvement in ECL efficiency. The ECL intensity of the SCT hydrogel / carboxylated multi-walled carbon nanotube / glassy carbon electrode system is 5.7 times stronger than that of the SCT hydrogel / glassy carbon electrode, demonstrating that carboxylated carbon nanotubes have more catalytically active sites and can significantly enhance the ECL signal of the SCT hydrogel. The ECL efficiency of the SCT hydrogel / carboxylated multi-walled carbon nanotube / glassy carbon electrode system is 1.87 times higher than that of the SCT hydrogel-persulfate (S2O82-) system alone; carboxylated single-walled carbon nanotubes are 1.58 times higher, single-walled carbon nanotubes are 1.51 times higher, and multi-walled carbon nanotubes are 1.24 times higher.

[0055] 3) Sensitivity to Staphylococcus aureus Different concentrations of Staphylococcus aureus samples were prepared: a: 5 CFU / mL, b: 10 CFU / mL, c: 10² CFU / mL, d: 10³ CFU / mL, e: 10⁴ CFU / mL, f: 10⁵ CFU / mL, g: 10⁶ CFU / mL, h: 10⁷ CFU / mL, i: ​​10⁸ CFU / mL, j: 2 × 10⁸ CFU / mL, and k: 5 × 10⁸ CFU / mL. The ECL signal after treatment with different concentrations of Staphylococcus aureus was detected. The results are as follows: Figure 4A As shown, within the concentration range of 10 CFU / mL to 10⁸ CFU / mL, the detected ECL signal exhibited a strong negative correlation with the logarithm of the Staphylococcus aureus concentration. The linear equation was I = 10²³⁹⁸ - 10¹⁸⁶lgc, R² = 0.999, (I: electrochemiluminescence intensity; c: Staphylococcus aureus concentration; R: correlation coefficient). Figure 4B The limit of detection (LOD) was 1.02 CFU / mL. Results are as follows: Figure 4A and Figure 4B As shown, ECL decreases with increasing Staphylococcus aureus concentration in a linear fashion, indicating that the constructed sensor has good sensitivity.

[0056] 4) Interference and stability studies To evaluate the selectivity of the constructed sensor, *Escherichia coli* (E. coli), *Porphyromonas gingivalis* (P. gingivalis), and *Lactobacillus rhamnosus* (X253) were selected as representative interfering bacteria, with *Staphylococcus aureus* as the target analyte. The concentration of all bacteria was uniformly set at 10⁵ CFU / mL. The following test samples were prepared: three single interfering bacteria, the target bacterium (*Staphylococcus aureus*), and a mixture of the target bacterium and the three interfering bacteria. Following the sensing method in Example 1, the working electrode and detection solution were prepared. 5 μL of the above sample was dropped onto the working electrode and incubated at room temperature in the dark for 40 minutes. The working electrode, after interaction with the interfering bacteria, was placed in a reaction chamber containing the detection solution, and the ECL signals of the interfering bacteria, *Staphylococcus aureus*, and the mixed bacterial solution were detected. The results are as follows: Figure 5A As shown, the ECL signal intensity of a single interfering substance is close to the initial value, and the ECL signal of Staphylococcus aureus mixed with the interfering substance is close to the measured value of Staphylococcus aureus, indicating that the constructed sensor has good selectivity.

[0057] Subsequently, the stability of the constructed sensor was tested using different concentrations (10³ CFU / mL, 10⁶ CFU / mL, and 10⁸ CFU / mL). Figure 5B As shown, the ECL signal recorded over 300 seconds was stable, with relative standard deviations (RSDs) of 1.09%, 1.60%, and 0.66%, respectively. The small RSD values ​​indicate that the sensing platform has good stability.

[0058] 5) Spike test Spiked recovery experiments of Staphylococcus aureus (10³ CFU / mL, 10⁵ CFU / mL, and 10⁷ CFU / mL) were conducted on actual samples (milk, water, and pork). Following the sensing detection method in Example 1, 5 μL of sample solution was dropped onto the working electrode and incubated at room temperature in the dark for 40 minutes. The working electrode was placed in a reaction cell containing the detection solution, and the corresponding ECL signal was detected. The recovery rate was calculated. As shown in Table 1, the recovery rate of this sensor ranged from 95.7% to 102%, and the relative standard deviation (RSD) ranged from 0.59% to 8.07%. In the detection of pork samples, the recovery rates were determined to be 95.7 ± 2.53% and 101.6 ± 7.95%. These results indicate that the proposed immunosensor is suitable for the accurate quantitative detection of Staphylococcus aureus in actual samples.

[0059] Table 1 Spike recovery experiments of actual samples

[0060] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing an electrochemiluminescence biosensor based on carbon-reinforced porphyrin hydrogel, characterized in that: The sensor includes a working electrode and a detection liquid, and the method includes the following steps: (1) Preparation of working electrode 1) Synthesis of SCT hydrogel Chitosan (CS) was dissolved in an aqueous acetic acid solution and stirred at room temperature to obtain CS hydrogel; sodium alginate (SA) was dissolved in NaCl solution and stirred at room temperature to obtain SA hydrogel; the prepared CS hydrogel was added to the SA hydrogel and mixed by magnetic stirring to obtain SA-CS hydrogel; 5,10,15,20-tetrakis(4-sulfonylphenyl)porphyrin (TPPS) was added to the prepared SA-CS hydrogel and stirred by magnetic stirring to obtain SCT hydrogel; 2) Preparation of Au NPs The tetrachloroauric acid (HAuCl4) solution was heated to a gentle boil; then, sodium citrate solution was rapidly added, and the mixture was refluxed at 90°C for 30 minutes; heating was stopped immediately when the solution color changed from colorless to red; the formation of gold nanoparticles (Au NPs) was confirmed by measuring its ultraviolet-visible absorption spectrum; the obtained Au NPs were stored below 4°C for later use. 3) Modified electrodes Carbon nanotube solution was drop-coated onto the electrode surface, followed by SCT hydrogel prepared in step 1). After drying, Au NPs prepared in step 2) were added, and then Staphylococcus aureus antibody solution was added and incubated for 4-8 hours. BSA (bovine serum albumin) solution was then used to incubate at 4°C for 15-30 minutes to obtain the working electrode. (2) Preparation of detection solution Dissolve and dilute K2S2O8 with PBS buffer solution to prepare K2S2O8 detection solution.

2. The method for preparing a carbon-reinforced porphyrin hydrogel electrochemiluminescence biosensor according to claim 1, characterized in that: In step 1), 350-400 mg of chitosan (CS) is weighed and dissolved in 20 mL of acetic acid aqueous solution, and stirred at room temperature for 4 hours to obtain CS hydrogel; 790-805 mg of sodium alginate (SA) is dissolved in 40 mL of NaCl solution and stirred for 4 hours to obtain SA hydrogel; the prepared CS hydrogel and SA hydrogel are mixed at a volume ratio of 1:0.5-2 and mixed by magnetic stirring to obtain SA-CS hydrogel; 0.5-2 mg of 5,10,15,20-tetra(4-sulfophenyl)porphyrin (TPPS) is added to 1 mL of the prepared SA-CS hydrogel and stirred by magnetic stirring to form a homogeneous state, thus obtaining SCT hydrogel.

3. The method for preparing a carbon-reinforced porphyrin hydrogel electrochemiluminescence biosensor according to claim 2, characterized in that: In step 1), the mass fraction of the acetic acid aqueous solution is 0.5%, and the concentration of the NaCl solution is 0.5 mol / L.

4. The method for preparing a carbon-reinforced porphyrin hydrogel electrochemiluminescence biosensor according to claim 1, characterized in that: In step 2), 100-150 mL of tetrachloroauric acid (HAuCl4) solution is placed on a magnetic stirrer and stirred, and heated to a gentle boil. Then, 3.0 mL of sodium citrate solution is quickly added, and the mixture is refluxed at 90 °C for 30 minutes. When the solution color changes from colorless to red, heating is stopped immediately. The formation of gold nanoparticles (Au NPs) is confirmed by measuring its ultraviolet-visible absorption spectrum. The obtained Au NPs are stored below 4 °C for later use.

5. The method for preparing a carbon-reinforced porphyrin hydrogel electrochemiluminescence biosensor according to claim 4, characterized in that: In step 2), the mass fraction of the tetrachloroauric acid solution is 0.01%; the mass fraction of the sodium citrate solution is 1%.

6. The method for preparing a carbon-reinforced porphyrin hydrogel electrochemiluminescence biosensor according to claim 1, characterized in that: In step 3), 5 μL of carbon nanotube solution is drop-coated onto the electrode surface, followed by 5 μL of SCT hydrogel prepared in step 1), and dried at 37°C; 5 μL of Au NPs prepared in step 2) is added, followed by 5 μL of Staphylococcus aureus antibody solution, and incubated at 4°C for 4–8 hours; 5 μL of BSA (bovine serum albumin) solution is used for incubation at 4°C for 15–30 minutes, and finally washed with PBS solution at pH 7.4, and excess water is absorbed with filter paper to obtain the working electrode.

7. The method for preparing a carbon-reinforced porphyrin hydrogel electrochemiluminescence biosensor according to claim 6, characterized in that: In step 3), the carbon nanotube solution is one of a multi-walled carbon nanotube solution, a carboxylated multi-walled carbon nanotube solution, a single-walled carbon nanotube solution, or a carboxylated single-walled carbon nanotube solution; the electrode is a glassy carbon electrode or a screen-printed electrode.

8. The method for preparing a carbon-reinforced porphyrin hydrogel electrochemiluminescence biosensor according to claim 6, characterized in that: In step 3), the concentration of the carbon nanotube solution is 1 mg / mL, the concentration of the Staphylococcus aureus antibody solution is 5 μg / mL, and the concentration of the BSA solution is 0.25 wt%.

9. The method for preparing a carbon-reinforced porphyrin hydrogel electrochemiluminescence biosensor according to claim 1, characterized in that: In step (2), 27-270 mg of K2S2O8 is weighed into a centrifuge tube, 10 mL of PBS solution with a pH of 7.4 is added, and the solution is sonicated to prepare a K2S2O8 detection solution with a concentration of 10-100 mmol / L.

10. The method for preparing a carbon-reinforced porphyrin hydrogel electrochemiluminescence biosensor according to claim 1, characterized in that: The sensor is used as follows: the sample to be tested is dropped onto the working electrode and incubated at room temperature in the dark for 40 to 60 minutes. Then, it is gently rinsed with PBS solution with a pH of 7.

4. The working electrode is then placed in a reaction cell containing the detection solution for ECL detection.

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