Dual-mode portable biosensor for detecting pseudomonas aeruginosa and application thereof

Abstract

The invention provides a dual-mode portable biosensor for detecting pseudomonas aeruginosa and application of the dual-mode portable biosensor, and belongs to the technical field of biosensors. The dual-mode portable biosensor comprises a closed bipolar electrode, magnetic beads for detecting pseudomonas aeruginosa, a methylene blue solution, a TCEP solution, a Ru (bpy) 3 < 2 + > solution, boron nitride quantum dots and a TPrA solution. The portable biosensor can rapidly and accurately detect the content of pseudomonas aeruginosa in a sample under low driving voltage, is high in sensitivity, good in specificity, high in anti-interference performance, good in repeatability and convenient to carry, and can be used for field detection. Pseudomonas aeruginosa adsorbed on the detected electrode is effectively killed by adopting copper ions, so that collaborative development of dual functions of detection and sterilization is realized, and environmental pollution is effectively avoided.

Description

Technical Field

[0001] The invention belongs to the technical field of biosensors, and in particular relates to a dual-mode portable biosensor for detecting Pseudomonas aeruginosa and an application thereof. Background Art

[0002] Pseudomonas aeruginosa (P.aeruginosa), formerly known as Pseudomonas aeruginosa, is a representative species of the genus Pseudomonas. It is widely distributed in nature and is one of the most common bacteria in the soil. This bacterium is found in various water, air, normal human skin, respiratory tract and intestines. Pseudomonas aeruginosa is a common conditional pathogen and an opportunistic infection pathogen that causes impaired immunity. Pseudomonas aeruginosa can cause foodborne diseases by contaminating food, especially packaged drinking water. It poses a greater health risk to people with weak resistance, such as the elderly, the weak, the sick, the young, and pregnant women, and is prone to causing diseases such as acute enteritis.

[0003] The testing principle of Pseudomonas aeruginosa is based on its specific growth, morphology, physiological and biochemical characteristics on a specific culture medium. First, a filter membrane with a pore size of 0.45 μm is used to intercept the microorganisms in 250 mL of water sample, and then the filter membrane is moved to CN agar culture medium and cultured at 36°C for 24 to 48 hours to allow the microorganisms on the filter membrane to grow; then, the suspected colonies are confirmed by combining the color and fluorescence characteristics of the colonies and biochemical tests such as acetamide broth and oxidase to determine whether Pseudomonas aeruginosa is detected and counted. The current testing standard in my country is "57 Pseudomonas aeruginosa" in GB 8538-2016 "National Food Safety Standard for Drinking Natural Mineral Water Testing Methods".

[0004] However, the above method is time-consuming and needs to be performed in a specific laboratory. It is not possible to perform rapid detection on site, nor is it possible to perform quantitative detection of Pseudomonas aeruginosa in samples. Summary of the invention

[0005] The purpose of the present invention is to provide a dual-mode portable biosensor for detecting Pseudomonas aeruginosa, which can quickly and accurately detect the content of Pseudomonas aeruginosa in a sample at a relatively low driving voltage, and is therefore easy to carry and conduct on-site detection.

[0006] The purpose of the present invention is achieved by the following technical solutions:

[0007] A dual-mode portable biosensor for detecting Pseudomonas aeruginosa, comprising a closed bipolar electrode, magnetic beads for detecting Pseudomonas aeruginosa, a methylene blue solution, a TCEP solution, and Ru(bpy)3 2+ solution, BN quantum dots and TPrA solution.

[0008] In the present invention, the magnetic beads used for detecting Pseudomonas aeruginosa carry Pseudomonas aeruginosa aptamers.

[0009] In the present invention, the magnetic beads for detecting Pseudomonas aeruginosa are prepared by amide reaction of Fe3O4 magnetic material wrapped with polydopamine and Pseudomonas aeruginosa aptamer with carboxyl group at the 5' end.

[0010] In the present invention, boron nitride quantum dots are prepared by the following method: boric acid, melamine and water are mixed and stirred, reacted at 190-210° C. for 18-22 hours, and cooled to room temperature to obtain boron nitride quantum dots.

[0011] In the present invention, the mass ratio of boric acid to melamine is 5:0.5-1.5.

[0012] In the present invention, the methylene blue solution is a 90-110 mM methylene blue aqueous solution, the TCEP solution is a 40-60 mM TCEP aqueous solution, and Ru(bpy)3 2+ The concentration of the solution is 0.5-1.5mM,

[0013] In the present invention, the concentration of TPrA solution is 4-6M.

[0014] The present invention also provides a method for detecting Pseudomonas aeruginosa using the biosensor, comprising the following steps:

[0015] (1) The sample is adsorbed by the magnetic beads for detecting Pseudomonas aeruginosa, and resuspended to obtain a test solution;

[0016] (2) A voltage of 2.9 V was applied to the closed bipolar electrode, a mixture of methylene blue solution, TCEP solution and the test solution was dripped onto the cathode surface, and Ru(bpy)3 was dripped onto the anode surface. 2+ The solution, the mixture of boron nitride quantum dots, TPrA solution and PBS buffer solution, the anode emits red light, and the ECL image is used to identify the R value of RGB, which is substituted into the standard curve to obtain the concentration of Pseudomonas aeruginosa;

[0017] (3) Use 808 nm near-infrared laser to irradiate 50 μL of the test solution for 3.5 min. Subtract the temperature before irradiation from the temperature after irradiation for 3.5 min. The resulting value is called ΔT. Substitute it into the standard curve to obtain the concentration of Pseudomonas aeruginosa.

[0018] In the present invention, after the detection is completed, a copper sulfate aqueous solution is added to the detected liquid and the detected magnetic beads for sterilization.

[0019] In the present invention, the concentration of the copper sulfate aqueous solution is 0.5-5 mM.

[0020] Beneficial effects of the present invention:

[0021] ① The closed bipolar electrode of the biosensor of the present invention, the cathode can trigger the reaction at a low voltage through the cascade reaction with MB, TCEP and PDA@MBs, thereby significantly improving the sensitivity of the sensor and avoiding a series of side reactions caused by high driving voltage, such as electrode passivation, battery electrical damage and hydrogen and oxygen evolution reactions. The anode effectively enhances the stability of luminescence by combining with the dual co-reactant TPrA and BNQDs. When Pseudomonas aeruginosa is present in the solution to be detected, the portable biosensor of the present invention, in the colorimetric mode, the red light emitted by the anode becomes darker. The higher the bacterial concentration, the smaller the RGB red value (R value). In the photothermal mode, the temperature change value ΔT of the sample being tested decreases as the bacterial concentration increases. Therefore, the RGB red value (R value) or the temperature change value ΔT can be used to detect Pseudomonas aeruginosa of different concentrations. The detection limit (LOD) of the biosensor of the present invention reaches 0.34CFU / mL and 0.40CFU / mL, respectively, with high sensitivity, good specificity, strong anti-interference and good repeatability.

[0022] ② The biosensor of the present invention does not need to be connected to an electrochemical workstation, and can be powered by a mobile device (such as a power bank with a USB interface, a laptop computer, and a mobile phone, etc.), and the voltage is controlled to 2.9V by connecting a voltage converter. Therefore, it is easy to carry, thereby achieving rapid and on-site detection.

[0023] ③After the detection is completed, copper ions are used to effectively kill Pseudomonas aeruginosa adsorbed on the electrode after detection, achieving the coordinated development of the dual functions of detection and sterilization, thereby effectively avoiding environmental pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The standard curve of the portable biosensor of the present invention is shown. Figure 1 (A) and (B) show the changes of R value (A) and ΔT (B) with the concentration of Pseudomonas aeruginosa (the insets are the ECL image and the thermometer image, respectively). The horizontal axis is the concentration of Pseudomonas aeruginosa in CFU / mL, and the vertical axis is the R value or ΔT (in °C), respectively. Figure 1 (C) is the standard curve of the portable biosensor of the present invention for detecting the standard solution, chicken breast processed solution (i.e., chicken breast matrix solution) or milk processed solution (i.e., milk matrix solution), the horizontal axis is lgC, C is the concentration of Pseudomonas aeruginosa in CFU / mL, and the vertical axis is the R value. Figure 1 (D) is the standard curve of the standard solution, chicken breast treated solution (i.e., chicken breast matrix solution) or milk treated solution (i.e., milk matrix solution) detected by the portable biosensor of the present invention, the abscissa is lgC, C is the concentration of Pseudomonas aeruginosa, the unit is CFU / mL, and the ordinate is ΔT, the unit is ℃.

[0025] Figure 2 Schematic diagram of the construction and detection process of the portable biosensor of the present invention.

[0026] Figure 3 (A) Schematic diagram of using the portable biosensor or qPCR of the present invention to detect Pseudomonas aeruginosa in actual samples. Figure 3 (B) Half violin plot of the error distribution of the portable biosensor and qPCR detection results of the present invention, A represents R value analysis, and B represents ΔT analysis. Figure 3 (C) Confusion matrix summarizes the discrimination performance of the portable biosensor of the present invention for positive samples (n=76) and negative samples (n=10).

[0027] Figure 4 This is the verification of the portable biosensor principle of the present invention. Figure 4 In (A), a is the bare GCE (2 mm diameter bare magnetic glassy carbon electrode) in the initial reaction system, and b is the Ru(bpy)3 2+ / GCE (reaction system: add 50 μL of 1 mM Ru(bpy)3 2+ solution), c is Ru(bpy)3 2+ / TPrA / GCE (reaction system: add 50 μL of 1 mM Ru(bpy)3 2+ solution and 5M TPrA solution), d is Ru(bpy)3 2+ / TPrA / BNQDs / GCE (reaction system: 50 μL of 1 mM Ru(bpy)3 was added dropwise to 3 mL of PBS buffer. 2+ a is the CV curve in solution, 5 M TPrA solution and BNQDs), e is the CV curve in BNQDs / GCE (reaction system: 50 μL of BNQDs was added dropwise to 3 mL of PBS buffer), and f is the CV curve in TPrA / GCE (50 μL of 5 M TPrA solution was added dropwise to 3 mL of PBS buffer). Figure 4In (B), a is MB / GCE (reaction system: 50 μL 100 mM MB was added to 3 mL PBS buffer), b is TCEP / GCE (reaction system: 50 μL 50 mM TCEP was added to 3 mL PBS buffer), c is PDA@MBs / GCE (10 μL of 0.7 mg / mL magnetic beads were added to the surface of the glassy carbon electrode, the initial reaction system), d is MB / TCEP / GCE (reaction system: 50 μL 100 mM MB and 50 mM TCEP were added to 3 mL PBS buffer), e is MB / TCEP / PDA@MBs / GCE (reaction system: 50 μL 100 mM MB and 50 mM TCEP were added to 3 mL PBS buffer, 10 μL 0.7 mg / mL magnetic beads were added to the surface of the glassy carbon electrode), and f is MB / TCEP / PA@PDA@MBs / 10 4 CV curve of CFU / mL Pseudomonas aeruginosa / GCE (reaction system: add 50 μL of 100 mM MB and 50 mM TCEP into 3 mL of PBS buffer, take 10 μL of 0.7 mg / mL magnetic beads bound to Pseudomonas aeruginosa and drop them on the surface of the glassy carbon electrode). Figure 4 (C) is the ECL luminescence mechanism of the anode. Figure 4 (D) is the cathode cascade effect mechanism. Figure 4 In (E), a is bare GCE (magnetic glassy carbon electrode), b is PDA@MBs / GCE (PDA@MBs dropped on glassy carbon electrode), c is PA@PDA@MBs / GCE (PA@PDA@MBs dropped on glassy carbon electrode), d is PA@PDA@MBs / 10 2 CFU / mL Pseudomonas aeruginosa / GCE (combined with 10 2 CFU / mL Pseudomonas aeruginosa magnetic beads were dropped on the glassy carbon electrode), e is PA@PDA@MBs / 10 6 CFU / mL Pseudomonas aeruginosa GCE (combined with 10 6 EIS curve of magnetic beads with CFU / mL of Pseudomonas aeruginosa dropped on a glassy carbon electrode. Figure 4 (F) (a) and (b) are the plate counts of the solution before and after adding copper sulfate, respectively.

[0028] Figure 5 (A) and 5(B) are the responses of different bacteria to the R value (A) and ΔT (B) of the portable biosensor of the present invention. Figure 5 (C) and 5 (D) are the responses of different matrices to the R value (A) and ΔT (B) of the portable biosensor of the present invention. Figure 5 (E) and (F) show the stability of the biosensor. Figure 5 E is the R value graph, Figure 5 F is ΔT. Figure 5 (G) and 5(H) show the reproducibility of the biosensor. Figure 5 (G) is the R value, Figure 5 (H) is ΔT.

[0029] Figure 6 This is a detection schematic diagram of a portable biosensor, where 1 is a power bank with a UBS interface, 2 is a voltage converter, 3 is a wire, 4 is a dark box, and 5 is a mobile phone.

[0030] The PBS buffer in the present invention has a pH of 7.2 and is an aqueous solution containing 136.89 mM NaCl, 2.67 mM KCl, 8.24 mM Na2HPO4 and 1.76 mM NaH2PO4.

[0031] Example 1 Dual-mode portable biosensor for detecting Pseudomonas aeruginosa

[0032] 1. Composition of the dual-mode portable biosensor for detecting Pseudomonas aeruginosa

[0033] A dual-mode portable biosensor for detecting Pseudomonas aeruginosa (hereinafter referred to as the portable biosensor of the present invention) comprises a closed bipolar electrode, magnetic beads for detecting Pseudomonas aeruginosa, a 100 mM MB (methylene blue) aqueous solution, a 50 mM TCEP aqueous solution, and a 1 mM Ru(bpy)3 2+ Solution, BNQDs, 5 M TPrA solution, PBS buffer.

[0034] (1) Preparation of magnetic beads for detecting Pseudomonas aeruginosa

[0035] 1.35 g FeCl3·6H2O, 3.6 g sodium acetate and 75 mL diethylene glycol were mixed and stirred for 1.5 h, and then reacted at 200°C for 16 h; cooled to room temperature, 2 mL of 25% ammonia water, 40 mL of ethanol, 90 mL of water and 0.5 g of dopamine hydrochloride were added, and stirred for 24 h. The final product, polydopamine-coated Fe3O4 magnetic material (denoted as PDA@MBs), was magnetically separated, washed three times with water, and stored in ethanol to obtain an ethanol suspension of PDA@MBs.

[0036] Prepare an activation solution: an aqueous solution containing 0.025 M NHS (N-hydroxysuccinimide) and 0.025 M EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride).

[0037] 10 μL of 10 μM PA (Pseudomonas aeruginosa aptamer with a carboxyl group at the 5' end) aqueous solution was mixed with 90 μL of activation solution and shaken at 20°C and 200 rpm for 1 hour to obtain an activated PA solution. 70 μL of 1 mg / mL PDA@MBs ethanol suspension was magnetically separated from PDA@MBs and resuspended in 100 μL of PBS buffer, then added to the activated PA solution and incubated at 20°C and 200 rpm for 1 hour. The carboxyl group of PA reacted with the amine group of PDA@MBs to obtain a magnetic material with aptamer fixed (referred to as PA@PDA@MBs), which was rinsed three times with PBS buffer after magnetic separation. 10 μL of 2 μg / mL BSA aqueous solution was added to PA@PDA@MBs for 2 h to block the active sites on PA@PDA@MBs and prevent nonspecific adsorption. After magnetic separation, the beads were rinsed three times with PBS buffer to obtain magnetic beads for detecting Pseudomonas aeruginosa.

[0038] The specific sequence of PA is as follows: 5'-COOH-TTTTTCCCCCGTTGCTTTCGCTTTTCCTTTCGCTTTTGTTCGTTTCGTCCCTGCTTCCTTTCTTG-3'. The nucleotide sequence of PA is shown in SEQ ID NO:1.

[0039] (2) Preparation of BNQDs

[0040] 0.15 g of boric acid, 0.035 g of melamine and 30 mL of water were mixed and stirred for 20 min, then reacted at 200° C. for 20 h, and cooled to room temperature to obtain boron nitride quantum dots (denoted as BNQDs).

[0041] (3) Others

[0042] Closed bipolar electrode (abbreviated as BPE), made of ITO conductive glass, was purchased from Yiyang Xiangcheng South China Optoelectronics Co., Ltd.

[0043] 100mM MB aqueous solution. MB is methylene blue.

[0044] 50 mM TCEP aqueous solution: TCEP is tris(2-carboxyethyl)phosphine, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0045] 1mM Ru(bpy)3 2+ Solution: The solvent was PBS buffer, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Ru(bpy)3 2+ : Terpyridine ruthenium chloride hexahydrate.

[0046] 5M TPrA solution: tri-n-propylamine solution, product number: T818828-250ml Lot#: C12109386, purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0047] PBS buffer: pH 7.2, an aqueous solution containing 136.89 mM NaCl, 2.67 mM KCl, 8.24 mM Na2HPO4 and 1.76 mM NaH2PO4.

[0048] 2. Method and principle of detecting Pseudomonas aeruginosa using the portable biosensor of the present invention

[0049] (1) Detection method

[0050] ① Make a standard curve

[0051] Pseudomonas aeruginosa was cultured in LB liquid medium until the logarithmic growth phase and then diluted to 10 in PBS buffer. 7 CFU / mL, and then dilute 10 times to 10 6 CFU / mL, 10 5 CFU / mL, 10 4 CFU / mL, 10 3 CFU / mL, 10 2 CFU / mL, 10 1 CFU / mL, and a series of standard solutions were obtained.

[0052] 70 μL of 1 mg / mL magnetic beads for detecting Pseudomonas aeruginosa were magnetically separated, washed three times with PBS buffer, and then 100 μL of each concentration of Pseudomonas aeruginosa (standard solution) were added, incubated at 20°C for 1 hour, and rinsed three times with PBS buffer after magnetic separation. The magnetic beads were resuspended in 100 μL PBS buffer to obtain the test solution, of which 50 μL of the test solution was used for electrochemical detection, and the remaining 50 μL of the test solution was used for photothermal detection analysis. Note: Each concentration of standard solution was adsorbed with 70 μL of 1 mg / mL magnetic beads for detecting Pseudomonas aeruginosa.

[0053] i) Electrochemical detection

[0054] A constant low voltage (2.9 V) was applied to the BPE (closed bipolar electrode). 10 μL of 100 mM MB aqueous solution, 10 μL of 50 mM TCEP aqueous solution and 10 μL of the test solution were mixed evenly. Then 10 μL was added to the cathode surface of the BPE. 10 μL of 1 mM Ru(bpy)3 2+The solution, 2μL BNQDs, 2μL 5M TPrA solution and 16μL PBS buffer were mixed evenly, and then 10μL was added to the anode surface. At this time, the anode emitted red light, and the luminescence stability time lasted for more than 1 minute. As the bacterial concentration in the standard solution increased, the red light dimmed. The luminescence video of the anode was shot in a dark box with a smartphone, and the ECL image in the stable luminescence state was captured to identify the R value (red light value) of RGB. The results are as follows: Figure 1 (A). Perform linear regression with x as the horizontal axis and y as the vertical axis (e.g. Figure 1 (C)), the standard curve equation is y = -11.604x + 208.621 (R 2 =0.996), where x is 1gC, C is the concentration of Pseudomonas aeruginosa in CFU / mL, and y is the red light value. The detection limit (LOD) of this electrochemical detection method reaches 0.34 CFU / mL.

[0055] ii) Photothermal detection

[0056] The photothermal experiment was carried out at room temperature of 25°C. 50 μL of the test solution was irradiated with an 808 nm near-infrared laser for 3.5 min. A portable digital thermometer was used to monitor the temperature change. The temperature after irradiation for 3.5 min was subtracted from the temperature before irradiation. The resulting value was the temperature difference, recorded as ΔT. The results are shown in Figure 1 (B). Perform linear regression with x as the horizontal axis and y as the vertical axis (the results are shown in Figure 1 (D)), the standard curve equation is y = -0.499x + 9.903 (R 2 =0.994), where x is lgC, C is the concentration of Pseudomonas aeruginosa in CFU / mL, and y is the temperature difference ΔT in ° C. The detection limit (LOD) of the photothermal detection method reaches 0.40 CFU / mL.

[0057] ②Sample testing

[0058] Meat product samples: 2.5 g of meat product was sampled, 22.5 mL of PBS buffer was added, the mixture was mixed and homogenized, 100 μL of the supernatant was taken, and the portable biosensor of the present invention was used for detection. The R value (red light value) of RGB was obtained according to the electrochemical detection method in the preparation of the standard curve (the supernatant after the meat product was processed was used instead of the standard solution), and the concentration of Pseudomonas aeruginosa in the sample was calculated by substituting it into the standard curve equation. The ΔT was obtained according to the photothermal detection method in the preparation of the standard curve (the supernatant after the meat product was processed was used instead of the standard solution), and the concentration of Pseudomonas aeruginosa in the sample was calculated by substituting it into the standard curve equation.

[0059] Milk sample: dilute 1 mL of milk 100 times with PBS buffer and take 100 μL for biosensor detection. The detection method is the same as that of meat samples.

[0060] (2) Detection principle

[0061] When Pseudomonas aeruginosa exists in the sample to be tested, Pseudomonas aeruginosa is connected to the surface of the magnetic beads for detecting Pseudomonas aeruginosa through the aptamer. Then, MB (methylene blue), TCEP, and PDA@MBs dripped on the BPE cathode undergo redox reactions, and Ru(bpy)3 dripped on the BPE anode undergoes redox reactions. 2+ At the same time, a redox reaction occurs with tripropylamine (TPrA) and BNQDs, and the anode emits red light. As the concentration of bacteria in the sample increases, the red light becomes dimmer. By detecting the relationship between the red light value and Pseudomonas aeruginosa, it is possible to detect whether Pseudomonas aeruginosa exists in the sample. In addition, the temperature change of the test liquid under 808nm near-infrared laser irradiation can be used to detect whether Pseudomonas aeruginosa exists in the sample. The construction and detection process of the portable biosensor of the present invention is shown in Figure 2 Using the system of the present invention, it was unexpectedly found that the driving voltage applied to the closed bipolar electrodes only needed to be 2.9V.

[0062] In order to obtain an excellent sensor for detecting Pseudomonas aeruginosa, a large number of experiments were conducted on the redox system of the cathode and the anode. For example, the substance dripped on the anode in the present invention was replaced with Ru(bpy)3 2+ and TPrA, the cathode remains unchanged, at this time the minimum luminescent voltage applied to the closed bipolar electrode is 3.2V and the luminescence is unstable; if only Prussian blue is added to the cathode, the anode remains unchanged, the minimum luminescent voltage applied to the closed bipolar electrode is 4.8V.

[0063] In order to facilitate on-site detection, a power bank 1 with a UBS interface is connected to a closed bipolar electrode through a voltage converter 2, and the closed bipolar electrode with the reaction solution added to the cathode and anode is placed in a dark box 4, and then a smart phone 5 is used to shoot a video of the anode light emission through the camera window of the dark box. The dark box is in a rectangular shape, including a top plate, a bottom plate, front and rear side plates and a right side plate, and there is no side plate on the left side, which is used to put in or take out the closed bipolar electrode. A camera window is provided on the top plate, and the camera of the smart phone is placed above the camera window to shoot the video.

[0064] Since the driving voltage of the portable biosensor of the present invention is lower than 3V, it does not need to be connected to an electrochemical workstation and can be powered by a mobile device (such as a power bank with a USB interface, a laptop computer and a mobile phone. The voltage can be controlled at 2.9V by connecting a voltage converter), so it is easy to carry. Figure 6 shown.

[0065] 3. Sterilization after detection of Pseudomonas aeruginosa using the portable biosensor of the present invention

[0066] 100 μL of 1 mM copper sulfate aqueous solution was added to the magnetic beads after electrochemical detection or the test solution after photothermal detection using the portable biosensor of the present invention, and then coated on solid agar medium and cultured at 37°C for 12 hours to observe the growth of the colony. Figure 4 (F), sterile reproduction. Therefore, the portable biosensor of the present invention is easy to sterilize and has high safety.

[0067] Example 2 Detection of Pseudomonas aeruginosa in actual samples using the portable biosensor of the present invention

[0068] Meat samples were procured from supermarkets and artificially contaminated to prepare actual samples.

[0069] Three types of meat products were used as models for testing and analysis: (1) raw meat products (chicken, beef, pork, duck, lamb, fish, crayfish, sausage); (2) pre-seasoned meat products (chicken, beef, pork, duck, lamb, fish, sausage); and (3) cooked meat products (chicken, beef, pork, duck, lamb, fish, crayfish, sausage).

[0070] Preparation of positive actual samples: First, Pseudomonas aeruginosa was cultured in LB liquid medium to the logarithmic growth phase, and then diluted to 10 in PBS buffer. 7 CFU / mL. Add 1 mL of 10 7 CFU / mL of Pseudomonas aeruginosa suspension, then cultured at 37°C for 6h, as a positive actual sample, 100 μL of supernatant was taken, and the portable biosensor of the present invention was used for detection, and 1 mL of supernatant was taken, DNA was extracted using a commercial DNA extraction kit, and the qPCR reagent of Beijing Qingke Biotechnology Co., Ltd. was used to detect the concentration of Pseudomonas aeruginosa by qPCR method using upstream primers (5'-ATGCGATACACCGAAGTG-3') and downstream primers (5'-AGCAGGTTGGGAATCTTG-3'), and 20 μL of system solution was prepared according to the instructions of the kit. Then RT-qPCR detection was performed under the following conditions: in the first stage, initial denaturation at 95°C for 5 minutes; in the second stage, 10 seconds at 95°C, 10 seconds at 45°C, 15 seconds at 72°C, 40 cycles; fluorescence signal was collected at 72°C. When the Ct value of qPCR was 10 to 35, Pseudomonas aeruginosa was present in the sample to be tested. A total of 76 positive actual samples were prepared. In addition, PBS buffer was used to replace the bacterial solution and added to each meat product, and the same method was used to obtain 10 negative actual samples. The negative actual samples were tested using the same method as above.

[0071] Figure 3 A is a flow chart of the above process. Figure 3As shown in Figure B, the actual concentration of each sample was calculated based on the qPCR data, and the error distribution of the portable biosensor and qPCR detection results of the present invention was summarized. Figure 3 As shown in Figure C, the results of 76 positive controls and 10 negative controls detected by the biosensor were 100% consistent with the qPCR data. Figure 3 It can be seen that the detection results of the portable biosensor of the present invention are completely consistent with the detection results of the qPCR method. The above results show that compared with the qPCR method, the portable biosensor of the present invention is a faster alternative method for detecting Pseudomonas aeruginosa.

[0072] Example 3 Performance of the portable biosensor of the present invention

[0073] 1. Principle verification of the portable biosensor of the present invention

[0074] In order to study the effect of the anode reaction system on luminescence, CV analysis was performed. A three-electrode system was used for CV measurement (potential from -0.7 to 0.7V, scan rate 0.1V / s, scan number 4), the working electrode was a bare magnetic glassy carbon electrode with a diameter of 2mm, the counter electrode was a platinum wire electrode, and the reference electrode was a saturated calomel electrode. All three electrodes were placed in the initial reaction system (3mL of PBS buffer). The results are shown in Figure 2. Figure 4 As shown in A, curve a shows a reversible behavior, indicating that the glassy carbon electrode functions normally. Then, 50 μL of 1 mM Ru(bpy)3 2+ Solution, 50 μL 1 mM Ru(bpy)3 2+ solution and 5M TPrA solution, 50μL 1mM Ru(bpy)3 2+ The corresponding CV measurements were performed for Ru(bpy)3 solution, 5M TPrA solution and BNQDs, 50μL BNQDs, and 50μL 5M TPrA solution. Curve b shows that Ru(bpy)3 2+ The potential is about 1.1 V. The oxidation peak in curve c is higher than that in curves b and f, indicating that TPrA is Ru(bpy)3 2+ It is worth noting that the oxidation peak in curve d exceeds that of other curves because BNQDs can promote the oxidation of Ru(bpy)3 2+ Electron transfer to improve TPrA / Ru(bpy)3 2+ ECL intensity of the system.

[0075] In order to explore the mechanism of cathode cascade effect reducing driving voltage, CV measurement was performed. The initial reaction system was 3mL of PBS buffer. A three-electrode system was used for CV measurement (potential from -0.8 to 0.8V, scan rate 0.1V / s, scan number 4), the working electrode was a bare magnetic glassy carbon electrode with a diameter of 2mm, the counter electrode was a platinum wire electrode, and the reference electrode was a saturated calomel electrode. All three electrodes were placed in the initial reaction system with 50μL 100mM MB added. Then, 50μL 50mM TCEP, 50μL 100mM MB and 50mM TCEP were added to the initial reaction system, and the corresponding CV measurements were performed. 10μL 0.7mg / mL magnetic beads were added to the surface of the glassy carbon electrode for CV measurement. 50 μL of 100 mM MB and 50 mM TCEP were added to the initial reaction system, and 10 μL of 0.7 mg / mL magnetic beads were added to the surface of the glassy carbon electrode for CV measurement. 50 μL of 100 mM MB and 50 mM TCEP were added to the initial reaction system, and 10 μL of 0.7 mg / mL magnetic beads bound to Pseudomonas aeruginosa were added to the surface of the glassy carbon electrode for CV measurement. The results are shown in Figure 2. Figure 4 As shown in Figure 1B, curve a shows a clear peak, while curves b and c do not. It is worth noting that the peak of curve d exceeds that of curve a due to the introduction of the chemical reducing agent TCEP. In addition, the peak of curve e exceeds that of curve d, which may be due to the fact that the magnetic Fe3O4 particles in PDA@MBs promote electron transfer. These particles promote the cascade effect involving TCEP and MB, further expanding the electrochemical response. Finally, in the presence of Pseudomonas aeruginosa, the bacteria attach to the Fe3O4 particles, hindering electron transfer and causing a decrease in current. This is why the peak of curve f is smaller than that of curve e. Figure 4 C and Figure 4 D illustrates this mechanism, and the reaction equation is as follows:

[0076] Reaction Scheme 1:

[0077] (1a)Ru(bpy)3 2+ -e - →Ru(bpy)3 3+

[0078] (1b)TPrA-e - →TPrA ·+

[0079] (1c)TPrA ·+ -H + →TPrA ·

[0080] (1d)Ru(bpy)33+ +TPrA · →Ru(bpy)3 2+ *+products

[0081] (1e)Ru(bpy)3 2+ *→Ru(bpy)3 2+ +hv

[0082] Reaction Scheme 2:

[0083] (2a)Ru(bpy)3 2+ -e - →Ru(bpy)3 3+

[0084] (2b)BNQDs-e - →BNQDs-NH ·+

[0085] (2c)BNQDs-NH ·+ -H + →BNQDs-NH ·

[0086] (2d)Ru(bpy)3 3+ +BNQDs-NH · →Ru(bpy)3 2+ *+products

[0087] (2e)Ru(bpy)3 2+ *→Ru(bpy)3 2+ +hv

[0088] Reaction line 3:

[0089] MB (red) -2e - →MB (ox)

[0090] MB (ox) +TCEP→MB (red) +TCEP=O

[0091] The reaction at the anode includes reaction circuits 1 and 2. In reaction circuit 1, the terpyridine ruthenium complex Ru(bpy)3 2+ It is oxidized at the electrode, loses an electron, and generates a trivalent Ru(bpy)3 3+ TPrA is oxidized near the electrode, losing an electron to generate a free radical cation (TPrA ·+ ). Oxidized TPrA ·+ By removing a proton (H +), forming a free radical (TPrA · ).Ru(bpy)3 3+ TPrA · An electron transfer reaction occurs. Free radical TPrA · It is a strong reducing agent that transfers an electron to the oxidized Ru(bpy)3 3+ , reducing it to Ru(bpy)3 2+ This step will produce an excited state of Ru(bpy)3 2+ *, and byproducts are generated by further reactions of TPrA. The excited state of terpyridine ruthenium complex (Ru(bpy)3 2+ ) returns to the ground state (Ru(bpy)3 2+ ). In this process, the energy of the excited state is released in the form of photons, emitting light of a specific wavelength (hν). In reaction route 2, Ru(bpy)3 2+ It loses an electron at the anode electrode and is oxidized to Ru(bpy)3 3+ BNQDs are oxidized near the anode electrode, losing an electron to generate a radical cation BNQDs-NH ·+ Radical cation BNQDs-NH ·+ By removing a proton (H + ), generating a neutral radical BNQDs-NH · Ru(bpy)3 3+ and radical BNQDs-NH · An electron transfer reaction occurs. In this process, Ru(bpy)3 3+ Received from BNQDs-NH · electrons, reduced to Ru(bpy)3 2+ , and enters the excited state Ru(bpy)3 2+ *. The byproducts of the reaction are generated by further reactions of BNQDs. The excited state of terpyridine ruthenium complex (Ru(bpy)3 2+ ) returns to the ground state (Ru(bpy)3 2+ ). In this process, the energy of the excited state is released in the form of photons, emitting light of a specific wavelength (hν).

[0092] At the cathode, the reduced state of methylene blue (MB(red)) loses two electrons and is oxidized to the oxidized state of methylene blue (MB(ox)). The oxidized state of methylene blue (MB(ox)) reacts with TCEP (reducing agent) and is reduced to the reduced state of methylene blue (MB(red)). At the same time, TCEP is oxidized to its oxidation product (TCEP=O).

[0093] Ru(bpy)3 2+ In the presence of co-reactant BNQDs, anodic ECL is exhibited. BNQDs containing abundant amino groups are introduced into TPrA / Ru(bpy)3 2+ system, forming BNQDs-NH ·+ Under alkaline conditions, BNQDs-NH ·+ Lose H + , generating the reducing intermediate BNQDs-NH · This intermediate reacts with Ru(bpy)3 3+ The reaction generates excited state Ru(bpy)3 2+* Then Ru(bpy)3 2+* Return to the ground state Ru(bpy)3 2+ , generating ECL signals for quantitative detection of Pseudomonas aeruginosa. Figure 4 D, MB red After the (reduced form of MB) molecule is oxidized at a specific potential, it can be reduced by TCEP, thereby restoring the electron transfer activity and enhancing the detection signal. In addition, the iron oxide in PDA@MBs promotes electron transfer, and a cascade effect is generated between MB, TCEP and PDA, which together provide additional voltage, thus significantly reducing the driving voltage of the closed bipolar electrode.

[0094] To determine the feasibility of biosensor preparation, an EIS test was conducted using a three-electrode system (initial voltage 0.194V, high frequency 100000Hz low frequency 0.1Hz, amplitude 0.005V, static time 2s). The working electrode was a bare magnetic glassy carbon electrode or a treated electrode with a diameter of 2mm, the counter electrode was a platinum wire electrode, the reference electrode was a saturated calomel electrode, and the reaction system was 3mL of ferrocyanide solution (an aqueous solution containing 5mM potassium ferrocyanide, 5mM potassium ferrocyanide, and 0.1M potassium chloride). The bare magnetic glassy carbon electrode, the magnetic glassy carbon electrode after adding 10μL of PDA@MBs, the magnetic glassy carbon electrode after adding 10μL of PA@PDA@MBs, and the magnetic glassy carbon electrode after adding 10μL of PDA@MBs were tested. 2 CFU / mL Pseudomonas aeruginosa magnetic beads were added to the glassy carbon electrode and 10 6 CFU / mL Pseudomonas aeruginosa was measured using a glassy carbon electrode behind magnetic beads. Figure 4As shown in Figure 1, the charge transfer resistance (Rct) value of bare GCE is the smallest (curve a). Secondly, the Rct value increases significantly after the immobilization of PDA@MBs (curve b), which is attributed to the fact that PDA is a large molecule and is not electrically active. This property makes it difficult for electroactive substances to pass through the membrane and reach the electrode surface where redox reactions occur. When the zygote is modified to the electrode surface (curve c), the semicircle diameter increases significantly, which is mainly due to the negative charge of the DNA phosphate backbone. Finally, after adding 10 2 CFU / mL Pseudomonas aeruginosa and 10 6 After the addition of CFU / mL Pseudomonas aeruginosa, Rct increased (curves d and e) because the bacteria hindered the ability of electron transfer, causing the resistance to increase.

[0095] The above results demonstrate that the portable biosensor of the present invention is feasible and successfully prepared.

[0096] 2. Anti-interference

[0097] Matrix effect (ME) refers to the fact that the coexistence of signal molecules and food matrix in the same system may affect the detection signal, which is a common and unavoidable challenge in food testing. In actual samples for the detection of foodborne pathogens, the number of interfering substances related to pathogens is very large, and the problems related to matrix effect are often overlooked. Therefore, in order to reduce the common false positives and false negatives in complex food samples, the ME formula is used to calculate the matrix linearity curve and standard curve.

[0098] In order to evaluate the potential of the portable biosensor of the present invention in rapid food safety detection, the effect of food matrix on the dual mode of the portable biosensor of the present invention was studied. Chicken breast and milk were selected as representative solid and liquid samples, respectively.

[0099] Add 4g of chicken breast to 40mL of sterile PBS buffer, put it into a sterile homogenization bag, and homogenize it for 1-2min using an HX-4 slapping sterile homogenizer (Shanghai Huxi Industrial Co., Ltd.). Then centrifuge at 12000rpm for 10min and collect the supernatant. Repeat this extraction process twice more. Combine the three supernatants and pass them through a 0.45μm syringe filter to obtain the final chicken breast treatment solution. For milk samples, mix 1mL of milk and 99mL of PBS buffer to obtain a milk treatment solution. Take 9mL of chicken breast treatment solution and 9mL of milk treatment solution respectively, and add 1mL of different concentrations of Pseudomonas aeruginosa (10 0 -10 7CFU / mL). Finally, 100 μL of each treatment solution added with Pseudomonas aeruginosa was taken to perform electrochemical and photothermal experiments using the portable biosensor of the present invention. The relationship between the R value and lgC of the chicken breast treatment solution and the milk treatment solution, and the relationship between ΔT and lgC were fitted respectively to obtain the standard curves of the chicken breast treatment solution and the milk treatment solution.

[0100] ME is calculated according to the following formula:

[0101] ME (%) = (K0 / K1-1) × 100%

[0102] Wherein, K0 is the slope of the standard curve of the matrix (chicken breast treated solution or milk treated solution), and K1 is the slope of the fitting curve of the standard solution (suspensions of Pseudomonas aeruginosa of various concentrations prepared with PBS buffer, prepared in the same way as the standard curve in Example 1).

[0103] When |ME|>50%, it indicates strong ME; 20%≤|ME|≤50%, it indicates moderate ME; and |ME|<20%, it indicates weak ME.

[0104] like Figure 1 As shown in C and D, the linear regression equations of the milk treatment solution are y = -13.514x + 195.839 (R 2 =0.983, y is the R value) and y = -0.571x + 10.892 (R 2 =0.974, y is ΔT). The linear regression equation of the chicken breast processed liquid (including R value and ΔT) is y=-10.560x+191.778 (R 2 =0.990, y is the R value) and y=-0.585x+10.096 (R 2 =0.993, y is ΔT). Compared with the standard solution, the R value and ΔT of the milk-treated solution were 3.8% and 14.4%, respectively. For the chicken breast-treated solution, the R value was 9.0% and ΔT was 17.2% compared with the standard solution. |ME|<20% indicates weak ME, that is, the R value of the food matrix is ​​equivalent to the blank value, indicating that the portable biosensor of the present invention has negligible effect on the luminescence of the food matrix. In summary, these research results show that the biosensor can effectively perform rapid food safety detection without matrix interference.

[0105] 3. Specificity

[0106] In order to verify the specificity of the portable biosensor of the present invention, several common coexisting bacteria and interfering substances in the matrix were studied. First, in order to verify the specificity of common coexisting bacteria, Escherichia coli, Salmonella typhimurium, Bacillus cereus and Clostridium perfringens were selected as interfering substances, and their contents were all 10 7CFU / mL. Four different bacteria, Pseudomonas aeruginosa (10 5 CFU / mL, blank group PBS buffer and mixture (10 5 CFU / mL Pseudomonas aeruginosa, 10 7 CFU / mL Escherichia coli, 10 7 CFU / mL Salmonella typhimurium, 10 7 CFU / mL Bacillus cereus and 10 7 CFU / mL of Clostridium perfringens) were detected using the portable biosensor of the present invention. The test results are shown in Figure 5 A and Figure 5 As shown in B, at 10 5 In the presence of CFU / mL aeruginosa, the R value and ΔT of the anode were the lowest. The mixture had similar results. The R values ​​and ΔT of other bacteria were similar to the blank.

[0107] In addition, 5 mg / mL yeast powder solution, 10 mg / mL peptone solution, 10 mg / mL sodium chloride solution, 10 mM calcium chloride solution, 10 mM calcium chloride solution vitamin B2 solution and 10 mM calcium chloride solution vitamin B6 solution were prepared with PBS buffer and tested using the portable biosensor of the present invention. The test results are shown in FIG. Figure 5 C and Figure 5 D, at 10 5 In the presence of CFU / mL aeruginosa, the R value and ΔT of the anode were the lowest. 5 The results of CFU / mL Pseudomonas aeruginosa and 5mg / mL yeast powder solution, 10mg / mL peptone solution, 10mg / mL sodium chloride solution, 10mM calcium chloride solution, 10mM vitamin B2 solution and 10mM vitamin B6 solution were consistent with the above. The R values ​​and ΔT of other interferents were similar to those of the blank control (PBS buffer).

[0108] The above results indicate that the portable biosensor of the present invention still exhibits good specificity despite the presence of interference at higher concentrations.

[0109] 4. Stability and repeatability

[0110] The PDA@MBs were stored at 4°C, and samples were taken every 3 days to prepare magnetic beads for detecting Pseudomonas aeruginosa. The portable biosensor of the present invention was prepared to detect the same biological sample. Figure 5 E and Figure 5 F. The relative standard deviations (RSD) of the biosensor indicators of the present invention are 1.48% and 2.04%, respectively, which further proves the good luminescence stability and photothermal stability.

[0111] Repeatability is another important indicator for evaluating biosensors. Under the same conditions, the repeatability of the biosensor platform was verified by intra-group and inter-group experiments. In the intra-group experiment, six luminescence and photothermal cycle tests were performed on the same group of samples. In the inter-group experiment, luminescence and photothermal tests were performed on six different groups of samples. The RSDs of the R value and ΔT in the intra-group experiment were 2.05% and 2.12%, respectively. In the inter-group experiment, the RSDs of the R value and ΔT were 2.46% and 2.55%, respectively. These results show that the portable biosensor platform of the present invention has excellent reproducibility.

Claims

1. A dual-mode portable biosensor for detecting Pseudomonas aeruginosa, characterized in that Includes closed bipolar electrode, magnetic beads for detection of Pseudomonas aeruginosa, methylene blue solution, TCEP solution, Ru(bpy)3 2+ solution, BN quantum dots and TPrA solution.

2. The biosensor according to claim 1, characterized in that The magnetic beads used for detecting Pseudomonas aeruginosa carry Pseudomonas aeruginosa aptamers.

3. The biosensor according to claim 1 or 2, characterized in that The magnetic beads for detecting Pseudomonas aeruginosa are prepared by amide reaction of Fe3O4 magnetic material wrapped with polydopamine and Pseudomonas aeruginosa aptamer with carboxyl group at the 5' end.

4. The biosensor according to claim 3, characterized in that The boron nitride quantum dots are prepared by the following method: boric acid, melamine and water are mixed and stirred, reacted at 190-210° C. for 18-22 hours, and cooled to room temperature to obtain boron nitride quantum dots.

5. The biosensor according to claim 4, characterized in that The mass ratio of boric acid to melamine is 5:0.5-1.

5.

6. The biosensor according to claim 5, characterized in that The methylene blue solution is a 90-110 mM methylene blue aqueous solution, the TCEP solution is a 40-60 mM TCEP aqueous solution, and Ru(bpy)3 2+ The concentration of the solution is 0.5-1.5mM, The concentration of TPrA solution is 4-6M.

7. A method for detecting Pseudomonas aeruginosa using the biosensor according to any one of claims 1 to 6, comprising the following steps: (1) The sample is adsorbed by the magnetic beads for detecting Pseudomonas aeruginosa, and resuspended to obtain a test solution; (2) A voltage of 2.9 V was applied to the closed bipolar electrode, a mixture of methylene blue solution, TCEP solution and the test solution was dripped onto the cathode surface, and Ru(bpy)3 was dripped onto the anode surface. 2+ The solution, the mixture of boron nitride quantum dots, TPrA solution and PBS buffer solution, the anode emits red light, and the ECL image is used to identify the R value of RGB, which is substituted into the standard curve to obtain the concentration of Pseudomonas aeruginosa; (3) Use 808 nm near-infrared laser to irradiate 50 μL of the test solution for 3.5 min. Subtract the temperature before irradiation from the temperature after irradiation for 3.5 min. The resulting value is called ΔT. Substitute it into the standard curve to obtain the concentration of Pseudomonas aeruginosa.

8. The method according to claim 7, characterized in that After the test is completed, a copper sulfate aqueous solution is added to the test solution and the magnetic beads after the test for sterilization.

9. The method according to claim 8, characterized in that The concentration of the copper sulfate aqueous solution is 0.5-5 mM.

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