Fluorescent biosensor, preparation method and application

Abstract

The invention discloses a fluorescent biosensor. The fluorescent biosensor comprises a lanthanide MOFs / bacteriophage composite material. Meanwhile, the invention also provides a corresponding preparation method and application thereof, which are the other purpose of the invention. The EuMOFs provided by the invention combines unique advantages of MOFs and luminescence characteristics of Eu < 3 + > ions, and has larger Stokes shift, higher quantum yield, longer fluorescence lifetime and more excellent fluorescence stability; euMOFs is chemically connected with bacteriophage to prepare the fluorescent biosensor, and the concentration of live pseudomonas aeruginosa can be rapidly and sensitively detected; and 2) the fluorescent biosensor has excellent performance of detecting pseudomonas aeruginosa, has the advantages of high sensitivity, fast detection, short time and wide detection range, has the detection limit as low as 2 CFU / mL, and can distinguish live and dead pseudomonas aeruginosa.

Description

Technical Field

[0001] The present invention belongs to the technical field of bacterial detection, and particularly relates to a fluorescence biosensor, a preparation method and an application thereof. Background Art

[0002] Pseudomonas aeruginosa is likely to cause various infectious diseases such as respiratory tract and urinary tract infections. It is one of the most widespread and serious problems in current hospital infections. In particular, it is prone to colonize in immunocompromised patients, leading to an increase in patient mortality. The emergence of its drug-resistant strains has seriously affected the treatment effect. Therefore, early, rapid and accurate detection is crucial for the treatment of infections caused by PA. In addition, the current pursuit of precision medicine, formulating diagnosis, treatment and prevention methods suitable for each patient, makes precise and rapid diagnosis necessary. In the field of pathogenic bacteria detection, previous technical means rarely focused on distinguishing live bacteria from dead bacteria. However, in fact, only live pathogenic bacteria can cause infection and disease. Therefore, not only qualitative and quantitative detection is important, but also distinguishing live and dead bacteria is worthy of attention. Being able to detect the fastest which pathogenic bacteria cause the infection and determine the amount of live bacteria can formulate the most suitable treatment plan for patients.

[0003] Currently, the gold standard method for detecting Pseudomonas aeruginosa is still the most traditional plate culture counting method. The detection result of this method is accurate and reliable, but it is time-consuming and laborious, taking 3 - 5 days. The newly emerged ELISA and PCR-related methods can greatly reduce the detection time (2 - 4h), but they require professional equipment and technical personnel, need complex sample pretreatment, and cannot distinguish live and dead bacteria, and are prone to false positive / false negative results. As an emerging bacterial detection platform, especially fluorescence biosensors, have received increasing attention due to their ultra-high sensitivity, high cost performance and rapid detection.

[0004] In order to achieve the specific detection of bacteria by biosensors, a suitable biorecognition element must be selected. Most of the recognition elements used in the currently reported biosensors are nucleic acids, antibodies and enzymes. These elements have high costs and lack the ability to distinguish live bacteria from dead bacteria. As a new type of recognition element, phage can not only specifically recognize host bacteria, but also has advantages such as high stability, low cost, easy large-scale amplification, and the ability to distinguish live bacteria from dead bacteria. Therefore, it is very meaningful to design and develop various bacterial detection biosensors based on phage.

[0005] At present, luminescent materials such as semiconductor quantum dots, carbon quantum dots, upconversion nanoparticles, and metal-organic frameworks (MOFs) have been used in the field of bacterial detection. As a network structure formed by the self-assembly of coordination bonds between metal nodes and organic ligands, MOFs have many excellent properties, such as diverse structures and compositions, adjustable pore sizes, high specific surface areas, adjustable active sites, and metal nodes and organic ligands that are easy to modify, and are prone to special interactions (van der Waals forces, hydrogen bonds, π-π stacking, etc.) with target analytes. Based on these characteristics, a variety of MOF sensing materials have been established, so the rational design of MOF materials for selective recognition of target analytes is crucial. Lanthanide MOFs, due to their excellent fluorescence properties, including long excited-state lifetimes, large Stokes shifts, and narrow emission bands, have been widely used in multiple fields such as ion detection, tumor therapy, fluorescence imaging, and miRNA sensing, attracting great attention. At present, the direct detection of pathogenic bacteria based on the fluorescence of MOFs is still in its initial stage. Summary of the Invention

[0006] Based on the above technical problems, the present invention provides a fluorescence biosensor, and it is another object of the present invention to provide its corresponding preparation method and application.

[0007] A fluorescence biosensor, the fluorescence biosensor comprising a lanthanide MOF (lanthanide metal-organic framework material) / phage composite material.

[0008] The lanthanide MOF / phage composite material is made by connecting lanthanide MOFs with phages.

[0009] The lanthanide MOF is EuMOF, and the phage is Pseudomonas aeruginosa phage.

[0010] A method for preparing a fluorescence biosensor, comprising the following steps:

[0011] 1) Preparation of lanthanide MOFs: Lanthanide MOFs are synthesized by a mixed solvent thermal method;

[0012] 2) Separation and preparation of phages;

[0013] 3) Purification of phages;

[0014] 4) Preparation of lanthanide MOFs / phage composite materials: Mix the lanthanide MOFs obtained in step 1) with water to prepare an aqueous solution of lanthanide MOFs at a concentration of 1 - 2 mg / mL. Mix 4 - 8 mL of the aqueous solution of lanthanide MOFs, 40 - 80 mg of EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide), and 40 - 80 mg of NHS (N-hydroxysuccinimide). Under room temperature conditions, after reacting for 30 - 40 min, centrifuge and discard the supernatant to obtain precipitate I.

[0015] Wash and resuspend precipitate I, and add 100 - 300 μL of the purified phage obtained in step 3) at a concentration of 10 9 -10 11 PFU / mL. Incubate and centrifuge, discard the supernatant to obtain precipitate II. Wash and resuspend precipitate II to obtain the lanthanide MOFs / phage composite material.

[0016] In step 4), in the washing and resuspension steps of precipitate I and precipitate II, PBS buffer is used. After washing precipitate I, resuspend it with 1 - 2 mL of PBS buffer; after washing precipitate II, resuspend it with 4 - 8 mL of PBS buffer. Incubation conditions: temperature 2 - 6 °C, incubation time 12 - 24 h.

[0017] In step 1), the lanthanide MOFs are EuMOFs;

[0018] The preparation method of EuMOFs includes the following steps: Add Al(NO3)3·9H2O, Eu(NO3)3, and H2BDC-NH2 to a mixed solvent, place it in a reaction kettle, react at 130 - 160 °C for 6 - 8 h, cool and centrifuge, collect the precipitate, wash and dry to obtain EuMOFs;

[0019] The dosage ratio of Al(NO3)3·9H2O, Eu(NO3)3, H2BDC-NH2, and the mixed solvent is (0.1 - 0.2) mmol∶(0.01 - 0.2) mmol∶(0.04 - 0.4) mmol∶(20 - 40) mL; the mixed solvent is made by mixing DMF, water, and absolute ethanol;

[0020] In steps 2) and 3), the phage is Pseudomonas aeruginosa phage.

[0021] In step 1), the volume ratio of DMF, water, and absolute ethanol is (15 - 25)∶(3 - 7)∶(3 - 7); centrifugation conditions: centrifuge at 8000 - 10000 rpm for 2 - 5 min; washing conditions: wash 3 - 7 times successively with distilled water and absolute ethanol; drying conditions: vacuum dry at 50 - 70 °C for 12 - 18 h.

[0022] In step 2), phages are separated and prepared: The untreated sewage sample from the hospital is centrifuged at 4000 - 6000 r / min for 10 - 20 min. After filtering the supernatant, a filtrate is obtained; 0.2 - 0.4 mL of the filtrate is mixed with 5 - 6 mL of Pseudomonas aeruginosa liquid in the logarithmic phase (10 6 -10 9 PFU / mL), added to 10 - 20 mL of 3×LB medium, and cultured at 37°C for 24 - 28 h. Then, it is centrifuged and filtered to obtain phages;

[0023] 3) Purify the phages: Dilute the phages obtained in step 2) to 10 2 -10 3 PFU / mL. Incubate 100 - 1000 μL of the diluted phages with 100 - 200 μL of Pseudomonas aeruginosa liquid in the logarithmic phase (10 6 -10 9 PFU / mL) for 5 - 15 min, and separate and purify them by the double-layer agar plate method.

[0024] Apply the fluorescent biosensor to the detection of living Pseudomonas aeruginosa.

[0025] The method for applying to the detection of living Pseudomonas aeruginosa includes the following steps:

[0026] 1) Establish a detection standard curve: Take the freshly cultured Pseudomonas aeruginosa liquid, centrifuge it, resuspend the precipitate with PBS buffer, and then dilute it into Pseudomonas aeruginosa liquid with different concentration gradients;

[0027] Take 100 - 200 μL of the lanthanide MOFs / phage composite material, add it to 700 - 800 μL of PBS buffer, and then add 100 μL of Pseudomonas aeruginosa liquid with different concentration gradients respectively. Incubate at room temperature for 5 - 15 min, detect the fluorescence intensity, fit the linear relationship between the fluorescence intensity and the concentration of Pseudomonas aeruginosa, and establish the detection standard curve;

[0028] 2) Apply the fluorescent biosensor to the detection of actual clinical samples containing Pseudomonas aeruginosa.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1) In the present invention, EuMOFs are synthesized by the mixed solvent thermal method. EuMOFs combines the unique advantages of MOFs and Eu 3+The luminescence characteristics of the ions have a larger Stokes shift, higher quantum yield, longer fluorescence lifetime, and better fluorescence stability; EuMOFs are chemically linked with phages to achieve covalent binding between the two, and a fluorescent biosensor is prepared, which can quickly and sensitively detect the concentration of living Pseudomonas aeruginosa;

[0031] 2) The fluorescent biosensor of the present invention has excellent performance in detecting Pseudomonas aeruginosa, with high sensitivity, fast detection, short time, wide detection range, detection limit as low as 2 CFU / mL, and can distinguish living and dead Pseudomonas aeruginosa, and can be applied to the detection of actual samples, with strong anti-interference ability, and can detect even trace amounts. Description of the Drawings

[0032] Figure 1 , Figure A is the ultraviolet absorption spectrum of Eu 3+ @MIL-53(Al); Figure B is the fluorescence absorption spectrum of Eu 3+ @MIL-53(Al) at different excitation wavelengths; Figure C is the X-ray diffraction pattern of MIL-53(Al) and Eu 3+ @MIL-53(Al); Figure D is the elemental analysis pattern of Eu 3+ @MIL-53(Al);

[0033] Figure 2 Among them, Figure A is the plaque formed by the isolated phage on the double-layer agar plate; Figure B is the transmission electron micrograph of the isolated phage;

[0034] Figure 3 Among them, Figures A and B are the scanning electron micrographs of Eu 3+ @MIL-53(Al) and Eu 3+ @MIL-53(Al) / phages respectively;

[0035] Figure 4 Among them, Figure A is the fluorescence spectrum of the fluorescent biosensor prepared in Example 1 when detecting different concentrations of Pseudomonas aeruginosa; Figure B is the linear relationship diagram between different concentrations of Pseudomonas aeruginosa and the change in fluorescence intensity; Figure C is the fluorescence change of the prepared fluorescent biosensor when detecting different bacteria; Figure D is the fluorescence change of the prepared fluorescent biosensor when detecting living, dead, and living-dead mixed Pseudomonas aeruginosa. Figure 5 is the reaction schematic diagram for preparing the fluorescent biosensor. Detailed Embodiments

[0036] The present invention will be further described below in conjunction with the detailed embodiments and the drawings.

[0037] Example 1

[0038] A fluorescent biosensor, the fluorescent biosensor comprising a EuMOFs (lanthanide metal-organic framework material) / phage composite material, the EuMOFs (europium metal-organic framework material) / phage composite material being made by connecting EuMOFs and phages, and the phage being a Pseudomonas aeruginosa phage.

[0039] A method for preparing a fluorescent biosensor, comprising the following steps:

[0040] 1) Preparation of EuMOFs: EuMOFs (Eu 3+ @MIL-53(Al)) was synthesized by a mixed solvent thermal method. 0.19 mmol of Al(NO3)3·9H2O, 0.2 mmol of Eu(NO3)3, and 0.04 mmol of H2BDC-NH2 were added to 30 mL of a mixed solvent (the volume ratio of DMF, water, and absolute ethanol was 20∶5∶5). After stirring evenly, it was placed in a polytetrafluoroethylene-lined reaction kettle and reacted at 130 °C for 8 h. After cooling to room temperature, it was centrifuged at 8000 rpm for 2 min, and the precipitate was collected. It was washed 7 times successively with distilled water and absolute ethanol; it was dried in vacuo at 60 °C for 12 h to obtain EuMOFs;

[0041] 2) Separation and preparation of phages: An untreated sewage sample from a hospital was centrifuged at 4000 r / min for 10 min. The supernatant was filtered through a 0.22 μm filter to obtain a filtrate; 0.2 mL of the filtrate was mixed with 5 mL of a freshly cultured Pseudomonas aeruginosa solution in the logarithmic phase at 10 8 CFU / mL and added to 10 mL of 3×LB medium. After culturing at 37 °C for 24 h, it was centrifuged at 4000 r / min for 10 min and filtered through a 0.22 μm filter to remove bacteria to obtain phages;

[0042] 3) Purification of phages: The phages in step 2) were serially diluted to 10 3 PFU / mL. 100 μL of the diluted phages was incubated with 100 μL of a freshly cultured Pseudomonas aeruginosa solution in the logarithmic phase at 10 8 CFU / mL for 10 min. The double-layer agar plate method was used for the test. A single plaque was selected from the double-layer plate cultured for 24 h, and the double-agar plate test was repeated 8 times to purify the phages;

[0043] 4) Preparation of EuMOFs / phage composite material: The EuMOFs in step 1) was mixed with water to prepare a 1 mg / mL EuMOFs aqueous solution. 4 mL of the EuMOFs aqueous solution, 40 mg of EDC, and 40 mg of NHS were mixed and reacted at room temperature for 30 min, then centrifuged, and the supernatant was discarded to obtain precipitate Ⅰ;

[0044] After washing the precipitate Ⅰ twice with PBS buffer, add 1 mL of PBS buffer to resuspend it, and then add 300 μL of 10 11 PFU / mL of the phage purified in step 3), incubate at 4 °C for 12 h, centrifuge, discard the supernatant, and obtain precipitate Ⅱ. Wash precipitate Ⅱ twice with PBS buffer, add 4 mL of PBS buffer to resuspend it, and obtain the EuMOFs / phage composite material. As Figure 5 shown.

[0045] Apply the fluorescence biosensor to the detection of living Pseudomonas aeruginosa.

[0046] Example 2

[0047] A fluorescence biosensor, the fluorescence biosensor includes a EuMOFs / phage composite material, the EuMOFs / phage composite material is made by connecting EuMOFs and phages, and the phage is a Pseudomonas aeruginosa phage.

[0048] A method for preparing a fluorescence biosensor, comprising the following steps:

[0049] 1) Preparation of EuMOFs: Synthesize EuMOFs (Eu 3+ @MIL-53(Al)) by the mixed solvent thermal method. Add 0.1 mmol of Al(NO3)3·9H2O, 0.1 mmol of Eu(NO3)3, and 0.4 mmol of H2BDC-NH2 to 30 mL of a mixed solvent (the volume ratio of DMF, water, and absolute ethanol is 16∶7∶7), place it in a polytetrafluoroethylene-lined reaction kettle, react at 150 °C for 7 h, cool to room temperature, centrifuge at 10000 rpm for 3 min, collect the precipitate, and wash it 5 times with distilled water and absolute ethanol in sequence; vacuum dry at 55 °C for 14 h to obtain EuMOFs;

[0050] 2) Separation and preparation of phages: Centrifuge the untreated sewage sample from the hospital at 6000 r / min for 15 min, filter the supernatant through a 0.22 μm filter to obtain a filtrate; take 0.4 mL of the filtrate and mix it with 6 mL of a fresh culture of Pseudomonas aeruginosa liquid in the logarithmic phase at 10 8 CFU / mL, add it to 20 mL of 3×LB medium, culture at 37 °C for 28 h, then centrifuge at 6000 r / min for 15 min, and filter with a 0.22 μm filter to remove bacteria to obtain phages;

[0051] 3) Purification of phages: Continuously dilute the phages obtained in step 2) to 10 2PFU / mL. Add 1000 μL of the diluted phage to 100 μL of freshly cultured Pseudomonas aeruginosa solution in the logarithmic growth phase at 10 8 CFU / mL and incubate for 15 min. Conduct the experiment using the double-layer agar plate method. Select a single plaque from the double-layer plate after 20 h of incubation and repeat the double-agar plate experiment 9 times to purify the phage;

[0052] 4) Preparation of EuMOFs / phage composite: Mix the EuMOFs obtained in step 1) with water to prepare a 2 mg / mL EuMOFs aqueous solution. Mix 8 mL of the EuMOFs aqueous solution, 80 mg of EDC, and 80 mg of NHS. React at room temperature for 40 min, then centrifuge and discard the supernatant to obtain precipitate Ⅰ;

[0053] Wash precipitate Ⅰ twice with PBS buffer, resuspend it in 1 mL of PBS buffer, add 200 μL of the purified phage from step 3) at 10 9 PFU / mL, incubate at 4 °C for 12 h, centrifuge and discard the supernatant to obtain precipitate Ⅱ. Wash precipitate Ⅱ twice with PBS buffer and resuspend it in 8 mL of PBS buffer to obtain the EuMOFs / phage (EuMOFs / phages) composite.

[0054] Apply the fluorescence biosensor to the detection of live Pseudomonas aeruginosa.

[0055] Example 3

[0056] A fluorescence biosensor, which includes the EuMOFs / phage composite. The EuMOFs / phage composite is made by connecting EuMOFs and phages, and the phage is a Pseudomonas aeruginosa phage.

[0057] A method for preparing a fluorescence biosensor, comprising the following steps:

[0058] 1) Preparation of EuMOFs: Synthesize EuMOFs (Eu 3+ @MIL-53(Al)) using the mixed solvent thermal method. Add 0.15 mmol of Al(NO3)3·9H2O, 0.05 mmol of Eu(NO3)3, and 0.2 mmol of H2BDC-NH2 to 30 mL of a mixed solvent (the volume ratio of DMF, water, and absolute ethanol is 24∶3∶3), place it in a Teflon-lined reaction kettle, react at 140 °C for 6 h, cool to room temperature, centrifuge at 9000 rpm for 5 min, collect the precipitate, and wash it 7 times with distilled water and absolute ethanol in sequence; Dry it under vacuum at 65 °C for 18 h to obtain EuMOFs;

[0059] 2) Isolation and preparation of phages: Centrifuge the untreated sewage sample from the hospital at 5000 r / min for 10 min. Filter the supernatant through a 0.22 μm filter to obtain a filtrate. Take 0.3 mL of the filtrate and mix it with 6 mL of freshly cultured Pseudomonas aeruginosa liquid in the logarithmic phase at 10 8 CFU / mL. Add the mixture to 20 mL of 3×LB medium and incubate at 37°C for 24 h. Then centrifuge at 6000 r / min for 15 min and filter through a 0.22 μm filter to remove bacteria, obtaining phages;

[0060] 3) Purification of phages: Continuously dilute the phages obtained in step 2) to 10 2 PFU / mL. Incubate 1000 μL of the diluted phages with 100 μL of freshly cultured Pseudomonas aeruginosa liquid in the logarithmic phase at 10 8 CFU / mL for 5 min. Conduct the experiment using the double-layer agar plate method. Select a single plaque from the double-layer plate cultured for 28 h and repeat the double-agar plate experiment 8 times to purify the phages;

[0061] 4) Preparation of EuMOFs / phage composite materials: Mix the EuMOFs obtained in step 1) with water to prepare a 1 mg / mL EuMOFs aqueous solution. Mix 6 mL of the EuMOFs aqueous solution, 60 mg of EDC, and 60 mg of NHS. React at room temperature for 30 min, then centrifuge and discard the supernatant to obtain precipitate Ⅰ;

[0062] Wash precipitate Ⅰ twice with PBS buffer, resuspend it in 1 mL of PBS buffer, add 100 μL of 10 9 PFU / mL of the phages purified in step 3), incubate at 4°C for 12 h, centrifuge, and discard the supernatant to obtain precipitate Ⅱ. Wash precipitate Ⅱ twice with PBS buffer and resuspend it in 6 mL of PBS buffer to obtain EuMOFs / phage (EuMOFs / phages) composite materials.

[0063] Apply the fluorescence biosensor to the detection of living Pseudomonas aeruginosa.

[0064] Example 4

[0065] 4.1 Characterization and analysis of EuMOFs (Eu 3+ @MIL-53(Al) or Eu 3+ @MIL-53(Al)-NH2), EuMOFs / phage (EuMOFs / phages) composite materials in Example 1

[0066] The fluorescence properties of the EuMOFs in Example 1 were investigated by a fluorescence spectrophotometer. At different excitation wavelengths, the position of the emission peak of the material remained unchanged, but the intensity changed. The excitation wavelength was set in the range of 320 - 380 nm, and the fluorescence spectrum scanning results were as shown in Figure 1 Figure A in Figure 1 Figure A in

[0067] showed that the intensity of the emission peak was more obvious under the excitation wavelength of 360 nm. 2、 The FT-IR spectra of H2BDC-NH 3+ EuMOFs (Eu Figure 1 @MIL-53(Al)) and EuMOFs / phages (EuMOFs / phages) are shown in Figure B in

[0068] As can be seen from Figure B in Figure 1 in the FT-IR spectrum of H2BDC-NH2, the doublet observed at 3506 cm -1 and 3392 cm -1 was due to the asymmetric and symmetric stretching vibrations of the amino group. The two sharp peaks near 1685 cm -1 and 1236 cm -1 were related to the asymmetric and symmetric vibrations of the carboxyl group respectively, revealing the presence of the dicarboxylic acid structure. The peak at 758 cm -1 was due to the bending vibration of the single bond H on the benzene ring. In the spectrum of EuMOFs, the broad absorption peak at 3427 cm -1 was caused by the stretching vibration of the N-H bond, while at 1255 cm -1 the absorption peak was caused by the stretching of the C-N bond. The stretching vibrations of the carboxyl group were at 1417 cm -1 and 1600 cm -1 , and the Al-O stretching vibration was at 600 cm -1 , which proved the successful preparation of EuMOFs. In the EuMOFs / phages spectrum, a stretching vibration peak (amide bond formation) at 1637 cm -1 verified the covalent binding of EuMOFs and phages. The above results further proved the successful preparation of EuMOFs and EuMOFs / phages.

[0069] The preparation of MIL-53(Al) was different from that in Example 1 in that Eu(NO3)3 was not added in step 1).

[0070] The XRD patterns of MIL-53(Al) and Eu 3+ @MIL-53(Al) are shown in Figure C in Figure 1 ​

[0071] As can be seen from Figure 1 Figure C, the sample without Eu 3+ completely conforms to the simulated MIL-53(Al) pattern, confirming the phase purity. After introducing Eu 3+ , most of the diffraction peaks remain unchanged, indicating that the MIL-53 framework is retained. In addition, the XRD patterns of the products synthesized in the system containing Eu 3+ are very similar to those of the products synthesized in the system without Eu 3+ , except for an emergent peak at about 30°, which indicates that the incorporation of Eu 3+ ions does not significantly change the phase of the coordination polymer. This new peak may be due to the addition of Eu 3+ ions into the structure.

[0072] The elemental analysis spectrum of Eu 3+ @MIL-53(Al) is shown in Figure 1 Figure D. Figure 1 Figure D shows the uniform co-distribution of Eu, Al, O, and C, confirming the successful binding of Eu 3+ , and the incorporation of Eu 3+ ions does not affect the structure of MIL-53(Al) composed of Al and ligands, without phase segregation.

[0073] 4.2 Transmission electron micrographs of plaques and phages in Example 1

[0074] In Example 1, the plaques formed by the isolated phages on the double-layer agar plate are shown in Figure 2 Figure A. The results show that the isolated phages have strong virulence and can lyse PA and produce progeny phages.

[0075] The morphology of the phages after negative staining was observed by transmission electron microscopy, as shown in Figure 2 Figure B. The phages exhibit an obvious polyhedral structure, similar to the phage head.

[0076] 4.3 Transmission electron micrographs of EuMOFs / phages in Example 1

[0077] The surface morphology of the materials was characterized by SEM. The scanning electron micrographs of EuMOFs (Eu 3+ @MIL-53(Al)) and the EuMOFs / phage composite material (Eu 3+ @MIL-53(Al) / phage) are shown in Figure 3 Figure A and Figure B, respectively.

[0078] Eu 3+The connection between @MIL-53(Al) and the phage is established through an amide bonding process that involves the carboxyl groups on Eu 3+ @MIL-53(Al) and the amine groups on the phage head. As can be seen from Figure 3 Figures A and B in 3+ Eu 3+ The SEM images of the Eu 3+ @MIL-53(Al) / phage composite show that the surface of Eu 3+ @MIL-53(Al) undergoes a transformation from smooth to rough after phage attachment, and the rough surface may indicate the formation of a phage layer, thus confirming the successful synthesis of the Eu

[0079] Example 5 Detection Test

[0080] 5.1 Specificity Test of Fluorescent Sensor

[0081] The method for applying the fluorescent biosensor of Example 1 to the detection of living Pseudomonas aeruginosa includes the following steps:

[0082] 1) Establishment of the detection standard curve: Take a freshly cultured Pseudomonas aeruginosa bacterial solution, centrifuge it, resuspend the precipitate with PBS buffer, and then dilute it into Pseudomonas aeruginosa bacterial solutions with different concentration gradients (10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 CFU / mL), and the Pseudomonas aeruginosa bacterial solution is 2.4×10 1 -2.4×10 6 CFU / mL;

[0083] Take 100 EuMOFs / phage composites, add them to 800 μL of PBS buffer, and then add 100 μL of Pseudomonas aeruginosa bacterial solutions with different concentration gradients respectively. Incubate at room temperature for 10 min, detect the fluorescence intensity (excitation wavelength is 360 nm, emission wavelength is 428 nm), fit the linear relationship between the fluorescence intensity and the concentration of Pseudomonas aeruginosa, and establish the detection standard curve; The fluorescence spectrum is as shown in Figure 4 Figure A in Figure 4 Figure B in

[0084] 2) Apply the fluorescent biosensor to the detection of actual clinical samples.

[0085] As can be seen from Figure 4As can be seen from Figures A and B, the fluorescence spectra and their corresponding standard curves show an obvious trend: the fluorescence intensity decreases proportionally with the bacterial concentration, and within the concentration range of 2.4×10 1 -2.4×10 6 CFU / mL, the distribution shows an obvious linear trend. These results clearly indicate that the EuMOFs / phage sensor can quickly and sensitively detect the concentration of PA. After the EuMOFs / phage composite reaches the target bacteria, a decrease in fluorescence intensity is observed, which may be due to the interaction between phages and bacteria. The limit of detection (LOD) of this biosensor for PA is 2 CFU / mL, which is determined by the formula LOD = 3SD / k, where SD is the standard deviation of 10 blank control measurements and k is the slope of the linear regression analysis.

[0086] To evaluate the specificity of the method, the EuMOFs / phage sensor prepared in Example 1 was used to detect a variety of bacteria, including Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Enterococcus faecalis, the host bacterium PA (Pseudomonas aeruginosa), and a mixture of all bacterial solutions. As Figure 4 shown in Figure C, the fluorescence biosensor showed obvious fluorescence changes when detecting the host bacterium, while the signal response to non-host bacteria was negligible.

[0087] In addition, live PA, dead PA, and a mixture of both were tested, and the test results are shown in Figure 4 Figure D. As can be seen from Figure 4 Figure D, the fluorescence biosensor of the present invention shows significant specificity and only responds to live PA. This also demonstrates the high specificity of the fluorescence biosensor for the target PA. The high specificity of this method stems from the phage as the recognition element, which has the unique ability to only recognize live target bacteria.

[0088] 5.2 Detection of actual clinical samples

[0089] Blood samples or urine samples from healthy people were taken, inoculated with different concentrations of Pseudomonas aeruginosa, and detected using the prepared fluorescence biosensor and the gold standard method (plate counting method) respectively, and the results were compared.

[0090] To evaluate the feasibility of this method, the fluorescence biosensor prepared in Example 1 was used to analyze urine and serum samples inoculated with PA. For comparison, plate culture counting was also used to detect these samples. Generally, different concentrations of PA were added to the urine samples and serum samples of healthy people. Subsequently, the prepared fluorescence biosensor and plate counting method were used for detection. The results are shown in Table 1.

[0091] Table 1 Detection of actual samples using the fluorescence biosensor and plate culture colony counting method

[0092]

[0093] As can be seen from Table 1, when using the fluorescent biosensor to detect clinical actual samples, the recovery rate is 96.75%-106.50%. When compared with the traditional plate culture medium method, the EuMOFs / phage biosensor shows acceptability and reliability in the analysis of actual samples.

[0094] Table 2 Performance comparison between the detection method of the fluorescent biosensor of the present invention and the existing methods for detecting Pseudomonas aeruginosa

[0095]

[0096]

[0097]

[0098] As can be seen from Table 2, compared with other detection methods, the fluorescent biosensor of the present invention has a shorter detection time of 20 min, a lower detection limit of 2 CFU / mL, and can distinguish between live and dead bacteria.

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Claims

1. A fluorescent biosensor, characterized in that: The fluorescent biosensor includes a lanthanide MOFs / bacteriophage composite material.

2. The fluorescent biosensor according to claim 1, characterized in that The lanthanide MOFs / bacteriophage composite material is prepared by connecting lanthanide MOFs with bacteriophages.

3. The fluorescent biosensor according to claim 2, characterized in that: The lanthanide MOFs are EuMOFs, and the bacteriophage is Pseudomonas aeruginosa phage.

4. A method for preparing the fluorescent biosensor according to any one of claims 1 to 3, characterized in that: The following steps are involved: 1) Preparation of lanthanide MOFs: Lanthanide MOFs were synthesized by mixed solvent thermal method; 2) Isolation and preparation of bacteriophages; 3) Purify phage; 4) Preparation of lanthanide MOFs / bacteriophage composite materials: Mix the lanthanide MOFs prepared in step 1) with water to prepare a 1-2 mg / mL lanthanide MOFs aqueous solution, mix 4-8 mL of the lanthanide MOFs aqueous solution, 40-80 mg of EDC, and 40-80 mg of NHS, react at room temperature for 30-40 minutes, centrifuge, discard the supernatant, and obtain precipitate I; Wash and resuspend the precipitate I, add 100-300 μL 10 9 -10 11 PFU / mL Step 3) Incubate and centrifuge the purified phage, discard the supernatant, obtain precipitate II, wash and resuspend precipitate II to obtain lanthanide MOFs / phage composite material.

5. The method for preparing a fluorescent biosensor according to claim 4, characterized in that: In step 4), PBS buffer is used in the washing and resuspension steps of precipitate I and precipitate II. After washing precipitate I, 1-2 mL PBS buffer is added and resuspended; after washing precipitate II, 4-8 mL PBS buffer is added and resuspended; incubation conditions: temperature 2-6°C, incubation time 12-24h.

6. The method for preparing a fluorescent biosensor according to claim 5, characterized in that: In step 1), the lanthanide MOFs are EuMOFs; The preparation method of EuMOFs comprises the following steps: adding Al(NO3)3·9H2O, Eu(NO3)3, and H2BDC-NH2 into a mixed solvent, placing the mixture in a reaction kettle, reacting at 130-160°C for 6-8 hours, cooling, centrifuging, collecting precipitates, washing, and drying to obtain EuMOFs; The usage ratio of Al(NO3)3·9H2O, Eu(NO3)3, H2BDC-NH2 and mixed solvent is (0.1-0.2)mmol: (0.01-0.2)mmol: (0.04-0.4)mmol: (20-40)mL; the mixed solvent is prepared by mixing DMF, water and anhydrous ethanol; In steps 2) and 3), the bacteriophage is a Pseudomonas aeruginosa phage.

7. The method for preparing a fluorescent biosensor according to claim 6, characterized in that: In step 1), the volume ratio of DMF, water and anhydrous ethanol is (15-25): (3-7): (3-7); centrifugation conditions: centrifugation at 8000-10000 rpm for 2-5 min; washing conditions: washing with distilled water and anhydrous ethanol for 3-7 times in sequence; drying conditions: vacuum drying at 50-70°C for 12-18 h.

8. The method for preparing a fluorescent biosensor according to claim 7, characterized in that: In step 2), the bacteriophage is isolated and prepared: the untreated sewage sample from the hospital is centrifuged at 4000-6000 r / min for 10-20 min, and the supernatant is filtered to obtain a filtrate; 0.2-0.4 mL of the filtrate is mixed with 5-6 mL of a logarithmic phase Pseudomonas aeruginosa solution, added to 10-20 mL of 3×LB culture medium, cultured at 37°C for 24-28 h, and then centrifuged and filtered to obtain the bacteriophage; 3) Purification of phage: dilute the phage from step 2) and incubate with the logarithmic phase Pseudomonas aeruginosa solution for 5-15 minutes, and purify using the double-layer agar plate method.

9. Use the fluorescent biosensor according to any one of claims 1 to 3 for detecting live Pseudomonas aeruginosa.

10. The use of the fluorescent biosensor according to claim 9, characterized in that: The detection method applied to live Pseudomonas aeruginosa comprises the following steps: 1) Establishment of the detection standard curve: Take freshly cultured Pseudomonas aeruginosa culture, centrifuge, resuspend the precipitate in PBS buffer, and then dilute it into different concentration gradients of Pseudomonas aeruginosa culture; Take 100-200 μL of lanthanide MOFs / phage composite material, add it to 700-800 μL PBS buffer, then add 100-200 μL of Pseudomonas aeruginosa bacterial solution with different concentration gradients, incubate at room temperature for 5-15 min, detect the fluorescence intensity, fit the linear relationship between fluorescence intensity and Pseudomonas aeruginosa concentration, and establish a standard curve for detection; 2) Apply the fluorescent biosensor to the detection of actual clinical samples containing P. aeruginosa.

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