CN115015338A - Composite material for separating and detecting klebsiella pneumoniae and application thereof - Google Patents
Composite material for separating and detecting klebsiella pneumoniae and application thereof Download PDFInfo
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- 241000588747 Klebsiella pneumoniae Species 0.000 title claims abstract description 48
- 239000002131 composite material Substances 0.000 title claims abstract description 26
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims abstract description 49
- 239000002086 nanomaterial Substances 0.000 claims abstract description 31
- WZPBZJONDBGPKJ-UHFFFAOYSA-N Antibiotic SQ 26917 Natural products O=C1N(S(O)(=O)=O)C(C)C1NC(=O)C(=NOC(C)(C)C(O)=O)C1=CSC(N)=N1 WZPBZJONDBGPKJ-UHFFFAOYSA-N 0.000 claims abstract description 24
- WZPBZJONDBGPKJ-VEHQQRBSSA-N aztreonam Chemical compound O=C1N(S([O-])(=O)=O)[C@@H](C)[C@@H]1NC(=O)C(=N/OC(C)(C)C(O)=O)\C1=CSC([NH3+])=N1 WZPBZJONDBGPKJ-VEHQQRBSSA-N 0.000 claims abstract description 24
- 229960003644 aztreonam Drugs 0.000 claims abstract description 24
- 239000000463 material Substances 0.000 claims abstract description 23
- 238000007885 magnetic separation Methods 0.000 claims abstract description 20
- 150000001875 compounds Chemical class 0.000 claims abstract description 15
- 229910021397 glassy carbon Inorganic materials 0.000 claims abstract description 11
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 4
- 230000008878 coupling Effects 0.000 claims abstract description 4
- 238000010168 coupling process Methods 0.000 claims abstract description 4
- 238000005859 coupling reaction Methods 0.000 claims abstract description 4
- 229910021389 graphene Inorganic materials 0.000 claims abstract description 4
- 239000008055 phosphate buffer solution Substances 0.000 claims description 43
- 239000000243 solution Substances 0.000 claims description 41
- 239000006228 supernatant Substances 0.000 claims description 23
- 238000001514 detection method Methods 0.000 claims description 19
- 238000005406 washing Methods 0.000 claims description 19
- 239000002122 magnetic nanoparticle Substances 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 15
- 238000000926 separation method Methods 0.000 claims description 14
- 239000006185 dispersion Substances 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 11
- 238000002360 preparation method Methods 0.000 claims description 11
- 238000002791 soaking Methods 0.000 claims description 8
- 239000011324 bead Substances 0.000 claims description 7
- 239000004793 Polystyrene Substances 0.000 claims description 6
- 230000003213 activating effect Effects 0.000 claims description 6
- 238000002474 experimental method Methods 0.000 claims description 6
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 claims description 6
- 229920002223 polystyrene Polymers 0.000 claims description 6
- 229940075397 calomel Drugs 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- ZOMNIUBKTOKEHS-UHFFFAOYSA-L dimercury dichloride Chemical compound Cl[Hg][Hg]Cl ZOMNIUBKTOKEHS-UHFFFAOYSA-L 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 230000010355 oscillation Effects 0.000 claims description 3
- 229910052697 platinum Inorganic materials 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- 238000009210 therapy by ultrasound Methods 0.000 claims description 3
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 3
- 239000012498 ultrapure water Substances 0.000 claims description 3
- 241000894006 Bacteria Species 0.000 claims description 2
- 230000004913 activation Effects 0.000 claims description 2
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- 101000730040 Klebsiella pneumoniae Arylsulfatase Proteins 0.000 claims 2
- 239000000725 suspension Substances 0.000 claims 1
- 101710116957 D-alanyl-D-alanine carboxypeptidase Proteins 0.000 abstract description 3
- 150000001732 carboxylic acid derivatives Chemical class 0.000 abstract description 3
- 210000000170 cell membrane Anatomy 0.000 abstract description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 abstract description 3
- 239000011540 sensing material Substances 0.000 abstract 1
- 239000008280 blood Substances 0.000 description 12
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- 239000000203 mixture Substances 0.000 description 9
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- 241000588921 Enterobacteriaceae Species 0.000 description 1
- 241000588748 Klebsiella Species 0.000 description 1
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- 206010041925 Staphylococcal infections Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
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- 208000015688 methicillin-resistant staphylococcus aureus infectious disease Diseases 0.000 description 1
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- 108700012359 toxins Proteins 0.000 description 1
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Abstract
The invention discloses a composite material for separating and detecting Klebsiella pneumoniae, which comprises a magnetic nano-material compound and an immunosensing material, wherein the magnetic nano-material compound is formed by coupling aztreonam and a magnetic nano-material; the immune sensing material consists of a Klebsiella pneumoniae monoclonal antibody, carboxyl functionalized graphene and a glassy carbon electrode, aztreonam is connected with carboxylic acid on the surface of a magnetic nano material through amino, and then can be combined with Penicillin Binding Protein (PBPs) on the surface of a Klebsiella pneumoniae cell membrane through beta-lactam bond of the aztreonam, so that the magnetic separation of the functionalized magnetic nano material on the Klebsiella pneumoniae is realized.
Description
Technical Field
The invention relates to the technical field of Klebsiella pneumoniae separation, and particularly relates to a material for separating Klebsiella pneumoniae and application thereof.
Background
Septicemia refers to acute systemic infection caused by various pathogenic bacteria invading blood circulation, growing and breeding in blood, and producing toxins. Klebsiella pneumoniae (k. pneumoniae) is the most important group of Klebsiella of enterobacteriaceae, and has pathogenicity, and the resulting diseases account for over 95% of Klebsiella infection. In China, Klebsiella pneumoniae is the second largest gram-negative pathogen of septicemia, and has become a serious challenge for clinical anti-infection treatment as the number of septicemia cases caused by K.
Currently, the method for detecting k. pneumoniale is usually time-consuming and the detection result is easily interfered due to the complex sample matrix.
Therefore, there is a need in the art to provide a material and a method for separation of klebsiella pneumoniae that can reduce interference and can be used for separation and detection.
Disclosure of Invention
In view of the above, the invention adopts aztreonam which is connected with carboxylic acid on the surface of the magnetic nano material through amino, and then can be combined with Penicillin Binding Protein (PBPs) on the surface of the cell membrane of Klebsiella pneumoniae through beta-lactam bond of the aztreonam, so as to realize the magnetic separation of the functionalized magnetic nano material on the Klebsiella pneumoniae; and the purpose of specificity detection is achieved by the specific combination of the immunosensing material and the Klebsiella pneumoniae.
In order to achieve the purpose, the invention adopts the following technical scheme:
a composite material for isolating and detecting klebsiella pneumoniae, the composite material comprising a magnetic nanomaterial complex and an immunosensor material; the magnetic nano material composite is formed by coupling aztreonam and a magnetic nano material; the immunosensing material consists of a Klebsiella pneumoniae monoclonal antibody, carboxyl functionalized graphene and a glassy carbon electrode.
As a preferable technical solution of the above technical solution, the magnetic nanomaterial is made of Fe 3 O 4 And polystyrene, wherein the polystyrene is wrapped in Fe 3 O 4 And (4) an outer layer.
As a preferable technical solution of the above technical solution, the preparation process of the magnetic nanomaterial composite comprises:
1) taking 1mg of carboxylated magnetic beads, washing the carboxylated magnetic beads for 3 times by using sterile PBS (phosphate buffer solution), and removing a supernatant after magnetic separation;
2) respectively dissolving EDC and NHSS in a sterile PBS solution to prepare a 10mg/mL solution, then respectively adding 29 mu L and 33 mu L to the solution obtained in the step 1), activating for 1h at room temperature, washing the magnetic nano material for 3 times, removing unreacted EDC and NHSS, and carrying out resuspension by using the sterile PBS to obtain a magnetic nano material solution after carboxyl activation;
3) and removing the supernatant after magnetic separation, re-suspending in 1mL of sterile PBS solution, adding 1mg of aztreonam, reacting for 4 hours on a rotary mixer at room temperature, removing the supernatant after magnetic separation, washing for 3 times, and re-suspending in 1mL of sterile PBS solution to obtain the aztreonam modified magnetic nanoparticle compound with the final concentration of 1 mg/mL.
As a preferable technical solution of the above technical solution, the preparation process of the immunosensing material comprises:
1) dispersing 1mg of Gr-COOH powder into 1mL of sterile ultrapure water, carrying out vortex oscillation for 30s, and carrying out ultrasonic treatment for 20min to obtain a Gr-COOH dispersion liquid;
2) sucking 5 mu L of Gr-COOH dispersion liquid, dripping the Gr-COOH dispersion liquid on the surface of a glassy carbon electrode, drying the glassy carbon electrode in an infrared oven, and cooling the glassy carbon electrode to room temperature to obtain a GCE/Gr-COOH electrode;
3) and (3) soaking the GCE/Gr-COOH electrode in 0.2M EDC/NHSS solution at room temperature for 45min to activate carboxyl, washing away unbound EDC/NHSS by using sterile PBS, soaking the electrode in 100 mu g/mL Ab solution, and incubating overnight at 4 ℃ to obtain the immunosensing material GCE/Gr-COOH/Ab.
The invention also provides application of the composite material in preparation of biochemical reagents for enriching, separating and detecting Klebsiella pneumoniae.
The invention also provides application of the composite material in preparation of a kit for enriching, separating and detecting Klebsiella pneumoniae.
The invention also discloses a method for separating and detecting Klebsiella pneumoniae by using the composite material, which is the same as the technical proposal of the invention, and the method is used for separating and detecting the Klebsiella pneumoniae in a sample to be detected.
As the preferable technical scheme of the technical scheme, the process for separating and detecting the Klebsiella pneumoniae in the sample to be detected comprises the following steps:
1) taking 0.9mL of a K.PNEUMONIAE-containing bacterial sample, adding 100 mu L of prepared AMBs, placing the bacterial sample in a shaking table, incubating for 45min at 37 ℃ under the condition of 180r/min, then placing the bacterial sample on a magnetic frame, separating for 4min, removing a supernatant, and re-suspending the K.Pneumoniale-AMBs compound by using 100 mu L of PBS;
2) soaking GCE/Gr-COOH/Ab in the K.Pneumoniale-AMBs compound for 2h, and then washing the surface of the electrode by PBS to obtain a GCE/Gr-COOH/Ab-K.Pneumoniale-AMBs structure;
3) and detecting the resistance of GCE/Gr-COOH/Ab-K.Pneumoniale-AMBs by using an electrochemical workstation, performing an impedance experiment, analyzing the result and drawing.
The electrochemical workstation is a three-electrode system, a glassy carbon electrode is used as a working electrode, a calomel electrode is used as a reference electrode, and a platinum electrode is used as a counter electrode; the impedance experiments were all at 5mM [ Fe (CN) ] containing 0.1M KCl 6 ] 3-/4- In solution.
Through the technical scheme, compared with the prior art, the invention has the following technical effects:
the invention adopts aztreonam which is connected with carboxylic acid on the surface of a magnetic nano material through amino and can be combined with Penicillin Binding Protein (PBPs) on the cell membrane surface of Klebsiella pneumoniae through beta-lactam bond of the aztreonam to realize the magnetic separation of the functionalized magnetic nano material on the Klebsiella pneumoniae, and then an immunosensing material GCE/Gr-COOH/Ab is incubated in a K.Pneuronium-AMBs compound for 2h to form a structure of GCE/Gr-COOH/K.Pneuronium-AMBs, thereby achieving the purpose of separation and detection of the invention;
the identification molecule adopted by the method is aztreonam, and compared with an antibody, the aztreonam has higher stability and lower cost, and has longer shelf life;
the magnetic nanoparticles with the particle size of 180nm are used for rapid enrichment of target bacteria in the matrix, so that the influence of interferents in other samples such as blood can be avoided, and separation and detection can be realized without other treatment;
the rapid detection method can realize rapid detection of Klebsiella pneumoniae, can realize quantitative detection compared with methods such as molecular biology method and the like, and has high reliability of result.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a flow chart of the method operations for magnetic separation and detection of Klebsiella pneumoniae in sepsis according to the present invention;
FIG. 2 is an SEM image of the functionalized magnetic nanomaterial and Klebsiella pneumoniae of the present invention;
FIG. 3 is a graph showing the separation effect of the present invention on Klebsiella pneumoniae with different concentrations in blood;
FIG. 4 is a test result of the present invention on the detection of Klebsiella pneumoniae with different concentrations in blood.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
Example 1
A composite material for separating and detecting Klebsiella pneumoniae is composed of a magnetic nano-material composite and an immunosensing material, wherein the magnetic nano-material composite is formed by coupling aztreonam and a magnetic nano-material; the magnetic nano material is made of Fe 3 O 4 And polystyrene, wherein the polystyrene is wrapped in Fe 3 O 4 An outer layer; the immunosensing material consists of a Klebsiella pneumoniae monoclonal antibody, carboxyl functionalized graphene and a glassy carbon electrode;
the preparation process of the magnetic nano material compound comprises the following steps:
0.1mL of magnetic nano-particle Fe is absorbed 3 O 4 (10mg/mL) is added into 0.9mL of sterile PBS solution with pH 7.4, then the magnetic nanoparticles are washed under the action of an external magnetic field, the washing is repeated for 3 times, and the washed magnetic nanoparticles are resuspended in 1mL of sterile pH solution with pH 7.4; weighing 0.29mg of EDC and 0.325mg of NHSS, adding into the washed magnetic nanoparticle solution, activating at 37 ℃ for 1h, washing the activated magnetic nanoparticles with sterile PBS for 3 times, and suspending in 1mL of sterile PBS solution; weighing 1mg of aztreonam, adding the aztreonam into the activated magnetic nanoparticle solution, reacting for 4h at room temperature, finally washing for 3 times by using sterile PBS, and suspending the aztreonam in 1mL of sterile PBS solution to obtain an aztreonam modified magnetic nanomaterial complex with the final concentration of 1mg/mL, wherein the aztreonam modified magnetic nanomaterial complex is used for enriching and separating Klebsiella pneumoniae; the preparation process of the immunosensor material comprises the following steps:
1) preparation of Gr-COOH dispersion: dispersing 1mg of Gr-COOH powder into 1mL of sterile ultrapure water, performing vortex oscillation for 30s, and performing ultrasonic treatment for 20min to obtain a Gr-COOH dispersion liquid;
2) preparation of GCE/Gr-COOH electrode: sucking 5 mu L of the Gr-COOH dispersion liquid, dripping the Gr-COOH dispersion liquid on the surface of a Glassy Carbon Electrode (GCE), drying the GCE/Gr-COOH dispersion liquid in an infrared oven, and cooling the GCE/Gr-COOH dispersion liquid to room temperature to obtain a GCE/Gr-COOH electrode;
3) preparation of immunosensing material GCE/Gr-COOH/Ab: the GCE/Gr-COOH electrode was immersed in 0.2M EDC/NHSS solution at room temperature for 45min to activate carboxyl groups, unbound EDC/NHSS was washed away with sterile PBS, and then immersed in 100. mu.g/mL Ab solution and incubated overnight at 4 ℃ to obtain GCE/Gr-COOH/Ab.
Example 2
K.pneumoniale was isolated with the composite of example 1, the principle is shown in figure 1;
taking 0.9mL of a K.PNEUMONIAE-containing bacterial sample, adding 100 mu L of prepared AMBs, placing the bacterial sample in a shaking table, incubating for 45min at 37 ℃ under the condition of 180r/min, then placing the bacterial sample on a magnetic frame, separating for 4min, removing a supernatant, and re-suspending the K.Pneumoniale-AMBs compound by using 100 mu L of PBS; then, soaking an immunosensing material GCE/Gr-COOH/Ab in the K.Pneumoniale-AMBs compound for 2 hours, and washing the surface of the electrode by PBS to obtain a GCE/Gr-COOH/Ab-K.PNEUMONIAE-AMBs structure; finally, an electrochemical workstation is adopted to detect the resistance of GCE/Gr-COOH/Ab-K.Pneumoniae-AMBs, and an impedance experiment is carried out, wherein the electrochemical workstation is a three-electrode system, a Glassy Carbon Electrode (GCE) is used as a working electrode, a calomel electrode is used as a reference electrode, and a platinum electrode is used as a counter electrode; impedance experiments were performed at 5mM [ Fe (CN) ] containing 0.1M KCl 6 ] 3-/4- The results of the analysis were performed in solution and plotted.
Material characterization
1) Taking magnetic nano particles Fe 3 O 4 1mg, washed 3 times with PBS, and the supernatant removed after magnetic separation; respectively dissolving EDC and NHSS in a sterile PBS solution, activating for 1h at room temperature, washing the magnetic beads again, removing unreacted EDC and NHSS, and re-suspending with PBS to obtain an activated magnetic nanoparticle solution; after magnetic separation, sucking out the supernatant, adding 1mg of aztreonam, reacting for 4 hours on a rotary mixer (15r/min) at room temperature, and removing the supernatant after magnetic separation;
2) adding 10 concentrations of the above-mentioned materials into blood samples 5 Pneumoniales 100. mu.L of CFU/mL, 100. mu.g of prepared AMBs and 800. mu.L of sterile PBS were addedPlacing the mixture in a shaking table, incubating the mixture for 45min at 37 ℃ and 180r/min, then placing the mixture in a magnetic frame for separation for 4min, placing supernatant in a new centrifugal tube, and resuspending K.Pneumoniale-AMBs compound by PBS;
3) the obtained AMBs and K.Pneumonium-AMBs complexes were characterized by SEM, respectively.
The characterization effects of each group are shown in fig. 2.
The results show that the AMBs have good dispersibility and that the complex of k. pneumoniale bound to the AMBs can be clearly seen, indicating that the material can be successfully enriched to MRSA.
Example 3
Magnetic separation effect verification of K.Pneumoniale with different concentrations in blood samples
1) Taking magnetic nano particles Fe 3 O 4 1mg, washed 3 times with PBS, and the supernatant removed after magnetic separation; respectively dissolving EDC and NHSS in a sterile PBS solution, activating for 1h at room temperature, washing the magnetic beads again, removing unreacted EDC and NHSS, and re-suspending with PBS to obtain an activated magnetic nanoparticle solution; sucking out the supernatant after magnetic separation, adding 1mg of aztreonam, reacting for 4 hours on a rotary mixer (15r/min) at room temperature, and removing the supernatant after magnetic separation;
2) adding 10 concentrations of the above-mentioned materials into blood samples 6 CFU/mL～10 1 0.1mL of CFU/mL K.PNEUMONIAE, adding 100 mu g of prepared AMBs and 800 mu L of PBS, placing the mixture in a shaking table, incubating the mixture for 45min at 37 ℃ at 180r/min, then placing the mixture in a magnetic frame for separation for 4min, placing the supernatant in a new centrifugal tube, and resuspending the K.Pneumoniale-AMBs compound by using the PBS;
4) the supernatant and the complex were diluted to 10 respectively 3 CFU/mL, 100. mu.L of each aliquot was pipetted and spotted, and after 12h of incubation in a 37 ℃ incubator, counted and calculated for enrichment efficiency (CE) according to the formula:
CE(％)＝[1-n/(N+n)]×100％
wherein N is the number of colonies in the separating medium, and N is the number of colonies in the supernatant;
the capture efficiency of each group is shown in FIG. 3.
The result shows that the method has ideal capture efficiency for the Klebsiella pneumoniae in the blood sample, and the scheme can efficiently enrich and separate the Klebsiella pneumoniae in the blood sample.
Example 4
Verification of detection effect of Klebsiella pneumoniae in blood sample
1) Taking 1mg of magnetic nanoparticles, washing the magnetic nanoparticles for 3 times by PBS (phosphate buffer solution), and removing a supernatant after magnetic separation; respectively dissolving EDC and NHSS in a sterile PBS solution, activating for 1h at room temperature, washing the magnetic beads again, removing unreacted EDC and NHSS, and re-suspending with PBS to obtain an activated magnetic nanoparticle solution; after magnetic separation, sucking out the supernatant, adding 1mg of aztreonam, reacting for 4 hours on a rotary mixer (15r/min) at room temperature, and removing the supernatant after magnetic separation;
2) adding 10 concentrations of the above-mentioned materials into blood samples 6 CFU/mL～10 1 0.1mL of CFU/mL K.Pneumoniae, adding 100 mu g of prepared AMBs and 800 mu L of PBS, placing the mixture in a shaking table, incubating the mixture for 45min at 37 ℃ under the condition of 180r/min, then placing the mixture in a magnetic frame for separation for 4min, removing supernatant, and then resuspending the K.Pneumoniae-AMBs compound by using 0.1mL of PBS;
4) soaking an immunosensor GCE/Gr-COOH/Ab in the K.Pneumoniale-AMBs compound for 2h, and then washing the surface of an electrode by PBS to obtain a GCE/Gr-COOH/Ab-K.Pneumoniale-AMBs structure;
5) and detecting the resistance of GCE/Gr-COOH/Ab-K.Pneumoniale-AMBs by adopting an electrochemical workstation, analyzing the result and mapping.
The detection effect of each group is shown in fig. 4.
The result shows that the kit has ideal detection effect on Klebsiella pneumoniae in blood samples, and the detection limit can reach 10 2 CFU/mL shows that the scheme can realize the detection of Klebsiella pneumoniae in septicemia samples.
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (9)
1. The composite material for separating and detecting Klebsiella pneumoniae comprises a magnetic nano-material composite and an immunosensing material, wherein the magnetic nano-material composite is formed by coupling aztreonam and a magnetic nano-material; the immunosensing material consists of a Klebsiella pneumoniae monoclonal antibody, carboxyl functionalized graphene and a glassy carbon electrode.
2. The composite material for separating and detecting Klebsiella pneumoniae of claim 1, wherein the magnetic nano material is Fe 3 O 4 And polystyrene, wherein the polystyrene is wrapped in Fe 3 O 4 And (4) an outer layer.
3. The composite material for separating and detecting klebsiella pneumoniae as claimed in claim 1, wherein the magnetic nano-material composite is prepared by a process comprising:
1) taking 1mg of carboxylated magnetic beads, washing the beads for 3 times by using sterile PBS (phosphate buffer solution), and removing a supernatant after magnetic separation;
2) respectively dissolving EDC and NHSS in a sterile PBS solution to prepare a 10mg/mL solution, then respectively adding 29 mu L and 33 mu L to the solution obtained in the step 1), activating for 1h at room temperature, washing the magnetic nano material for 3 times again, removing unreacted EDC and NHSS, and carrying out heavy suspension by using sterile PBS to obtain a magnetic nano material solution after carboxyl activation;
3) and removing the supernatant after magnetic separation, suspending in 1mL of sterile PBS solution, adding 1mg of aztreonam, reacting for 4 hours on a rotary mixer at room temperature, removing the supernatant after magnetic separation, washing for 3 times, and suspending in 1mL of sterile PBS solution to obtain the aztreonam modified magnetic nanoparticle composites AMBs with the final concentration of 1 mg/mL.
4. The composite material for separating and detecting klebsiella pneumoniae of claim 1, wherein the immunosensing material is prepared by a process comprising:
1) dispersing 1mg of Gr-COOH powder into 1mL of sterile ultrapure water, performing vortex oscillation for 30s, and performing ultrasonic treatment for 20min to obtain a Gr-COOH dispersion liquid;
2) sucking 5 mu L of Gr-COOH dispersion liquid, dripping the Gr-COOH dispersion liquid on the surface of the GCE, drying the GCE in an infrared oven, and cooling to room temperature to obtain a GCE/Gr-COOH electrode;
3) and (3) soaking the GCE/Gr-COOH electrode in a 0.2MEDC/NHSS solution at room temperature for 45min to activate carboxyl, washing away unbound EDC/NHSS by using sterile PBS, soaking the electrode in a 100 mu g/mL Ab solution, and incubating overnight at 4 ℃ to obtain the immunosensing material GCE/Gr-COOH/Ab.
5. The use of the composite material for separation and detection of klebsiella pneumoniae of claim 1 or 2 in the preparation of biochemical reagents for enrichment separation and detection of klebsiella pneumoniae.
6. The use of the composite material for separation and detection of klebsiella pneumoniae of claim 1 or 2 in the preparation of a kit for enrichment separation and detection of klebsiella pneumoniae.
7. A method for separating and detecting klebsiella pneumoniae, which is characterized in that the composite material of claim 1 or 2 is used for separating and detecting klebsiella pneumoniae in a sample to be detected.
8. The method for separating and detecting klebsiella pneumoniae of claim 7, wherein the separation of klebsiella pneumoniae in a sample to be tested by using the composite material of claim 2 comprises:
1) taking 0.9mL of K.Pneumoniae bacteria sample, adding 100 mu L of prepared AMBs, placing the AMBs in a shaking table to incubate for 45min at 37 ℃ under the condition of 180r/min, then placing the AMBs on a magnetic frame to separate for 4min, removing supernatant, and resuspending the K.Pneumoniae-AMBs compound by using 100 mu L of PBS;
2) soaking GCE/Gr-COOH/Ab in the K.Pneumoniale-AMBs compound for 2h, and then washing the surface of the electrode by PBS to obtain a GCE/Gr-COOH/Ab-K.Pneumoniale-AMBs structure;
3) and detecting the resistance of GCE/Gr-COOH/Ab-K.Pneumoniale-AMBs by using an electrochemical workstation, performing an impedance experiment, analyzing the result and drawing.
9. The method for separating and detecting klebsiella pneumoniae as claimed in claim 8, wherein the electrochemical workstation is a three-electrode system, a glassy carbon electrode is used as a working electrode, a calomel electrode is used as a reference electrode, and a platinum electrode is used as a counter electrode; the impedance experiments were all at 5mM [ Fe (CN) ] containing 0.1M KCl 6 ] 3-/4- In solution.
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