CN111458516A - Electrochemical luminescence biosensor for detecting bacterial drug resistance and preparation method thereof - Google Patents

Electrochemical luminescence biosensor for detecting bacterial drug resistance and preparation method thereof Download PDF

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CN111458516A
CN111458516A CN202010056277.1A CN202010056277A CN111458516A CN 111458516 A CN111458516 A CN 111458516A CN 202010056277 A CN202010056277 A CN 202010056277A CN 111458516 A CN111458516 A CN 111458516A
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drug resistance
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coli
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CN111458516B (en
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马芬
刘家玮
陈玉
孙利娜
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Northwestern University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4724—Lectins
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    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • G01N2400/50—Lipopolysaccharides; LPS

Abstract

The invention discloses an electrochemiluminescence biosensor for detecting bacterial drug resistance and a preparation method thereof, comprising the step of preparing NH2The invention provides an electrochemical luminescence biosensor which can quickly, simply, reliably and effectively detect drug resistance, and utilizes an electrochemical luminescence technology as a signal output mode, wherein the technology has extremely high sensitivity, and the electrochemical luminescence biosensor provided by the invention is used for detecting fine particlesWhether the bacteria have drug resistance or not and can be used for quantitatively detecting the concentration of the escherichia coli.

Description

Electrochemical luminescence biosensor for detecting bacterial drug resistance and preparation method thereof
Technical Field
The invention relates to the technical field of electrochemical luminescence, in particular to an electrochemical luminescence biosensor for detecting bacterial drug resistance and a preparation method thereof.
Background
The extremely strong drug resistance of NDM-1 superbacteria greatly increases the treatment difficulty of infections caused by the superbacteria, and the common drug-resistant bacteria cannot "pass" the drug-resistant genes to other bacteria, while the NDM-1 gene can be transferred among different bacteria to transfer resistance.
The currently established means for detecting whether bacteria express NDM-1 resistance genes are broth dilution and disc diffusion, which involve multiple time-consuming steps including (1) culturing the bacteria to a detectable bacterial density (24-48h), (2) incubating the bacteria and antibiotics (24-48h) in 96-well plates or dishes, (3) detecting bacterial growth by uv absorption spectroscopy or visual detection.
Therefore, a method which is rapid, simple and low in cost is developed to detect the drug resistance of bacteria, so that targeted medication is achieved, and the method has important significance for clinical application of antibiotics.
Disclosure of Invention
In order to solve the technical problems, the invention provides a preparation method of an electrochemiluminescence biosensor for detecting bacterial drug resistance
The invention provides a preparation method of an electrochemiluminescence biosensor for detecting bacterial drug resistance, which is characterized by comprising the following steps:
step 1: preparation of NH2-MI L-53 (Al) nanoplates;
step 1.1: 0.7243g of 3mmol AlCl3·6H2Dissolving O in 15m L deionized water;
step 1.2: 0.5435g of 3mmol 2-amino-1, 4-phthalic acid is slowly added under the stirring condition, and the stirring is continued for 30 minutes to obtain a mixed solution 1;
step 1.3, dropwise adding 15m of L6 mmol of urea aqueous solution into the mixed solution 1, and continuously stirring for 30 minutes to obtain a mixed solution 2;
step 1.4, transferring the mixed solution 2 into a 50m L polytetrafluoroethylene high-pressure reaction kettle, and standing and reacting for 5 hours at the temperature of 150 ℃ to obtain a mixed solution 3;
step 1.5: slowly cooling the mixed solution 3 to room temperature, sucking and filtering to obtain milk white yellowish precipitate, and washing with a large amount of deionized water to obtain a mixed solution 4;
step 1.6, dispersing the mixed solution 4 in 20m L N, N-dimethylformamide solution, and stirring for 24 hours at room temperature;
step 1.7: replacing N, N-dimethylformamide solution with methanol of the same volume, continuing stirring for 24 hours, removing methanol after the reaction is finished, and vacuum drying at 70 ℃ overnight to obtain NH2-MI L-53 (Al) nanoplates;
step 2, preparing a probe solution for detecting bacterial drug resistance:
preparing 50 mu L100 mu mol of bis (2,2 '-bipyridyl) -4' -methyl-4-carboxybipyridyl-ruthenium (N-succinimidyl ester) -bis (hexafluorophosphate) solution by using N, N-dimethylformamide solution, adding the solution into 1.5m L10 mu mol of hemilactoglobulin (Con A) solution prepared by buffer solution, and slowly stirring for 6 hours at 25 ℃ in the dark;
step 3, assembling an electrochemical luminescence sensor for detecting bacterial drug resistance;
step 3.1, the NH synthesized in step 12Ultrasonically dispersing an-MI L-53 (Al) nano sheet in a DMF (dimethyl formamide) solution, dripping the dispersed solution on the surface of a cleanly treated working electrode, and drying to obtain NH2-MI L-53 (Al)/GCE modified electrode;
step 3.2, adding EDC and NHS into the probe solution obtained in the step 2 for activation, and immersing the modified electrode obtained in the step 3.1 into the activated probe solution for modification to obtain Con A-Ru/NH2-MI L-53 (Al)/GCE modified electrode;
step 3.3, Bovine Serum Albumin (BSA) is dissolved in the binding buffer solution and dripped into the Con A-Ru/NH obtained in step 3.22And (4) sealing the surface of the-MI L-53 (Al)/GCE modified electrode to obtain the electrochemiluminescence biosensor for detecting the bacterial drug resistance.
In the step 2, the buffer solution is: containing 1mM CaCl2,1mM MnCl210mM Tris-hydrochloric acid solution, buffer pH 8.0.
In a further embodiment, in step 3.1, NH modified to the surface of the working electrode is added2The dispersion concentration of the-MI L-53 (Al) nanosheets is 0.1-1mg/m L.
In a further scheme, in the step 3.1, the working electrode is one of a glassy carbon electrode, a graphite electrode, an ITO electrode and a noble metal electrode.
In a further scheme, in the step 3.2, the concentrations of EDC and NHS are respectively 2 mg/L and 5 mg/L, the concentration of the probe solution ConA-Ru is 0.1-10 mu M, and the modification time is 2-4 h.
In a further embodiment, in step 3.3, the concentration of the BSA solution is 0.1-1%, the volume dropped on the electrode surface is 10. mu. L, and the blocking time is 30-60 min.
The mechanism of the electrochemical luminescence biosensor for detecting the drug resistance of bacteria is as follows:
the lectin is a structure with various proteins, can be specifically combined with polysaccharide and shows high affinity, and has the characteristics of easy production and marking so as to provide a good platform for designing the biosensor.
The invention constructs an electrochemiluminescence biosensor for detecting bacterial drug resistance based on the specific combination of lectin (Con A) and lipopolysaccharide (L PS). firstly, NH is used2The electrode is modified by taking MI L-53 (Al) nanosheets as a substrate material, a porous interface can be provided to increase the surface area of the electrode, amino groups on the nanosheets can react with carboxyl groups on a Con A probe and can be used for fixing the probe, lectin (Con A) serves as a specific recognition element to recognize lipopolysaccharide (L PS) on the surface of escherichia coli (E.coli), bipyridine ruthenium (Con A-Ru) marked on the specific recognition element provides an electrochemiluminescence signal, when the sensor is combined with bacteria, the electrochemiluminescence signal is weakened, and the sensor has specific response to the escherichia coli, when the sensor is used for detecting bacteria (E.coli B L21) without drug resistance, if the E.coli B L21 in a liquid to be detected does not act with antibiotics, the concentration of the escherichia coli living in the liquid is high, the Con A protein on the surface of the sensor can be specifically combined with a large number of bacteria, and the luminescence of Ru complexes is inhibited, so that the electrochemiluminescence intensity is detectedMeasuring the rate of change delta I/I before and after the bacteria0Large (Δ I ═ I)0–I,I0And I is the electrochemiluminescence signal before and after the detection of the bacteria by the sensor), if e.coli B L21 reacts with various antibiotics, the concentration of escherichia coli surviving in the solution will decrease, the number of bacteria specifically bound to the sensor surface will be less, and the luminescence inhibition degree of the Ru complex will be lower, resulting in a change rate Δ I/I of the electrochemiluminescence intensity before and after the detection of the bacteria0Is smaller. Therefore, when detecting bacteria without drug resistance, the change rate delta I/I of the detection signal of the sensor is between the bacteria to be detected and the antibiotics0From big to small, when the sensor is used for detecting drug-resistant bacteria (NDM-1E. coli B L21), if NDM-1E. coli B L21 interacts with β -lactam antibiotics (cefpirome, imipenem), the concentration of NDM-1E. coli B L21 in the solution cannot be changed because β -lactam antibiotics can be decomposed by β -lactamase expressed by NDM-1E. coli B L21 and then are ineffective, so that when detecting NDM-1E. coli B L21, no matter whether the bacteria interact with β -lactam antibiotics or not, the signal change rate delta I/I of the sensor before and after detecting the bacteria is detected0In addition, if NDM-1E. coli B L21 interacts with non- β -lactam antibiotics (levofloxacin and tetracycline), and β -lactamase expressed by bacteria in the test solution cannot decompose the antibiotics, NDM-1E. coli B L21 can cause death due to the antibiotics, and the concentration of the bacteria is reduced, so that the bacteria do not react with the non- β -lactam antibiotics by delta I/I0Larger, Delta I/I after action with non- β -lactam antibiotics0Is smaller. I.e. based on the detected Δ I/I0Coli B L21/NDM-1E. coli B L21 were distinguished to determine whether or not the bacteria had drug resistance (expression of M βL s gene).
Compared with the related art, the invention has the following beneficial effects:
(1) the electrochemical luminescence biosensor has high sensitivity, detects whether bacteria have drug resistance or not, and can be used for quantitatively detecting the concentration of escherichia coli.
(2)Ru(bpy)3 2+System ofHas good stability, higher EC L quantum yield and biocompatibility, Ru (bpy)3 2+The fixation on the electrode surface can not only reduce the usage amount of expensive reagents, but also enhance the EC L signal intensity and simplify the experimental process.
(3) The signal is stable, the Con A-Ru probe is modified on the surface of the electrode in a covalent connection mode, and the phenomenon that an Au-S bond is broken when the potential of the traditional Au-S self-assembly is +0.9V is avoided.
(4) The multi-load performance of the MOF material is utilized, more probe connection sites are provided, and the signal amplification effect is achieved.
(5) Low cost and less reagent amount.
Drawings
FIG. 1 is a schematic diagram of an electrochemiluminescence biosensor according to the present invention for detecting bacterial drug resistance;
fig. 2a shows the electrochemiluminescence signals corresponding to different concentrations of e.coli B L21, and fig. 2B shows the linear relationship between the luminescence intensity value and the concentration of e.coli B L21;
FIG. 3 shows that the electrochemiluminescence biosensor detects the corresponding delta I/I of E.coli B L21/NDM-1 E.coli B L21 after the action of various antibiotics0A value from which it can be determined whether the bacteria have drug resistance;
fig. 4 is a graph of the stability results of the electrochemiluminescence biosensor detecting e.coli B L21.
Detailed Description
The invention will be further explained with reference to the drawings and the embodiments.
A preparation method of an electrochemiluminescence biosensor for detecting bacterial drug resistance takes the detection of whether bacteria express M βL s as an example, and the selected bacteria are E.coli B L21/NDM-1 E.coli, and the specific preparation steps are as follows:
step 1, preparation of NH2-MI L-53 (Al) nanoplates;
mixing AlCl3·6H2O (3mmol,0.7243g) was dissolved in 15m L g of deionized water, 2-amino-1, 4-benzenedicarboxylic acid (3mmol,0.5435g) was added slowly with stirring, stirring was continued for another 30 minutes, and then 15m L of urea (6mmol O)l,0.3604g) aqueous solution is dropwise added into the mixed solution and is continuously stirred for 30 minutes, then the mixture is transferred into a 50m L polytetrafluoroethylene high-pressure reaction kettle and is kept stand at 150 ℃ for reaction for 5 hours, after the reaction is finished, the mixture is slowly cooled to room temperature, is sucked and filtered to obtain milk white yellowish precipitate, and is washed clean by a large amount of deionized water, then the product is dispersed in 20m L N, N-Dimethylformamide (DMF) solution and is stirred for 24 hours at room temperature, finally, methanol with the same volume is used for replacing the DMF solution, the stirring is continuously carried out for 24 hours, after the reaction is finished, the methanol is removed, and the NH is obtained after vacuum drying is carried out at 70 ℃ overnight2-MI L-53 (Al) nanoplates.
Step 2, preparing a probe solution for detecting bacterial drug resistance:
50 μ L100 μmol of bis (2,2 '-bipyridyl) -4' -methyl-4-carboxybipyridyl-ruthenium (N-succinimidyl ester) -bis (hexafluorophosphate) solution was prepared in DMF solution, and added to a buffer solution (10mM Tris-HCl,1mM CaCl)2,1mMMnCl2pH 7.4) to 1.5m L10 μmol hemistaphyloccludin (Con a) solution, and slowly stirring at 25 ℃ under dark conditions for 6 h.
Step 3, assembling an electrochemical luminescence sensor for detecting bacterial drug resistance;
step 3.1, the NH synthesized in step 12Ultrasonically dispersing an-MI L-53 (Al) nano sheet in a DMF (dimethyl formamide) solution, dripping the dispersed solution on the surface of a cleanly treated working electrode, and drying to obtain NH2-MI L-53 (Al)/GCE modified electrode;
step 3.2, adding EDC and NHS into the probe solution obtained in the step 2 for activation, and immersing the modified electrode obtained in the step 3.1 into the probe solution for modification to obtain Con A-Ru/NH2-MI L-53 (Al)/GCE modified electrode;
step 3.3, Bovine Serum Albumin (BSA) is dissolved in the binding buffer solution and dripped into the Con A-Ru/NH obtained in step 3.22And (4) sealing the surface of the-MI L-53 (Al)/GCE modified electrode to obtain the electrochemiluminescence biosensor for detecting the bacterial drug resistance.
The use method of the electrochemical luminescence biosensor for detecting the drug resistance of bacteria specifically comprises the following steps:
(1) constructing a three-electrode system by using the electrochemical luminescence biosensor for detecting the drug resistance of bacteria as a working electrode, an Ag/AgCl electrode as a reference electrode (saturated KCl) and a platinum wire electrode as a counter electrode;
(2) in the electrochemiluminescence test solution, the electrochemiluminescence intensity I is tested0The electrochemical method adopted is as follows: cyclic voltammetry; scanning range: 0.2V-1.35V; scanning rate: 0.1 V.S-1；
(3) Preparing a bacteria solution to be detected and the bacteria solution to be detected after the bacteria solution to be detected and the antibiotics act.
Step 1, preparing an antibacterial drug stock solution;
the antibacterial drug stock solution is prepared by sterilized distilled water, the concentration of the antibacterial drug stock solution is not less than 1000 mu g/m L, and the prepared antibacterial drug stock solution is stored at-60 ℃.
Step 2, preparing L uria-Bertani (L B) culture medium;
dissolving 10g tryptone, 5g yeast extract and 10g NaCl in deionized water, stirring until solute is dissolved, adjusting pH to 7.0 with 5 mol/L NaOH, diluting to 1L with deionized water, and sterilizing at 121 deg.C for 20 min.
Step 3, preparing a bacterial solution to be detected;
step 3.1, inoculating 10 mu L escherichia coli (E.coli B L21, without drug resistance) bacterial suspension into 10m L L B culture medium, carrying out overnight shaking culture under the conditions of 37 ℃ and 180rmp, then taking 100 mu L of the cultured bacterial solution to transfer into 10m L L B culture medium, continuing to culture until the OD value is 0.6, centrifuging the cultured bacteria under the conditions of 4000rmp and 4 ℃ for 10min, then carrying out heavy suspension by using a combined buffer solution, repeating for three times, and diluting the bacteria into different concentrations by using the combined buffer solution, thus obtaining the E.coli B L21 solution to be detected without drug resistance.
Step 3.2, inoculating 10 mu L Escherichia coli (NDM-1E. coli B β 121 with drug resistance) capable of synthesizing β -lactamase into 10m L L B culture medium containing 25 mu g/L kanamycin, shaking and culturing overnight at 37 ℃ and 180rmp, taking 100 mu L of the cultured bacterial liquid, transferring the bacterial liquid into 10m L culture medium containing 25 mu g/L kanamycin L B, culturing until the OD value is 0.6, adding 100m mu m isopropyl- β 0-D-thiogalactoside (IPTG) for induction, continuing culturing for 2h, centrifuging the cultured bacteria at 4000rmp and 4 ℃ for 10min, then re-suspending with a combined buffer solution, repeating for three times, and finally diluting with the combined buffer solution to different concentrations to obtain the NDM-1E. coli B L21 solution to be tested with drug resistance.
Step 4, preparing a solution to be tested for E.coli B L21/NDM-1 E.coli B L21 after interaction with antibiotics;
diluting the bacterial solution to be tested prepared in the step 3 by using a binding buffer solution until the OD value is 0.2, then respectively adding 2mg/m L of antibiotic solution, carrying out shake incubation for 3h at 37 ℃ and 180rmp, and diluting to obtain the bacterial solution to be tested after the antibiotic action.
(4) Immersing a working electrode in a 50 mu L E.coli B L21 test solution with known concentration, washing with a buffer solution after 60min to remove adsorbed substances, testing in an electrochemiluminescence test solution to obtain electrochemiluminescence intensity corresponding to the E.coli B L21 concentration, repeating the steps to obtain a plurality of groups of electrochemiluminescence intensity data corresponding to different E.coli B L21 concentrations, wherein the E.coli B L21 concentration range is 5 × 10-5 × 107cells/m L, and testing according to the sequence of E.coli B L21 concentration from small to large;
(5) fitting according to the multiple groups of electrochemiluminescence intensity data corresponding to different E.coli B L21 concentrations obtained in the step (4) to obtain a fitting curve between the bacterial concentration and the electrochemiluminescence intensity;
(6) immersing the working electrode in 50 mu L E.coli B L21/NDM-1 E.coli B L21 solution to be tested and the solution to be tested after the interaction with various antibiotics for 60min, washing with buffer solution (pH 8.0) to remove adsorbed substances, testing the electrochemical luminescence intensity I in the electrochemical luminescence test solution, and finally, testing the signal change rate delta I before and after the bacteria solution to be tested (delta I is I)0-I/I0,I0Electrochemiluminescence intensity before detecting bacteria, I is electrochemiluminescence intensity after detecting bacteria) to judge whether the bacteria is NDM-1E. coli B L21 with drug resistance.
The electrochemical luminescence test solution contains 50mMTripropylamine (TPA) buffer solution (10mM Tris-HCl,1mM CaCl)2,1mM MnCl2，pH＝7.4)。
The electrochemiluminescence biosensor can detect β -lactam antibiotic-resistant bacteria, the steps are simple during testing, after the electrochemiluminescence biosensor is assembled, the electrochemiluminescence biosensor can be combined with a to-be-detected escherichia coli solution for 60min to be detected for one-step detection, and the fast detection of the drug resistance of the bacteria is facilitated.
Example 1
A preparation method of an electrochemiluminescence biosensor for detecting bacterial drug resistance takes the example of detecting whether bacteria express M βL s gene, the selected bacteria are E.coli B L21/NDM-1 E.coli, and the specific preparation steps are as follows:
step 1, preparation of NH2-MI L-53 (Al) nanoplates;
mixing AlCl3·6H2O (3mmol,0.7243g) was dissolved in 15m L g of deionized water, and 2-amino-1, 4-benzenedicarboxylic acid (NH) was slowly added with stirring2-H2BDC,3mmol and 0.5435g), stirring for 30 minutes, then dropwise adding a 15m L of urea (6mmol and 0.3604g) aqueous solution into the mixed solution, stirring for 30 minutes, transferring the mixture into a 50m L polytetrafluoroethylene high-pressure reaction kettle, standing for reaction for 5 hours at 150 ℃, slowly cooling the mixture to room temperature after the reaction is finished, sucking and filtering to obtain milk white yellowish precipitate, washing with a large amount of deionized water, dispersing the product into a 20m L N, N-Dimethylformamide (DMF) solution, stirring for 24 hours at room temperature, replacing the DMF solution with methanol of the same volume, stirring for 24 hours, removing the methanol after the reaction is finished, and drying in vacuum at 70 ℃ for overnight to obtain NH2-MI L-53 (Al) nanoplates.
Step 2, preparing a probe solution for detecting bacterial drug resistance:
50 μ L100 μmol of bis (2,2 '-bipyridyl) -4' -methyl-4-carboxybipyridyl-ruthenium (N-succinimidyl ester) -bis (hexafluorophosphate) solution was prepared in DMF solution, and added to a buffer solution (10mM Tris-HCl,1mM CaCl)2,1mMMnCl2pH 7.4) of 1.5m L10 μmol half cutter ballThe protein (Con A) solution was stirred slowly at 25 ℃ in the dark for 6 h. After the reaction is finished, a probe solution (Con A-Ru) for detecting the drug resistance of the bacteria can be obtained.
Step 3, assembling an electrochemical luminescence sensor for detecting bacterial drug resistance;
step 3.1, the NH synthesized in step 12Ultrasonically dispersing an-MI L-53 (Al) nanosheet in a DMF solution to obtain a final concentration of 0.1mg/m L, dripping 10 mu L of the dispersion onto the surface of a cleanly treated working electrode, and drying to obtain NH2-MI L-53 (Al)/GCE modified electrode;
step 3.2, adding 2 mg/L EDC and 5 mg/L NHS into the probe solution obtained in the step 2 for activation, immersing the modified electrode obtained in the step 3.1 into the activated probe solution with the thickness of 30 mu L0.1.1 mu M for modification for 2h, and washing with a buffer solution to obtain Con A-Ru/NH2-MI L-53 (Al)/GCE modified electrode;
step 3.3, Bovine Serum Albumin (BSA) is dissolved in the binding buffer solution, the concentration is 0.1%, 10 mu L is dripped into the Con A-Ru/NH obtained in the step 3.22And sealing the surface of the-MI L-53 (Al)/GCE modified electrode for 30min, and washing with a buffer solution to obtain the electrochemiluminescence biosensor for detecting the bacterial drug resistance.
Example 2
A preparation method of an electrochemiluminescence biosensor for detecting bacterial drug resistance takes the detection of whether bacteria express M βL s gene as an example, the selected bacteria are E.coli B L21/NDM-1 E.coli, and the specific preparation steps are as follows:
step 1, preparation of NH2-MI L-53 (Al) nanoplates;
mixing AlCl3·6H2O (3mmol,0.7243g) was dissolved in 15m L g of deionized water, and 2-amino-1, 4-benzenedicarboxylic acid (NH) was slowly added with stirring2-H2BDC,3mmol,0.5435g) and stirring for a further 30 minutes, then, 15m of L g of aqueous urea (6mmol,0.3604g) was added dropwise to the above mixture and stirring was continued for a further 30 minutes, after which the above mixture was transferred to a 50m L teflon autoclave and allowed to stand at 150 ℃ for 5 hours, after completion of the reaction, the mixture was stirredSlowly cooling to room temperature, sucking and filtering to obtain milk white yellowish precipitate, washing with a large amount of deionized water, dispersing the product in 20m L N, N-Dimethylformamide (DMF) solution, stirring at room temperature for 24h, finally, replacing the DMF solution with methanol with the same volume, continuing stirring for 24h, removing the methanol after the reaction is finished, and vacuum-drying at 70 ℃ overnight to obtain NH2-MI L-53 (Al) nanoplates.
Step 2, preparing a probe solution for detecting bacterial drug resistance:
50 μ L100 μmol of bis (2,2 '-bipyridyl) -4' -methyl-4-carboxybipyridyl-ruthenium (N-succinimidyl ester) -bis (hexafluorophosphate) solution was prepared in DMF solution, and added to a buffer solution (10mM Tris-HCl,1mM CaCl)2,1mMMnCl2) The prepared 1.5m L10 mu mol hemistaphyloccludin (Con A) solution is slowly stirred for 6h at 25 ℃ in the dark, and a probe solution (Con A-Ru) for detecting the drug resistance of bacteria can be obtained after the reaction is finished.
Step 3, assembling the electrochemical luminescence sensor;
step 3.1, the NH synthesized in step 12Ultrasonically dispersing an-MI L-53 (Al) nanosheet in a DMF solution to obtain a final concentration of 0.5mg/m L, dripping 10 mu L of the dispersion onto the surface of a cleanly treated working electrode, and drying to obtain NH2-MI L-53 (Al)/GCE modified electrode;
step 3.2, adding 2 mg/L EDC and 5 mg/L NHS as activators into the probe solution obtained in the step 2, immersing the modified electrode obtained in the step 3.1 into the activated probe solution with the particle size of 30 mu L7 mu M for modification for 3h, and washing with a buffer solution to obtain Con A-Ru/NH2-MI L-53 (Al)/GCE modified electrode;
step 3.3, Bovine Serum Albumin (BSA) is dissolved in the binding buffer solution, the concentration is 0.5%, 10 mu L is dripped into the Con A-Ru/NH obtained in the step 3.22And sealing the surface of the-MI L-53 (Al)/GCE modified electrode for 40min, and washing with a buffer solution to obtain the electrochemiluminescence biosensor for detecting the bacterial drug resistance.
Example 3
A preparation method of an electrochemiluminescence biosensor for detecting bacterial drug resistance takes the example of detecting whether bacteria express M βL s gene, the selected bacteria are E.coli B L21/NDM-1 E.coli, and the specific preparation steps are as follows:
step 1, preparation of NH2-MI L-53 (Al) nanoplates;
mixing AlCl3·6H2O (3mmol,0.7243g) was dissolved in 15m L g of deionized water, and 2-amino-1, 4-benzenedicarboxylic acid (NH) was slowly added with stirring2-H2BDC,3mmol and 0.5435g), stirring for 30 minutes, then dropwise adding a 15m L of urea (6mmol and 0.3604g) aqueous solution into the mixed solution, stirring for 30 minutes, transferring the mixture into a 50m L polytetrafluoroethylene high-pressure reaction kettle, standing for reaction for 5 hours at 150 ℃, slowly cooling the mixture to room temperature after the reaction is finished, sucking and filtering to obtain milk white yellowish precipitate, washing with a large amount of deionized water, dispersing the product into a 20m L N, N-Dimethylformamide (DMF) solution, stirring for 24 hours at room temperature, replacing the DMF solution with methanol of the same volume, stirring for 24 hours, removing the methanol after the reaction is finished, and drying in vacuum at 70 ℃ for overnight to obtain NH2-MI L-53 (Al) nanoplates.
Step 2, preparing a probe solution for detecting bacterial drug resistance:
50 μ L100 μmol of bis (2,2 '-bipyridyl) -4' -methyl-4-carboxybipyridyl-ruthenium (N-succinimidyl ester) -bis (hexafluorophosphate) solution was prepared in DMF solution, and added to a buffer solution (10mM Tris-HCl,1mM CaCl)2,1mMMnCl2) The prepared 1.5m L10 mu mol hemistaphyloccludin (Con A) solution is slowly stirred for 6h at 25 ℃ in the dark, and a probe solution (Con A-Ru) for detecting the drug resistance of bacteria can be obtained after the reaction is finished.
Step 3, assembling the electrochemical luminescence sensor;
step 3.1, the NH synthesized in step 12Ultrasonically dispersing an-MI L-53 (Al) nanosheet in a DMF solution to obtain a final concentration of 1mg/m L, dripping 10 mu L of the dispersion liquid onto the surface of a cleanly treated working electrode, and drying to obtain NH2-MI L-53 (Al)/GCE modified electrode;
step 3.2 inAdding 2 mg/L EDC and 5 mg/L NHS as activators into the probe solution obtained in the step 2, immersing the modified electrode obtained in the step 3.1 into the probe solution activated by 30 mu L10 mu M for modification for 4h, and washing the modified electrode with a buffer solution to obtain Con A-Ru/NH2-MI L-53 (Al)/GCE modified electrode;
step 3.3, Bovine Serum Albumin (BSA) is dissolved in the binding buffer solution, the concentration is 1 percent, 10 mu L is dripped into the Con A-Ru/NH obtained in the step 3.22And sealing the surface of the-MI L-53 (Al)/GCE modified electrode for 60min, and washing with a buffer solution to obtain the electrochemiluminescence biosensor for detecting the bacterial drug resistance.
Examples of the applications
Example 1 detection of bacterial concentration
The use method of the electrochemical luminescence biosensor for detecting the bacterial concentration is to detect E.coli B L21 as the following steps:
(1) constructing a three-electrode system by using the electrochemical luminescence biosensor for detecting bacterial drug resistance prepared in the preparation example 1 as a working electrode, an Ag/AgCl electrode as a reference electrode and a platinum wire electrode as a counter electrode;
(2) in the electrochemiluminescence test solution, the electrochemiluminescence intensity I is tested0The electrochemical method adopted is as follows: cyclic voltammetry; scanning range: 0.2V-1.35V; scanning rate: 0.1 V.S-1(ii) a The electrochemiluminescence test solution is a buffer solution (10mM Tris-HCl,1mM CaCl) containing 50mM Tripropylamine (TPA)2,1mM MnCl2)。
(3) Preparation of a solution of bacteria to be tested
Step 1, preparing L uria-Bertani (L B) culture medium;
dissolving 10g tryptone, 5g yeast extract and 10g NaCl in deionized water, stirring until solute is dissolved, adjusting pH to 7.0 with 5 mol/L NaOH, diluting to 1L with deionized water, and sterilizing at 121 deg.C for 20 min.
Step 3, preparing a bacterial solution to be tested for E.coli B L21;
step 3.1, 10. mu. L E.coli (E.coli B L21, non-resistant) suspension was inoculated into 10m L L B medium at 37 ℃ and 180rCulturing under mp overnight shaking, transferring 100 μ L of cultured bacterial liquid into 10m L L B culture medium, and culturing to OD600The value is 0.6, the bacteria obtained by culture are centrifuged for 10min at 4000rmp and 4 ℃, then the bacteria are re-suspended by the combined buffer solution, the operation is repeated for three times, and the bacteria are diluted into different concentrations by the combined buffer solution, so that the solution to be tested of E.coli B L21 can be obtained.
(4) The working electrode prepared in example 1 was then immersed in the above solution (3) in the order of 50. mu. L concentration of 5 × 10cells/m and 5 × 102cells/mL、5×103cells/mL、5×104cells/mL、5×105cells/mL、5×106cells/mL、5×107In a solution to be tested of e.coli B L21 of cells/m L, after 60min, the solution is washed with a buffer solution (pH 8.0), and in an electrochemiluminescence test solution, the electrochemiluminescence intensity I (corresponding to e.coli B L21 of different concentrations, as shown in fig. 2 a) is tested at different concentrations.
(5) According to the test results, the electrochemiluminescence intensity I is found to be 5 × 10 cells/m-5 × 10 at the concentration of E.coli B L214Linear relationship I ═ 454.89lg [ e.coli B L21 in the cells/m L range]+2204.9, R2 ═ 0.9798 (as shown in fig. 2 b).
(5) The working electrode prepared in example 1 was immersed in 50 μ L test solution containing e.coli B L21, washed with buffer solution (pH 7.4) after 60min, and the electrochemiluminescence intensity I was measured in the electrochemiluminescence test solution, and the concentration C of escherichia coli was obtained from the linear relationship between the electrochemiluminescence intensity and the concentration of escherichia coliE.coliThe unit is cell/m L.
From example 1, it can be seen that the electrochemiluminescence intensity value and the E.coli concentration are 5 × 10 cells/m-5 × 104The concentration range of cells/m L is linear.
Example 2 detection of bacterial resistance
An application method of an electrochemiluminescence biosensor for detecting bacterial drug resistance, taking the example of detecting whether bacteria express M βL s gene, the selected bacteria is E.coli B L21/NDM-1 E.coli B L21, and comprises the following steps:
(1) constructing a three-electrode system by using the electrochemical luminescence biosensor for detecting bacterial drug resistance prepared in the preparation example 1 as a working electrode, an Ag/AgCl electrode as a reference electrode and a platinum wire electrode as a counter electrode;
(2) in the electrochemiluminescence test solution, the electrochemiluminescence intensity I is tested0The electrochemical method adopted is as follows: cyclic voltammetry; scanning range: 0.2V-1.35V; scanning rate: 0.1 V.S-1(ii) a The electrochemiluminescence test solution is a buffer solution (10mM Tris-HCl,1mM CaCl) containing 50mM Tripropylamine (TPA)2,1mM MnCl2)。
Step 1, preparing an antibacterial drug stock solution;
the antibacterial drug stock solution is prepared by sterilized distilled water, the concentration of the antibacterial drug stock solution is not less than 1000 mu g/m L, and the prepared antibacterial drug stock solution is stored at-60 ℃.
Step 2, preparing L uria-Bertani (L B) culture medium;
dissolving 10g tryptone, 5g yeast extract and 10g NaCl in deionized water, stirring until solute is dissolved, adjusting pH to 7.0 with 5 mol/L NaOH, diluting to 1L with deionized water, and sterilizing at 121 deg.C for 20 min.
Step 3, preparing a bacterial solution to be detected;
step 3.1, inoculating 10 mu L Escherichia coli (E.coli B L21, without drug resistance) bacterial suspension into 10m L L B culture medium, carrying out overnight shaking culture at 37 ℃ and 180rmp, taking 100 mu L of the cultured bacterial solution, transferring the bacterial solution into 10m L L B culture medium, continuing to culture until the OD value is 0.6, centrifuging the cultured bacteria at 4000rmp and 4 ℃ for 10min, then re-suspending with binding buffer solution, repeating for three times, diluting with binding buffer solution until the OD value is OD600At 0.2, further dilution 104And (5) doubling to obtain the solution to be tested of E.coli B L21.
Step 3.2, 10 mu L Escherichia coli (NDM-1E. coli B L21, with drug resistance) suspension capable of synthesizing β -lactamase is inoculated into 10m L L B culture medium containing 25 mu g/L kanamycin, and is subjected to overnight shaking culture at 37 ℃ and 180rmp, then 100 mu L of the cultured bacterial liquid is taken out and is transferred into 10m L culture medium containing 25 mu g/L kanamycin L B, the culture is carried out until OD value is 0.6, and then 100m mu m isopropyl- β 0 is added-D-thiogalactoside (IPTG) induction and further culture for 2 h. The cultured bacteria were centrifuged at 4000rmp at 4 ℃ for 10min and resuspended in binding buffer three times. Finally, the mixture was diluted to OD with binding buffer600At 0.2, further dilution 104Multiplying to obtain the solution to be tested of NDM-1E. coli B L21.
Step 4, preparing a solution to be tested for E.coli B L21/NDM-1 E.coli B L21 after interaction with antibiotics;
diluting the bacterial solution to be tested prepared in the step 3 with a binding buffer solution until the OD value is 0.2, then respectively adding 2mg/m L tetracycline, levofloxacin, cefpirome and imipenem solutions, shaking and incubating for 3h at 37 ℃ and 180rmp, and then diluting with the binding buffer solution for 104And (4) multiplying to obtain the E.coli B L21/NDM-1 E.coli B L21 to-be-detected solution after the interaction with the antibiotics.
(3) The working electrode prepared in example 1 was immersed in 50 μ L of e.coli B L21/NDM-1 e.coli B L21 prepared in (3) above and a test solution after the treatment with antibiotics, respectively, and after 60min, the working electrode was washed with a binding buffer solution (pH 7.4), and the electrochemiluminescence intensity I was measured at different concentrations in an electrochemiluminescence test solution.
(4) Respectively calculating delta I/I according to the test results0The results are shown in FIG. 3.
From this example 2, it can be seen that if the test bacterium does not have drug resistance (E.coli B L21), after interaction with any of the four antibiotics, Δ I/I is compared to that without the antibiotic0Obviously reducing the difference, if the bacteria to be detected have drug resistance (NDM-1E. coli B L21), if NDM-1E. coli B L21 interacts with antibiotics (tetracycline, levofloxacin) of non- β -lactam, β -lactamase expressed by the bacteria can not hydrolyze the antibiotics, and compared with the delta I/I of the antibiotics which do not act, the delta I/I of the antibiotics can not be hydrolyzed by the antibiotics0If NDM-1E. coli B L21 interacts with β -lactam antibiotics (cefpirome, imipenem), the antibiotics are hydrolyzed by β -lactamase expressed by bacteria and are ineffective, compared with delta I/I without the antibiotics0No obvious change. Therefore, Δ I/I can be determined from the analysis0To determine whether the bacteria haveHas drug resistance.
Example 3 stability test
The electrochemiluminescence biosensor for detecting bacterial drug resistance prepared in the preparation example 1 is used as a working electrode, the experimental conditions are the same as those of the above example 1, and the EC L biosensor and the EC 5 × 10 biosensor are the same as those of the example 13After cell/m L E. coli B L interaction, 10 circles of EC L response signals are continuously scanned at 0.2V-1.35V, and the result is shown in FIG. 4. the EC L signal intensity of the EC L biosensor is continuously scanned for 10 circles of relative standard deviation of 2.8%.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by using the contents of the present specification and the accompanying drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.

Claims (7)

1. A preparation method of an electrochemiluminescence biosensor for detecting bacterial drug resistance is characterized by comprising the following steps:
step 1: preparation of NH2-MI L-53 (Al) nanoplates;
step 1.1: 0.7243g of 3mmol AlCl3·6H2Dissolving O in 15m L deionized water;
step 1.2: 0.5435g of 3mmol 2-amino-1, 4-phthalic acid is slowly added under the stirring condition, and the stirring is continued for 30 minutes to obtain a mixed solution 1;
step 1.3, dropwise adding 15m of L6 mmol of urea aqueous solution into the mixed solution 1, and continuously stirring for 30 minutes to obtain a mixed solution 2;
step 1.4, transferring the mixed solution 2 into a 50m L polytetrafluoroethylene high-pressure reaction kettle, and standing and reacting for 5 hours at the temperature of 150 ℃ to obtain a mixed solution 3;
step 1.5: slowly cooling the mixed solution 3 to room temperature, sucking and filtering to obtain milk white yellowish precipitate, and washing with a large amount of deionized water to obtain a mixed solution 4;
step 1.6, dispersing the mixed solution 4 in 20m L N, N-dimethylformamide solution, and stirring for 24 hours at room temperature;
step 1.7: replacing N, N-dimethylformamide solution with methanol of the same volume, continuing stirring for 24 hours, removing methanol after the reaction is finished, and vacuum drying at 70 ℃ overnight to obtain NH2-MI L-53 (Al) nanoplates;
step 2, preparing a probe solution for detecting bacterial drug resistance:
preparing 50 mu L100 mu mol of bis (2,2 '-bipyridyl) -4' -methyl-4-carboxybipyridyl-ruthenium (N-succinimidyl ester) -bis (hexafluorophosphate) solution by using N, N-dimethylformamide solution, adding the solution into 1.5m L10 mu mol of hemilactoglobulin (Con A) solution prepared by buffer solution, and slowly stirring for 6 hours at 25 ℃ in the dark;
step 3, assembling an electrochemical luminescence sensor for detecting bacterial drug resistance;
step 3.1, the NH synthesized in step 12Ultrasonically dispersing an-MI L-53 (Al) nano sheet in a DMF (dimethyl formamide) solution, dripping the dispersed solution on the surface of a cleanly treated working electrode, and drying to obtain NH2-MI L-53 (Al)/GCE modified electrode;
step 3.2, adding EDC and NHS into the probe solution obtained in the step 2 for activation, and immersing the modified electrode obtained in the step 3.1 into the activated probe solution for modification to obtain Con A-Ru/NH2-MI L-53 (Al)/GCE modified electrode;
step 3.3, Bovine Serum Albumin (BSA) is dissolved in the binding buffer solution and dripped into the Con A-Ru/NH obtained in step 3.22And (4) sealing the surface of the-MI L-53 (Al)/GCE modified electrode to obtain the electrochemiluminescence biosensor for detecting the bacterial drug resistance.
2. The method according to claim 1, wherein in step 2, the buffer solution is: containing 1mM CaCl2,1mM MnCl210mM Tris-hydrochloric acid solution, buffer pH 7.4.
3. Root of herbaceous plantThe method of claim 2, wherein in step 3.1, the NH modified on the surface of the working electrode is used to prepare the electrochemiluminescence biosensor for detecting bacterial drug resistance2The dispersion concentration of the-MI L-53 (Al) nanosheets is 0.1-1mg/m L.
4. The method according to claim 3, wherein in step 3.1, the working electrode is one of a glassy carbon electrode, a graphite electrode, an ITO electrode and a noble metal electrode.
5. The method of claim 4, wherein in step 3.2, the concentrations of EDC and NHS are 2 mg/L and 5 mg/L, respectively, the concentration of the probe solution Con A-Ru is 0.1-10 μ M, and the modification time is 2-4 h.
6. The method of claim 5, wherein in step 3.3, the concentration of BSA solution is 0.1-1%, the volume dropped on the electrode surface is 10 μ L, and the blocking time is 30-60 min.
7. An electrochemiluminescence biosensor for detecting bacterial drug resistance, wherein the electrochemiluminescence biosensor is prepared by the preparation method of any one of claims 1 to 6.
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