CN111458516B - 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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CN111458516B
CN111458516B CN202010056277.1A CN202010056277A CN111458516B CN 111458516 B CN111458516 B CN 111458516B CN 202010056277 A CN202010056277 A CN 202010056277A CN 111458516 B CN111458516 B CN 111458516B
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马芬
刘家玮
陈玉
孙利娜
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Abstract

The invention discloses an electrochemiluminescence biosensor for detecting bacterial drug resistance and a preparation method thereof, comprising the step of preparing NH2Assembly of MIL-53(Al) nanoplates, preparation of a probe solution for detection of bacterial resistance and an electrochemiluminescence sensor for detection of bacterial resistance. The electrochemical luminescence biosensor provided by the invention can quickly, simply, reliably and effectively detect drug resistance. The invention uses the electrochemical luminescence technology as a signal output mode, the technology has extremely high sensitivity, and the electrochemical luminescence biosensor detects whether bacteria have drug resistance and can be used for quantitatively detecting the concentration of 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 drug resistance of pathogenic bacteria to antibiotics is continuously increased, and the public attention is gradually attracted by 'super bacteria' reported by media. New Delhi metallo-beta-lactamase-1 (NDM-1) is a drug resistance gene carried by superbacteria that neutralizes beta-lactam antibiotics, rendering the bacteria resistant to almost all antibiotics. The extremely strong drug resistance of the NDM-1 superbacteria greatly increases the treatment difficulty of infection caused by the superbacteria, common drug-resistant bacteria cannot transmit drug-resistant genes to other bacteria, and the NDM-1 genes can be transferred among different bacteria to transmit resistance. NDM-1 found at present is mainly present in Escherichia coli and Klebsiella pneumoniae, which are not pathogenic under normal conditions, but when the NDM-1 gene is obtained, the NDM-1 gene is shaken to become a super bacterium which is almost indescribable. In view of the above problems, there is an urgent need for a reliable and effective detection means for detecting whether bacteria have drug resistance.
Currently established means for detecting whether bacteria express NDM-1 resistance gene include broth dilution and disc diffusion, which involve multiple time-consuming steps including (1) culturing the bacteria to a detectable density (24-48h), (2) incubating the bacteria and antibiotics in 96-well plates or dishes (24-48h), and (3) detecting bacterial growth by UV absorption spectroscopy or visual inspection. In addition, these testing methods typically require the collection of large patient samples for analysis, such as blood, sputum, or urine. The use of molecular genetic techniques (e.g., polymerase chain reaction and gene chips) is common, but these are based on direct detection of beta-lactamase isolated from clinical samples. The direct detection method is a method for directly detecting hydrolysis of beta-lactam in a cell extract by using a spectrophotometric method, which is a time-consuming and labor-consuming task and thus cannot be used for conventional detection. Matrix assisted laser desorption ionization-time of flight (MALDI-TOF) Mass Spectrometry (MS) has been widely used in recent years for the study of antimicrobial drug resistance mechanisms, but relatively small antibiotics (1000 Da) are difficult to analyze because of their interaction with the matrix which produces higher noise levels. In addition, the price of the apparatus and the maintenance cost thereof are additional problems. Therefore, physicians often adopt empirical therapy in the face of unclear whether patients are infected with drug-resistant bacteria, resulting in abuse of antibiotics and increased bacterial resistance.
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-MIL-53(Al) nanoplates;
step 1.1: 0.7243g of 3mmol AlCl3·6H2Dissolving O in 15mL of 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 15mL of 6mmol 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 50mL polytetrafluoroethylene high-pressure reaction kettle, and standing and reacting for 5h at 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 20mL of N, N-dimethylformamide solution, and stirring at room temperature for 24 h;
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-MIL-53(Al) nanoplates;
step 2, preparing a probe solution for detecting bacterial drug resistance:
preparing 50 mu L of 100 mu mol of bis (2,2 '-bipyridyl) -4' -methyl-4-carboxyl bipyridyl-ruthenium (N-succinimidyl ester) -bis (hexafluorophosphate salt) solution by using an N, N-dimethylformamide solution, adding the solution into 1.5mL of 10 mu mol of hemiscissors globulin (Con A) solution prepared by using a buffer solution, and slowly stirring the solution 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-MIL-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-MIL-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-MIL-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 (3) sealing the surface of the-MIL-53 (Al)/GCE modified electrode to obtain the electrochemiluminescence biosensor for detecting the drug resistance of bacteria.
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-MIL-53 (Al) nano-sheets is 0.1-1 mg/mL.
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 2mg/L and 5mg/L, the concentration of the probe solution Con A-Ru is 0.1-10 mu M, and the modification time is 2-4 h.
In a further scheme, in the step 3.3, the concentration of the BSA solution is 0.1-1%, the volume of the BSA solution dropped on the surface of the electrode is 10 μ 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:
metal Organic Frameworks (MOFs), also known as porous Coordination Polymers (CPs), are an organic-inorganic hybrid material with intramolecular pores formed by self-assembly of organic ligands and metal ions or clusters through coordination bonds. The nano-porous material has a unique structure with large surface area and uniform nano-cavity, and excellent chemical properties such as molecular pore size, structure and good catalytic activity, and is a popular research material since the beginning of the century. Lectins are structurally diverse proteins that bind specifically to polysaccharides and exhibit high affinity, and their ease of production and labeling provide a good platform for designing biosensors. The electrochemical luminescence (ECL) biosensor has the advantages of both electrochemical and chemiluminescent biosensors, has the advantages of high sensitivity, easy design and the like, and has wide application prospect.
The invention constructs an electrochemiluminescence biosensor for detecting bacterial drug resistance based on the specific combination of lectin (Con A) and Lipopolysaccharide (LPS). First, with NH2The MIL-53(Al) nanosheet is used as a substrate material for modifying an electrode, a porous interface can be provided to increase the surface area of the electrode, and the amino groups on the nanosheet can react with the carboxyl groups on the Con A probe, so that the electrode can be used for immobilizing the probe. Lectin (Con a) as a specific recognition element recognizes Lipopolysaccharide (LPS) on the surface of escherichia coli (e.coli), and bipyridyl ruthenium (Con a-Ru) labeled above provides an electrochemiluminescence signal. When the sensor binds to bacteria, the electrochemiluminescence signal is reduced, indicating that the sensor has specific response to escherichia coli. When the sensor is used for detecting bacteria (E.coli BL21) without drug resistance, if E.coli BL21 in a solution to be detected does not act with antibiotics, the concentration of escherichia coli surviving in the solution is high, Con A protein on the surface of the sensor can be specifically combined with a large number of bacteria to inhibit the luminescence of a Ru compound, so that the change rate delta I/I of the electrochemical luminescence intensity before and after the detection of the bacteria is caused0Large (Δ I ═ I)0–I,I0And I are the electrochemiluminescence signals before and after the sensor detects the bacteria), respectively). Coli BL21 surviving in solution after action of various antibioticsThe concentration is reduced, the number of bacteria specifically bound with the surface of the sensor is less, and the luminescence inhibition degree of the Ru compound is lower, so that the change rate delta I/I of the electrochemical luminescence intensity before and after the detection of the bacteria is reduced0Is 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 large to small. When the sensor is used for detecting drug-resistant bacteria (NDM-1E. coli BL21), if NDM-1E. coli BL21 interacts with beta-lactam antibiotics (cefpirome, imipenem), the concentration of NDM-1E. coli BL21 in the solution cannot be changed because the beta-lactam antibiotics can be decomposed by beta-lactamase expressed by NDM-1E. coli BL21 and become ineffective, so that the signal change rate delta I/I of the sensor before and after detecting the bacteria is detected when the NDM-1E. coli BL21 is detected, no matter whether the bacteria interact with the beta-lactam antibiotics or not0There was no significant change. In addition, if NDM-1E. coli BL21 interacts with non-beta-lactam antibiotics (levofloxacin and tetracycline), and beta-lactamase expressed by bacteria in the solution to be tested cannot decompose the antibiotics, NDM-1E. coli BL21 can cause death of the antibiotics and reduce the concentration of the bacteria. So that the bacteria have no delta I/I effect on non-beta-lactam antibiotics0Larger, delta I/I after action with non-beta-lactam antibiotics0Is smaller. I.e. based on the detected Δ I/I0Coli BL21/NDM-1E. coli BL21 was distinguished to determine whether or not the bacteria had drug resistance (expressed M.beta.ls 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+The system has good stability, higher ECL quantum yield and biocompatibility, and Ru (bpy)3 2+The fixing on the electrode surface can not only reduce the usage amount of expensive reagents, but also enhance the ECL signal intensity and simplify the experimentThe 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 BL21, and fig. 2b shows the linear relationship between the luminescence intensity value and the concentration of e.coli BL 21;
FIG. 3 shows that the electrochemical luminescence biosensor detects the corresponding delta I/I of E.coli BL21/NDM-1E.coli BL21 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 BL 21.
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 example of whether bacteria express M beta Ls or not, and the selected bacteria are E.coli BL21/NDM-1E.coli, and the specific preparation steps are as follows:
step 1, preparation of NH2-MIL-53(Al) nanoplates;
mixing AlCl3·6H2O (3mmol,0.7243g) was dissolved in 15mL of deionized water, and 2-amino-1, 4-benzenedicarboxylic acid (3mmol,0.5435g) was slowly added with stirring, and stirring was continued for another 30 minutes. Subsequently, 15mL of an aqueous solution of urea (6mmol,0.3604g) was added dropwise to the above mixture and stirring was continued for 30 minutes. Thereafter, the mixture obtained above was transferred to a 50mL polytetrafluoroethylene autoclave and allowed to stand at 150 ℃ for 5 hours. After the reaction was complete, the mixture was slowly cooled to room temperatureWarm, suction filter to get milk white yellowish precipitate, and rinse with a large amount of deionized water. Then, the product was dispersed in 20mL of N, N-Dimethylformamide (DMF) solution and stirred at room temperature for 24 h. Finally, replacing DMF solution with methanol with the same volume, continuing stirring for 24 hours, removing methanol after the reaction is finished, and vacuum drying at 70 ℃ overnight to obtain NH2-MIL-53(Al) nanoplates.
Step 2, preparing a probe solution for detecting bacterial drug resistance:
50 μ L of 100 μmol bis (2,2 '-bipyridine) -4' -methyl-4-carboxybipyridine-ruthenium (N-succinimidyl ester) -bis (hexafluorophosphate) solution was prepared in DMF solution, and added to a buffer solution (10mM Tris-HCl,1mM CaCl)2,1mM MnCl2pH 7.4) to 10 μmol hemiglobin (Con a) solution at 25 ℃ under dark conditions for 6 h. After the reaction is finished, the 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-MIL-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-MIL-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-MIL-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 (3) sealing the surface of the-MIL-53 (Al)/GCE modified electrode to obtain the electrochemiluminescence biosensor for detecting the drug resistance of bacteria.
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;
and (3) preparing an antibacterial medicament stock solution with sterilized distilled water, wherein the concentration of the antibacterial medicament stock solution is not less than 1000 mu g/mL. The prepared antibacterial drug stock solution is stored at-60 ℃.
Step 2, preparing a Luria-Bertani (LB) 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 5mol/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, 10. mu.L of Escherichia coli (E.coli BL21, without drug resistance) suspension was inoculated into 10mL of LB medium and shake-cultured overnight at 37 ℃ under 180 rmp. Then 100. mu.L of the cultured bacterial liquid is taken and transferred into 10mL LB culture medium, and the culture is continued until the OD value is 0.6. The cultured bacteria were centrifuged at 4000rmp at 4 ℃ for 10min and resuspended in binding buffer solution three times. And diluting the mixture into different concentrations by using a combined buffer solution to obtain the solution to be tested of E.coli BL21 without drug resistance.
Step 3.2, 10 u L can synthesize beta lactamase Escherichia coli (NDM-1E. coli BL21, with drug resistance) bacterial suspension inoculated into 10mL containing 25 u g/L kanamycin LB medium, at 37 degrees C, 180rmp conditions overnight shake culture. Then, 100. mu.L of the cultured bacterial liquid is transferred into 10mL LB culture medium containing 25. mu.g/L kanamycin, the culture is carried out until the OD value is 0.6, then 100 μm mu m isopropyl-beta-D-thiogalactoside (IPTG) is added for induction, and the culture is continued for 2 h. The cultured bacteria were centrifuged at 4000rmp at 4 ℃ for 10min and resuspended in binding buffer three times. And finally, diluting the bacterial solution into different concentrations by using a combined buffer solution to obtain the NDM-1E.
Step 4, preparing a solution to be tested for E.coli BL21/NDM-1E.coli BL21 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/mL antibiotic solutions, 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 the working electrode into 50 mu L of E.coli BL21 test solution with known concentration, washing with buffer solution to remove adsorbed substances after 60min, and testing in the electrochemiluminescence test solution to obtain the electrochemiluminescence intensity corresponding to the E.coli BL21 concentration; repeating the steps to obtain a plurality of groups of electrochemiluminescence intensity data corresponding to different E.coli BL21 concentrations; concentration range of E.coli BL21 is 5 × 10-5 × 107cells/mL, and the concentration of E.coli BL21 is tested from small to large;
(5) fitting according to the multiple groups of electrochemiluminescence intensity data corresponding to different E.coli BL21 concentrations obtained in the step (4) to obtain a fitting curve between the bacterial concentration and the electrochemiluminescence intensity;
(6) the working electrode was immersed in 50 μ L of e.coli BL21/NDM-1e.coli BL21 test solution and test solution after interaction with various antibiotics for 60min, the adsorbed substances were removed by washing with a buffer solution (pH 8.0), and the electrochemiluminescence intensity I was measured in an electrochemiluminescence test solution. Finally, according to the signal change rate delta I before and after detecting the bacterial solution to be detected (delta I ═ I)0-I/I0,I0The electrochemiluminescence intensity before detecting the bacteria, and I is the electrochemiluminescence intensity after detecting the bacteria) to judge whether the bacteria is NDM-1E.
The electrochemiluminescence test solution is a buffer solution (10mM Tris-HCl,1mM CaCl) containing 50mM Tripropylamine (TPA)2,1mM MnCl2，pH＝7.4)。
The electrochemical luminescence biosensor can detect bacteria resistant to beta-lactam antibiotics, the steps are simple during testing, after the electrochemical luminescence biosensor is assembled, the bacteria can be detected in one step after being combined with escherichia coli solution to be detected for 60min, and the rapid 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 detection of whether bacteria express M beta Ls gene as an example, the selected bacteria are E.coli BL21/NDM-1E.coli, and the specific preparation steps are as follows:
step 1, preparation of NH2-MIL-53(Al) nanoplates;
mixing AlCl3·6H2O (3mmol,0.7243g) was dissolved in 15mL of deionized water, and 2-amino-1, 4-benzenedicarboxylic acid (NH) was slowly added with stirring2-H2BDC,3mmol,0.5435g) and stirring continued for an additional 30 minutes. Subsequently, 15mL of an aqueous solution of urea (6mmol,0.3604g) was added dropwise to the above mixture and stirring was continued for 30 minutes. Thereafter, the mixture obtained above was transferred to a 50mL polytetrafluoroethylene autoclave and allowed to stand at 150 ℃ for 5 hours. After the reaction was complete, the mixture was slowly cooled to room temperature, filtered with suction to give a milky yellowish precipitate, and rinsed with copious amounts of deionized water. Then, the product was dispersed in 20mL of N, N-Dimethylformamide (DMF) solution and stirred at room temperature for 24 h. Finally, replacing DMF solution with methanol with the same volume, continuing stirring for 24 hours, removing methanol after the reaction is finished, and vacuum drying at 70 ℃ overnight to obtain NH2-MIL-53(Al) nanoplates.
Step 2, preparing a probe solution for detecting bacterial drug resistance:
50 μ L of 100 μmol bis (2,2 '-bipyridine) -4' -methyl-4-carboxybipyridine-ruthenium (N-succinimidyl ester) -bis (hexafluorophosphate) solution was prepared in DMF solution, and added to a buffer solution (10mM Tris-HCl,1mM CaCl)2,1mM MnCl2pH 7.4) to 10 μmol hemiglobin (Con a) solution at 25 ℃ under dark conditions 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-MIL-53 (Al) nano sheet in a DMF solution to obtain a final concentration of 0.1mg/mL, dripping 10 mu L of the dispersion liquid on the surface of a cleanly treated working electrode, and drying to obtain NH2-MIL-53(Al)/GCE modified electrode;
step 3.2, adding 2mg/L EDC and 5mg/L NHS into the probe solution obtained in the step 2 for activation, immersing the modified electrode obtained in the step 3.1 into 30 mu L of the activated probe solution with 0.1 mu M for modification for 2h, and washing with a buffer solution to obtain Con A-Ru/NH2-MIL-53(Al)/GCE modified electrode;
step 3.3, Bovine Serum Albumin (BSA) is dissolved in the binding buffer solution with the concentration of 0.1 percent, 10 mu L of solution is dripped into the Con A-Ru/NH obtained in the step 3.22And sealing the surface of the-MIL-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 beta Ls gene as an example, the selected bacteria are E.coli BL21/NDM-1E.coli, and the specific preparation steps are as follows:
step 1, preparation of NH2-MIL-53(Al) nanoplates;
mixing AlCl3·6H2O (3mmol,0.7243g) was dissolved in 15mL of deionized water, and 2-amino-1, 4-benzenedicarboxylic acid (NH) was slowly added with stirring2-H2BDC,3mmol,0.5435g) and stirring continued for an additional 30 minutes. Subsequently, 15mL of an aqueous solution of urea (6mmol,0.3604g) was added dropwise to the above mixture and stirring was continued for 30 minutes. Thereafter, the mixture obtained above was transferred to a 50mL polytetrafluoroethylene autoclave and allowed to stand at 150 ℃ for 5 hours. After the reaction was complete, the mixture was slowly cooled to room temperature, filtered with suction to give a milky yellowish precipitate, and rinsed with copious amounts of deionized water. Then, the product was dispersed in 20mL of N, N-Dimethylformamide (DMF) solution and stirred at room temperature for 24 h. Finally, equal volume of methanol was used instead of DMF solutionStirring for 24 hours, removing methanol after the reaction is finished, and drying overnight in vacuum at 70 ℃ to obtain NH2-MIL-53(Al) nanoplates.
Step 2, preparing a probe solution for detecting bacterial drug resistance:
50 μ L of 100 μmol bis (2,2 '-bipyridine) -4' -methyl-4-carboxybipyridine-ruthenium (N-succinimidyl ester) -bis (hexafluorophosphate) solution was prepared in DMF solution, and added to a buffer solution (10mM Tris-HCl,1mM CaCl)2,1mM MnCl2) Prepared 1.5mL of 10. mu. mol hemistaphyloccludin (Con A) solution was stirred slowly at 25 ℃ for 6h in the absence of light. 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 the electrochemical luminescence sensor;
step 3.1, the NH synthesized in step 12Ultrasonically dispersing an-MIL-53 (Al) nano sheet in a DMF solution to obtain a final concentration of 0.5mg/mL, dripping 10 mu L of the dispersion liquid on the surface of a cleanly treated working electrode, and drying to obtain NH2-MIL-53(Al)/GCE modified electrode;
step 3.2, adding 2mg/L EDC and 5mg/L NHS as activators into the probe solution obtained in the step 2, immersing the modified electrode obtained in the step 3.1 into 30 mu L of the activated probe solution with 7 mu M for modification for 3h, and washing the modified electrode with a buffer solution to obtain Con A-Ru/NH2-MIL-53(Al)/GCE modified electrode;
step 3.3, Bovine Serum Albumin (BSA) is dissolved in the binding buffer solution with the concentration of 0.5 percent, 10 mu L of solution is dripped into the Con A-Ru/NH obtained in the step 3.22And sealing the surface of the-MIL-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 detection of whether bacteria express M beta Ls gene as an example, the selected bacteria are E.coli BL21/NDM-1E.coli, and the specific preparation steps are as follows:
step 1, preparation of NH2-MIL-53(Al) nanoplates;
mixing AlCl3·6H2O (3mmol,0.7243g) was dissolved in 15mL of deionized water, and 2-amino-1, 4-benzenedicarboxylic acid (NH) was slowly added with stirring2-H2BDC,3mmol,0.5435g) and stirring continued for an additional 30 minutes. Subsequently, 15mL of an aqueous solution of urea (6mmol,0.3604g) was added dropwise to the above mixture and stirring was continued for 30 minutes. Thereafter, the mixture obtained above was transferred to a 50mL polytetrafluoroethylene autoclave and allowed to stand at 150 ℃ for 5 hours. After the reaction was complete, the mixture was slowly cooled to room temperature, filtered with suction to give a milky yellowish precipitate, and rinsed with copious amounts of deionized water. Then, the product was dispersed in 20mL of N, N-Dimethylformamide (DMF) solution and stirred at room temperature for 24 h. Finally, replacing DMF solution with methanol with the same volume, continuing stirring for 24 hours, removing methanol after the reaction is finished, and vacuum drying at 70 ℃ overnight to obtain NH2-MIL-53(Al) nanoplates.
Step 2, preparing a probe solution for detecting bacterial drug resistance:
50 μ L of 100 μmol bis (2,2 '-bipyridine) -4' -methyl-4-carboxybipyridine-ruthenium (N-succinimidyl ester) -bis (hexafluorophosphate) solution was prepared in DMF solution, and added to a buffer solution (10mM Tris-HCl,1mM CaCl)2,1mM MnCl2) Prepared 1.5mL of 10. mu. mol hemistaphyloccludin (Con A) solution was stirred slowly at 25 ℃ for 6h in the absence of light. 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 the electrochemical luminescence sensor;
step 3.1, the NH synthesized in step 12Ultrasonically dispersing an-MIL-53 (Al) nano sheet in a DMF solution to obtain a final concentration of 1mg/mL, dripping 10 mu L of the dispersion liquid on the surface of a cleanly treated working electrode, and drying to obtain NH2-MIL-53(Al)/GCE modified electrode;
step 3.2, adding 2mg/L EDC and 5mg/L NHS as activators into the probe solution obtained in the step 2, immersing the modified electrode obtained in the step 3.1 into 30 mu L of 10 mu M activated probe solution for modification for 4h, and washing with buffer solution to obtain Con A-Ru/NH2-MIL-53(Al)/GCE modified electrode;
step 3.3, Bovine Serum Albumin (BSA) is dissolved in the binding buffer solution with the concentration of 1 percent, 10 mu L of solution is dripped into the Con A-Ru/NH obtained in the step 3.22And sealing the surface of the-MIL-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 BL21 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 a Luria-Bertani (LB) 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 5mol/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 BL 21;
step 3.1, 10. mu.L of Escherichia coli (E.coli BL21, without drug resistance) suspension was inoculated into 10mL of LB medium and shake-cultured overnight at 37 ℃ under 180 rmp. Then taking 100 mu L of the cultured bacterial liquid, transferring the bacterial liquid into 10mL LB culture medium, and continuously culturing until OD is reached600The value was 0.6. Centrifuging the cultured bacteria at 4000rmp and 4 deg.C for 10min, and resuspending with binding buffer solutionThis was repeated three times. And diluting the mixture into different concentrations by using a combined buffer solution to obtain the solution to be tested for E.coli BL 21.
(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 5X 10cells/m, respectively2cells/mL、5×103cells/mL、5×104cells/mL、5×105cells/mL、5×106cells/mL、5×107In a cell/mL solution to be tested of e.coli BL21, after 60min, the sample was washed with a buffer solution (pH 8.0), and in an electrochemiluminescence test solution, the electrochemiluminescence intensity I at different concentrations (the electrochemiluminescence signal corresponding to e.coli BL21 at different concentrations, as shown in fig. 2 a) was measured.
(5) According to the test results, the electrochemiluminescence intensity I is found to be 5 × 10cells/m to 5 × 10cells/m at the concentration of E.coli BL214Linear relationship I-454.89 lg [ e.coli BL21 in cells/mL 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 of a test solution containing e.coli BL21, washed with a buffer solution (pH 7.4) after 60min, and tested for electrochemiluminescence intensity I in an electrochemiluminescence test solution; obtaining the concentration value C of the Escherichia coli according to the linear relation between the electrochemiluminescence intensity and the concentration of the Escherichia coliE.coliThe unit is cell/mL.
From example 1, it can be seen that the electrochemiluminescence intensity value and the Escherichia coli concentration are 5X 10cells/m to 5X 104The concentration range of cells/mL 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 beta Ls gene, the selected bacteria is E.coli BL21/NDM-1E.coli BL21, 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 electrochemiluminescence testingIn liquid, measuring the electrochemiluminescence intensity I0The 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;
and (3) preparing an antibacterial medicament stock solution with sterilized distilled water, wherein the concentration of the antibacterial medicament stock solution is not less than 1000 mu g/mL. The prepared antibacterial drug stock solution is stored at-60 ℃.
Step 2, preparing a Luria-Bertani (LB) 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 5mol/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, 10. mu.L of Escherichia coli (E.coli BL21, without drug resistance) suspension was inoculated into 10mL of LB medium and shake-cultured overnight at 37 ℃ under 180 rmp. Then 100. mu.L of the cultured bacterial liquid is taken and transferred into 10mL LB culture medium, and the culture is continued until the OD value is 0.6. The cultured bacteria were centrifuged at 4000rmp at 4 ℃ for 10min and resuspended in binding buffer solution three times. Dilution to OD with binding buffer600At 0.2, further dilution 104And multiplying to obtain the solution to be tested of E.coli BL 21.
Step 3.2, 10 u L can synthesize beta lactamase Escherichia coli (NDM-1E. coli BL21, has drug resistance) bacterial suspension inoculated into 10mL containing 25 u g/L kanamycin LB medium, at 37 degrees C, 180rmp conditions overnight shake culture. Then, 100. mu.L of the cultured bacterial liquid is transferred into 10mL LB culture medium containing 25. mu.g/L kanamycin, the culture is carried out until the OD value is 0.6, then 100 μm mu m isopropyl-beta-D-thiogalactoside (IPTG) is added for induction, and the culture is continued 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 buffer600Is 0.2, and still furtherDilution 104And multiplying to obtain the solution to be tested of NDM-1E.
Step 4, preparing a solution to be tested for E.coli BL21/NDM-1E.coli BL21 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/mL tetracycline, levofloxacin, cefpirome and imipenem solutions, shaking and incubating for 3h at 37 ℃ and 180rmp, and diluting with the binding buffer solution for 104And (5) doubling to obtain the E.coli BL21/NDM-1E.coli BL21 test solution after interaction with the antibiotic.
(3) The working electrode prepared in example 1 was immersed in 50 μ L of each of e.coli BL21/NDM-1e.coli BL21 prepared in (3) above and a test solution after the treatment with antibiotics, washed with a binding buffer solution (pH 7.4) after 60min, and tested for the electrochemiluminescence intensity I 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 BL21), after interaction with any of the four antibiotics, Δ I/I is compared to that without the antibiotic0Is obviously reduced. If the bacteria to be detected have drug resistance (NDM-1E. coli BL21), if NDM-1E. coli BL21 interacts with the antibiotics of non-beta-lactam class (tetracycline, levofloxacin), the beta-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 hydrolyze the antibiotics0Is obviously reduced. Coli BL21 interacts with beta-lactam antibiotics (cefpirome, imipenem), which are then hydrolyzed by the bacterially expressed beta-lactamase to a degree that is less effective than the antibiotic, and a delta I/I0No obvious change. Therefore, Δ I/I can be determined from the analysis0To determine whether the bacteria have 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, and experimental conditions are adoptedAs in example 1 above, the ECL biosensor was used in combination with a 5X 10 biosensor3After cell/mL e.coli BL21 interaction, 10 consecutive scans of ECL response signals were performed at 0.2V-1.35V, and the results are shown in fig. 4. ECL signal intensity of ECL biosensor relative standard deviation of 2.8% for 10 consecutive scans. The result shows that the ECL biosensor prepared by the invention has good stability.
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 (5)

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-MIL-53(Al) nanoplates;
step 1.1: 0.7243g of 3mmol AlCl3·6H2Dissolving O in 15mL of 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 15mL of 6mmol 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 50mL polytetrafluoroethylene high-pressure reaction kettle, and standing and reacting for 5h at 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 20mL of N, N-dimethylformamide solution, and stirring at room temperature for 24 h;
step 1.7: replacing the N, N-dimethylformamide solution with methanol of the same volume, continuing stirring for 24 hours, removing the methanol after the reaction is finished,vacuum drying at 70 deg.C overnight to obtain NH2-MIL-53(Al) nanoplates;
step 2, preparing a probe solution for detecting bacterial drug resistance:
preparing 50 mu L of 100 mu mol of bis (2,2 '-bipyridyl) -4' -methyl-4-carboxyl bipyridyl-ruthenium (N-succinimidyl ester) -bis (hexafluorophosphate salt) solution by using an N, N-dimethylformamide solution, adding the solution into 1.5mL of 10 mu mol of hemiscissors globulin Con A solution prepared by using a buffer solution, and slowly stirring the solution for 6 hours at 25 ℃ in the dark; wherein the buffer solution contains 1mM CaCl2,1mM MnCl2The pH of the buffer solution is 7.4;
step 3, assembling an electrochemical luminescence sensor for detecting bacterial drug resistance;
step 3.1, the NH synthesized in step 12Ultrasonically dispersing an-MIL-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-MIL-53(Al)/GCE modified electrode; wherein NH modified to the surface of the working electrode2-MIL-53(Al) nanoplates at a dispersed concentration of 0.1-1 mg/mL;
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-MIL-53(Al)/GCE modified electrode;
step 3.3, bovine serum albumin BSA is dissolved in the binding buffer solution and is dripped into the Con A-Ru/NH obtained in the step 3.22And (3) sealing the surface of the-MIL-53 (Al)/GCE modified electrode to obtain the electrochemiluminescence biosensor for detecting the drug resistance of bacteria.
2. The method of claim 1, 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.
3. The method for preparing an electrochemiluminescence biosensor for detecting bacterial drug resistance as claimed in claim 2, wherein in step 3.2, the concentrations of EDC and NHS are 2mg/L and 5mg/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.
4. The method of claim 3, wherein in step 3.3, the concentration of BSA solution is 0.1-1%, the volume dropped on the surface of the electrode is 10 μ L, and the blocking time is 30-60 min.
5. 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 4.
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CN112649417B (en) * 2020-12-01 2021-10-29 西北大学 Electrochemical luminescence biosensor for detecting MMP-14 and preparation method thereof
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101907556A (en) * 2010-07-19 2010-12-08 湖南大学 Method for detecting the colon bacillus by combining magnetic nanoparticle enrichment with bi-color flow cytometry
EP2491048B1 (en) * 2009-10-23 2015-12-23 IFP Energies nouvelles Novel mil-53-al-n3 organic/inorganic hybrid solid provided with an azide function, and method for manufacturing same
CN106093021A (en) * 2016-06-03 2016-11-09 浙江省农业科学院 The escherichia coli visualization bio-sensing method of acidity regulation and control and agglutinin identification
CN108918856A (en) * 2018-07-31 2018-11-30 济南大学 A kind of preparation method and application of double MOFs material quenching type electrochemiluminescimmunosensor immunosensors
CN109908879A (en) * 2019-04-26 2019-06-21 江南大学 A method of detection tetracycline antibiotics

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7745001B2 (en) * 2004-03-23 2010-06-29 University Of New Orleans Research And Technology Foundation, Inc. Synthesis of nanoassemblies containing luminescent quantum dots and magnetic nanoparticles

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2491048B1 (en) * 2009-10-23 2015-12-23 IFP Energies nouvelles Novel mil-53-al-n3 organic/inorganic hybrid solid provided with an azide function, and method for manufacturing same
CN101907556A (en) * 2010-07-19 2010-12-08 湖南大学 Method for detecting the colon bacillus by combining magnetic nanoparticle enrichment with bi-color flow cytometry
CN106093021A (en) * 2016-06-03 2016-11-09 浙江省农业科学院 The escherichia coli visualization bio-sensing method of acidity regulation and control and agglutinin identification
CN108918856A (en) * 2018-07-31 2018-11-30 济南大学 A kind of preparation method and application of double MOFs material quenching type electrochemiluminescimmunosensor immunosensors
CN109908879A (en) * 2019-04-26 2019-06-21 江南大学 A method of detection tetracycline antibiotics

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Amino-Functionalized Metal-Organic Frameworks Nanoplates-Based Energy Transfer Probe for Highly Selective Fluorescence Detection of Free Chlorine;Ting Lu等;《Analytical Chemisty》;20160224;第88卷;第3414页 *
以凝集素为识别物的细菌/细胞电化学发光生物传感器的研究;杨海英;《中国博士学位论文电子期刊 工程科技Ⅰ辑》;20140315(第3期);正文第23-40页 *
基于MIL-53(Fe) 的微生物传感器在抗生素检测中的应用;陈立志 等;《化学试剂》;20200212;第42卷(第4期);第354页 *
金属-有机框架结构材料的制备及其在电化学生物传感中的应用;曹阳;《中国硕士学位论文电子期刊 工程科技Ⅰ辑》;20180115(第1期);B014-851 *

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