CN114428070A - Molecularly imprinted fluorescent nanoparticle for detecting gram-negative bacteria and preparation method thereof - Google Patents

Molecularly imprinted fluorescent nanoparticle for detecting gram-negative bacteria and preparation method thereof Download PDF

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CN114428070A
CN114428070A CN202111622929.4A CN202111622929A CN114428070A CN 114428070 A CN114428070 A CN 114428070A CN 202111622929 A CN202111622929 A CN 202111622929A CN 114428070 A CN114428070 A CN 114428070A
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molecularly imprinted
fluorescent
gram
negative bacteria
bacteria
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李前进
金�雨
王婷婷
王萌
王奋英
王龙凤
李建林
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Nanjing Normal University
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Nanjing Normal University
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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
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6402—Atomic fluorescence; Laser induced fluorescence

Abstract

The invention discloses a molecularly imprinted fluorescent nanoparticle for detecting gram-negative bacteria and a preparation method thereof, belongs to the field of analytical chemistry, and relates to the analytical detection of gram-negative bacteria and the synthesis of a molecularly imprinted polymer. The molecularly imprinted fluorescent nanoparticle can specifically identify gram-negative bacteria such as Escherichia coli O157: H7 and pseudomonas fluorescens, and can perform quantitative analysis on bacteria through fluorescence signal change. The synthesis method comprises the following steps: firstly, pre-assembling template bacterium liquid with a functional monomer and a fluorescent monomer, and then sequentially adding a cross-linking agent and an initiator to carry out polymerization reaction; and after the reaction is finished, removing the polymerized nano particles from the template to obtain the molecularly imprinted fluorescent nano particles of the bacteria. The method adopts a one-pot stirring mode to prepare the molecularly imprinted fluorescent nanoparticles of the gram-negative bacteria, is simple to operate, and can specifically and quickly identify the gram-negative bacteria.

Description

Molecularly imprinted fluorescent nanoparticle for detecting gram-negative bacteria and preparation method thereof
Technical Field
The invention belongs to the field of analytical chemistry, relates to detection and analysis of gram-negative bacteria and synthesis of a molecularly imprinted polymer, and particularly relates to a molecularly imprinted fluorescent nanoparticle for detecting gram-negative bacteria and a preparation method thereof.
Background
Gram-negative bacteria refer to bacteria that cannot be stained with crystal violet when bacteria identification is performed using a gram staining method, such as escherichia coli and pseudomonas fluorescens; and bacteria capable of being stained by crystal violet, are known as gram positive bacteria, such as staphylococcus aureus. The cell wall of gram-negative bacteria has a thin peptidoglycan layer, and a large amount of carbohydrate conjugates such as lipopolysaccharide and glycoprotein are distributed on the surface of the cell membrane, while the carbohydrate conjugates on the surface of gram-positive bacteria are less distributed. Gram-negative bacteria are a large group of pathogens that infect all eukaryotes from plants to mammals [ Current Opinion in Immunology,38(2016) 8-17], the main pathogenic capacity of which is lipopolysaccharide on the outer membrane, also known as endotoxin. After a human body is infected by gram-negative bacteria, endotoxin on the surface of the bacteria can react with an immune system to cause functional disorder, so that food-borne diseases are caused. Therefore, the development of a rapid and sensitive method for detecting gram-negative bacteria has important significance and value in the fields of clinical diagnosis and food safety. At present, gram-negative bacteria detection and analysis methods mainly comprise a colony counting method, a Polymerase Chain Reaction (PCR), enzyme-linked immunoassay (ELISA) and the like, and although the methods have high sensitivity and accuracy, the methods have the defects of complex operation, time and labor waste, high analysis cost and the like. The fluorescence detection analysis method based on the molecular imprinting fluorescent nanoparticles can overcome the defects of the method, is used for the specific rapid analysis of the target object, and has simple operation and low analysis cost [ RSC adv, 11(2021)7732-7737; Sci. Rep, 10 (2020) 9924 ].
A Molecular Imprinted Polymer (MIP) is a high molecular Polymer with special molecular recognition capability, and the formation principle of the selective recognition capability is as follows: firstly, forming a compound by a template molecule and a functional monomer through covalent bond or non-covalent interaction, and then adding a cross-linking agent and an initiator to obtain a molecular imprinting pre-polymerization solution; then, initiating a polymerization reaction to obtain a high molecular polymer containing template molecules; finally, the template molecule in the polymer is removed, so that an imprinting cavity with a shape complementary to that of the template molecule is left in the polymer, and a functional group of the functional monomer is reserved in the imprinting cavity, so that the imprinting cavity has selective recognition capability on the template molecule. Due to the simple preparation, low cost, high stability, long service life and large-scale production of the molecularly imprinted polymer, the molecularly imprinted polymer has shown important application value in the fields of separation science, biosensing, drug delivery, mimic enzyme catalysis and the like [ chem. Soc. Rev., 45 (2016) 2137-2211 ]. Especially, the molecular imprinting fluorescent nanoparticles not only show strong advantages in the aspect of fluorescence imaging analysis, but also show great potential and prospect in disease diagnosis and treatment [ chem. Rev., 120 (2020) 9554-389582; Angew. chem. int. Ed., 60 (2021) 3858-3869 ]. The method is characterized in that a molecularly imprinted polymer is designed and synthesized by taking microorganisms as a research target, and the used template molecules are mainly protein or peptide fragments, but the template molecules are difficult to purify and difficult to obtain high-purity template molecules, so that the research on the molecularly imprinted polymer is greatly limited; in addition, the molecularly imprinted polymers prepared by using these proteins or peptide fragments as template molecules have low selectivity and specificity, which results in insufficient related research of microbial molecularly imprinted polymers in the fields of food safety and medical diagnosis [ chem. Soc. Rev., 47 (2018) 5574-5587 ].
Disclosure of Invention
The method is characterized in that a molecularly imprinted polymer is designed and synthesized by taking microorganisms as research targets, and proteins, peptide fragments or protein compounds and the like on the surfaces of the microorganisms are commonly used as templates, but the preparation and purification of the template molecules are difficult; in addition, molecularly imprinted polymers prepared using biomolecules such as these proteins as templates have poor selectivity and specificity because the purified configuration of these molecules is different from the configuration of the molecules on the surface of microorganisms, resulting in poor specificity. In order to solve the problems, the invention provides a method for preparing the molecularly imprinted fluorescent nanoparticles of the gram-negative bacteria by directly using the gram-negative bacteria as a template, which can overcome the defect that biomolecules such as protein, peptide fragments or protein compounds purified from the surface of the microorganism are used as the template, and a phenylboronic acid silanization reagent is used as a functional monomer, can selectively react with sugar chains (such as lipopolysaccharide, which are abundantly present on the surface of the gram-negative bacteria) on the surface of the microorganism, and adopts a one-pot method at normal temperature based on the sol-gel polymerization reaction of the silanization reagent.
In order to solve the problems of the prior art, the invention adopts the technical scheme that:
a preparation method of molecularly imprinted fluorescent nanoparticles for detecting gram-negative bacteria comprises the following steps:
step one, the concentration is 106-108 Adding CFU/mL template bacteria into the solution, and then adding a functional monomer with the concentration of 4.7-9.4 mu mol/mL and a fluorescent monomer with the concentration of 0.1-0.4 mu mol/mL for preassembling to obtain a molecular imprinting pre-aggregation solution, wherein the template bacteria are gram-negative bacteria;
step two, sequentially adding a 1000 mM cross-linking agent with the concentration of 100 and an initiator with the mass concentration of 2-10% into the molecularly imprinted pre-polymerizing solution prepared in the step one, and stirring the polymerizing solution to generate molecularly imprinted fluorescent nanoparticles, wherein the nanoparticles are adsorbed on the surface of the template bacteria;
and step three, removing the template bacteria in the step two by using an elution solution to obtain the molecularly imprinted fluorescent nanoparticles capable of specifically identifying the target bacteria.
Preferably, in the first step, the template bacteria are Escherichia coli O157: H7 or Pseudomonas fluorescens, the solution is a mixed solution of phosphate buffer and ethanol, and the volume fraction of the ethanol is 5-15%.
Preferably, in the first step, the concentration of the functional monomer is 4.7 [ mu ] mol/mL, the concentration of the fluorescent monomer is 0.2 [ mu ] mol/mL, and the concentration of the template bacteria is 0.42 x 108CFU/mL, phosphate buffer and BThe volume fraction of ethanol in the alcohol mixture was 15%.
Preferably, in the second step, the concentration of the cross-linking agent is 849 mM, and the mass fraction of the initiator is 6%.
Preferably, in the second step, the cross-linking agent is tetraethoxysilane or tetramethoxysilane, the initiator is ammonia water, and the stirring speed is 700-1500 rpm.
Preferably, the functional monomer in the step one is a phenylboronic acid silanization reagent, and the functional monomer is obtained by one-step reaction of aldehyde phenylboronic acid and an amino silanization reagent in ethanol according to a molar ratio of 1: 1; the fluorescent monomer can be obtained by one-step reaction of fluorescent dye containing isocyanate or isothiocyanate and amino silanization reagent in ethanol according to the molar ratio of 1:1, and the obtained functional monomer and the fluorescent monomer do not need to be purified.
Preferably, the aldehyde phenylboronic acid is any one of 3-formyl phenylboronic acid, 2-formyl phenylboronic acid, 4-formyl phenylboronic acid, 2-fluoro-3-aldehyde phenylboronic acid, 4-fluoro-3-aldehyde phenylboronic acid, or 3-fluoro-4-aldehyde phenylboronic acid.
Preferably, the aminosilane reagent is 3-aminopropyl-triethoxysilane, 3-aminopropyl-trimethoxysilane, 3- (2-aminoethylamino) propyl-trimethoxysilane, or diethylenetriaminopropyl-trimethoxysilane.
Preferably, the fluorescent dye is an isocyanate fluorescein dye, an isothiocyanate fluorescein dye, or an isothiocyanate rhodamine dye.
The molecularly imprinted fluorescent nanoparticle for detecting gram-negative bacteria is prepared by any one of the preparation methods.
Has the advantages that:
the invention directly takes gram-negative bacteria as a template, overcomes the defect that biomolecules such as protein and the like are taken as the template, takes phenylboronic acid silanization reagent as a functional monomer, adopts a one-pot method to prepare the molecularly imprinted fluorescent nanoparticles of the gram-negative bacteria based on the sol-gel polymerization reaction of the silanization reagent, has simple preparation and low cost, and can carry out specific identification and detection analysis on target bacteria. The invention provides a stable molecular recognition material with low preparation cost for developing a rapid detection and analysis method of gram-negative bacteria, particularly pathogenic bacteria therein, and has good practical application value and prospect.
1. The gram-negative bacteria is directly used as a template, the phenylboronic acid silanization reagent is used as a functional monomer, the defects of conventional template molecules such as protein can be overcome, the molecular imprinting efficiency is obviously improved, the specific molecular recognition capability of the molecular imprinting nanoparticles is improved, and the biological antibody is hopeful to be replaced.
2. The method adopts the sol-gel polymerization reaction of the silane reagent, has mild reaction conditions, is carried out at room temperature, avoids the polymerization reaction initiated by heating or ultraviolet irradiation, ensures the stability of the template bacteria in the imprinting process, and is beneficial to obtaining the high-specificity molecularly imprinted nanoparticles.
3. Compared with the prior art, the invention utilizes the one-step synthesis of the phenyl boronic acid silanization reagent by the reaction of the amino silanization reagent and Schiff base of aldehyde phenyl boronic acid, can be used for polymerization reaction without purification, and has the advantages of simple operation and time saving.
4. The fluorescent monomer used in the invention is synthesized by one-step reaction of an amino silanization reagent and a fluorescent dye containing isothiocyanate or isocyanate, the preparation is simple, the fluorescent monomer can be used for polymerization reaction without purification, and the time and labor are saved.
5. The molecularly imprinted fluorescent nanoparticle prepared by the invention has higher specificity to target gram-negative bacteria and high response speed, can be used for constructing a rapid fluorescence detection method of the gram-negative bacteria, and can also be used for fluorescence imaging analysis of the gram-negative bacteria.
Drawings
FIG. 1 is a schematic diagram of the synthesis of a gram-negative bacterium molecularly imprinted fluorescent nanoparticle;
FIG. 2 is a scanning electron microscope image of gram-negative bacteria molecular imprinting fluorescent nanoparticle MIP-1-1 (A, B) and a control fluorescent nanoparticle NIP-1-1 (C, D), wherein scale marks in the image are 20 mu m (A, C) and 1 mu m (B, D) respectively;
FIG. 3 is the fluorescence response analysis (excitation wavelength 471nm, emission wavelength 513 nm) of gram-negative bacteria molecular imprinting fluorescent nanoparticles and their contrast fluorescent nanoparticles (MIP-1-MIP-5) to target template bacteria;
FIG. 4 shows the fluorescence response analysis (excitation wavelength 471nm, emission wavelength 513 nm) of gram-negative bacteria molecularly imprinted fluorescent nanoparticles (MIP-5) on other bacteria (Escherichia coli O157: H7, Escherichia coli, Pseudomonas fluorescens, Staphylococcus aureus);
fig. 5 is a fluorescence response isotherm (excitation wavelength 471nm, emission wavelength 513 nm) of gram-negative bacteria molecular imprinting fluorescent nanoparticles (MIP-5) to target template bacteria e.coli O157: H7 of different concentrations;
FIG. 6 is the fluorescence response analysis (excitation wavelength 471nm, emission wavelength 520 nm) of gram-negative bacteria molecular imprinting fluorescent nanoparticle (MIP-6) and its control fluorescent nanoparticle (NIP-6) for Pseudomonas fluorescens of target template bacteria;
FIG. 7 is a fluorescence response isotherm (excitation wavelength 471nm, emission wavelength 520 nm) of gram-negative bacteria molecularly imprinted fluorescent nanoparticles (MIP-6) for different concentrations of target template bacteria Pseudomonas fluorescens;
FIG. 8 is the fluorescence response analysis (excitation wavelength 471nm, emission wavelength 520 nm) of gram-positive bacteria molecular fluorescent nanoparticles (MIP-7) and control fluorescent nanoparticles (NIP-7) to Staphylococcus aureus (a target template bacteria);
FIG. 9 is a fluorescence response isotherm (excitation wavelength 471nm, emission wavelength 520 nm) of gram-positive bacteria molecular imprinting fluorescence nanoparticles (MIP-7) on control target template bacteria Staphylococcus aureus of different concentrations;
FIG. 10 is the fluorescence response analysis (excitation wavelength 545nm, emission wavelength 580 nm) of gram-negative bacteria molecularly imprinted fluorescent nanoparticle (MIP-8) and its control fluorescent nanoparticle (NIP-8) to target template bacteria E.coli O157: H7;
FIG. 11 is the fluorescence response isotherm (excitation wavelength 545nm, emission wavelength 580 nm) of gram-negative bacteria molecularly imprinted fluorescent nanoparticles (MIP-8) to different concentrations of target template bacteria E.coli O157: H7.
Detailed Description
The following examples are presented to enable one of ordinary skill in the art to more fully understand the present invention and are not intended to limit the invention in any way.
Coli O157: H7, Pseudomonas fluorescens and Staphylococcus aureus (ATCC 25923) used in the examples of the present invention were purchased from China center for culture Collection; the general E.coli used was E.coli O6 (ATCC 25922) purchased from American type culture Collection (American type culture collection).
Example 1
The phenylboronic acid silylation reagent was prepared as follows: dissolving 3-formylphenylboronic acid (FPBA, 75 mg, 0.5 mmol) in 10 mL ethanol, adding 3-aminopropyltriethoxysilane (APTES, 120 mu L, 0.5 mmol), stirring and reacting at 25 ℃ for 12h, and removing solvent ethanol through a rotary evaporator under vacuum condition to obtain phenylboronic acid silylation reagent FPBA-APTES which can be used as a functional monomer for preparing gram-negative bacteria molecular imprinting fluorescent nanoparticles without purification.
Example 2
The fluorescence silylation reagent is prepared by dissolving fluorescein isothiocyanate (FITC, 6.6 mg, 17 μmol) in 1.5 mL of absolute ethanol, adding APTES (4 μ L, 17 μmol), and shaking at 25 ℃ for 12h to obtain an ethanol solution of the fluorescein silylation reagent FITC-APTES. 200 μ L of FITC-APTES was taken for use.
Example 3
The fluorescent silanization reagent is prepared by dissolving rhodamine isothiocyanate B (RBITC, 9.1 mg, 17 [ mu ] mol) in 1.5 mL of absolute ethanol, adding APTES (4 [ mu ] L, 17 [ mu ] mol), and shaking at 25 ℃ for 12h to obtain an ethanol solution of the rhodamine silanization reagent RBITC-APTES. 200. mu.L of RBITC-APTES was taken for use.
Example 4
0.5mL of bacterial suspension E.coli O157: H7 (10) was added to the round-bottomed flask8CFU/mL), 4mL PBS (10 mM, pH = 7.4), using a magnetic stirrer to stir at 1000rpm, and then rapidly adding 0.2mL of the ethanol solution of BA-APTES and 0.2mL of the ethanol solution of LFITC-APTES and stirring for 40 min. Will be provided withTEOS (675 μ L, 3.2 mmol) was dissolved in 1mL of anhydrous ethanol, 2.55 mL of PBS (10 mM, pH = 7.4) was added and transferred to a round bottom flask, and mixed for 3min with stirring at 1000 rpm. 1.35mL of 25% aqueous ammonia was added to a co-solvent of 0.8mL ethanol and 2mL PBS and transferred to a round bottom flask, stirred at 1500rpm for 5min and then at 900rpm for 12 h. The template molecules were removed with an elution solution (40% 100 mM acetic acid/methanol solution (v/v)) to give E.coli O157: H7 molecularly imprinted fluorescent nanoparticles. The elution solution used was a solution.
The yield was calculated according to the following formula:
yield (%) = mass of product/mass of raw material (including functional monomer, fluorescent monomer, crosslinking agent) × 100%,
the gram-negative bacteria molecularly imprinted fluorescent nanoparticle prepared in this example was named MIP-1-1. The yield of MIP-1-1 was 23%.
Example 5
The difference from example 4 is that PBS was used at a pH of 6.0.
The gram-negative bacteria molecularly imprinted fluorescent nanoparticle prepared in this example was named MIP-1-2. MIP-1-2 was prepared in 22% yield.
Example 6
The difference from example 5 is that PBS was used at a pH of 8.5.
The gram-negative bacteria molecularly imprinted fluorescent nanoparticle prepared in this example was named MIP-1-3. MIP-1-3 was prepared in 24% yield.
Comparative example 1
The difference from example 4 is that no template E.coli O157: H7 was added.
The fluorescent nanoparticles prepared in this comparative example were named NIP-1-1 with a yield of 21%.
Comparing the fluorescent nanoparticles prepared in example 4 and comparative example 1, and observing morphological characteristics, specifically as shown in fig. 2, fig. 2A and 2B are scanning electron micrographs of the fluorescent nanoparticle MIP-1-1 prepared in example 1, and fig. 2C and 2D are scanning electron micrographs of the fluorescent nanoparticle NIP-1-1, and it can be seen from the images that the molecularly imprinted fluorescent nanoparticle (MIP-1-1) prepared by the method of the present invention and the control fluorescent nanoparticle (NIP-1-1) both show a random polymer nanoparticle structure, and the nanoparticles are easily agglomerated together to form micron-sized particles.
Comparative example 2
The difference from example 5 is that no template E.coli O157: H7 was added.
The fluorescent nanoparticles prepared in this comparative example were named NIP-1-2, and the yield was 20%.
Comparative example 3
The difference from example 6 is that no template E.coli O157: H7 was added.
The fluorescent nanoparticles prepared in this comparative example were named NIP-1-3, with a yield of 21%.
Example 7
Same as example 4 except that e.coli O157: H7 was used at a concentration of 0.5 x 107CFU/mL。
The gram-negative bacteria molecularly imprinted fluorescent nanoparticle prepared in this example was named MIP-2-1. MIP-2-1 was prepared in 22% yield.
Example 8
Same as example 4 except that e.coli O157: H7 was used at a concentration of 0.5 x 109CFU/mL。
The gram-negative bacteria molecularly imprinted fluorescent nanoparticle prepared in this example was named MIP-2-2. MIP-2-2 was prepared in 24% yield.
Comparative example 4
The difference from example 7 is that no template E.coli O157: H7 was added.
The fluorescent nanoparticles prepared in this comparative example were named NIP-2-1 with a yield of 21%.
Comparative example 5
The difference from example 8 is that no template E.coli O157: H7 was added.
The fluorescent nanoparticles prepared in this comparative example were named NIP-2-2 with a yield of 20%.
According to different concentrations of added templates, the molecularly imprinted fluorescent nanoparticles prepared in examples 7-8 and comparative examples 4-5 and the control nanoparticles thereof are selected, and the change of the fluorescence response signal of the fluorescent nanoparticles to E.coli O157: H7 is respectively determined, the used excitation wavelength is 471nm, and the emission wavelength is 513nm, and the result is shown in FIG. 3, and as can be seen from the graph, the change value of the fluorescence response signal of MIP-2-2 is the highest, and is not only higher than that of NIP-2-2, but also higher than that of MIP-2-1, which indicates that MIP-2-2 has good selectivity and the selectivity is higher than that of MIP-2-1.
Example 9
The difference from example 4 is that TEOS was used in a volume of 337.5. mu.L.
The gram-negative bacteria molecularly imprinted fluorescent nanoparticle prepared in this example was named MIP-3-1. MIP-3-1 was prepared in 20% yield.
Example 10
The difference from example 4 is that TEOS was used in a volume of 1350. mu.L.
The gram-negative bacteria molecularly imprinted fluorescent nanoparticle prepared in this example was named MIP-3-2. MIP-3-2 was prepared in a 26% yield.
Comparative example 6
The difference from example 9 is that no template E.coli O157: H7 was added.
The fluorescent nanoparticles prepared in this comparative example were named NIP-3-1 with a yield of 21%.
Comparative example 7
The difference from example 10 is that no template E.coli O157: H7 was added.
The fluorescent nanoparticle prepared in this comparative example was named NIP-3-2, and the yield was 20%.
Example 11
The difference from example 4 is that FITC-APTES was used in an amount of 1.33. mu. mol.
The gram-negative bacterium molecular imprinting fluorescent nanoparticle prepared in the example is named MIP-4-1. MIP-4-1 was prepared in 23% yield.
Example 12
The difference from example 4 is that FITC-APTES was used in an amount of 5.32. mu. mol.
The gram-negative bacterium molecular imprinting fluorescent nanoparticle prepared in the example is named MIP-4-2. MIP-4-2 was prepared in 22% yield.
Comparative example 8
The difference from example 11 is that no template E.coli O157: H7 was added.
The fluorescent nanoparticles prepared in this comparative example were named NIP-4-1 with a yield of 21%.
Comparative example 9
The difference from example 12 is that no template E.coli O157: H7 was added.
The fluorescent nanoparticles prepared in this comparative example were named NIP-4-2 with a yield of 22%.
Example 13
Same as example 4 except that e.coli O157: H7 was used at a concentration of 0.5 x 109CFU/mL, PBS used pH 8.5.
The gram-negative bacteria molecularly imprinted fluorescent nanoparticle prepared in this example was named MIP-5. MIP-5 was prepared in 25% yield.
The specificity of the gram-negative bacteria molecularly imprinted fluorescent nanoparticles prepared in example 13 was determined.
2mL of 0.025mg/mL MIP-5 was added to a 4mL fluorescent cuvette and then added to the cuvette to a final concentration of 102Stirring different bacterium solutions (pseudomonas fluorescens or common escherichia coli or staphylococcus aureus) of CFU/mL for 6min, and measuring a fluorescence signal by using a fluorescence spectrophotometer (excitation wavelength 471nm and emission wavelength 513 nm) and marking as F; calculating to obtain a fluorescence response change value ((F)0-F)/F0). As shown in FIG. 4, the response change value of MIP-5 to E.coli O157: H7 is higher than that to other bacteria, which indicates that MIP-5 has good fluorescence response specificity to the template bacteria liquid E.coli O157: H7.
The response of the gram-negative bacteria molecularly imprinted fluorescent nanoparticle prepared in example 13 is linearly measured.
Adding 2mL of 0.025mg/mL MIP-5 into 4mL fluorescence cuvette, adding Escherichia coli O157: H7 bacterial liquid with different concentrations into the cuvette, stirring for 6min, and measuring fluorescence with fluorescence spectrophotometer (excitation wavelength 471nm, emission wavelength 513 nm)An optical signal, denoted as F; calculating to obtain a fluorescence response change value ((F)0-F)/F0). As shown in FIG. 5, MIP-5 is at 102-106Good linearity in the CFU/mL range.
Comparative example 10
The difference from example 13 is that no template E.coli O157: H7 was added.
The fluorescent nanoparticles prepared in this comparative example were designated NIP-5 with a yield of 23%.
Example 14
The difference from example 13 is that Pseudomonas fluorescens is used as template.
The gram-negative bacteria molecularly imprinted fluorescent nanoparticle prepared in this example was named MIP-6. MIP-6 was prepared in 25% yield.
The response of the gram negative bacteria molecular imprinting fluorescent nanoparticles prepared in example 14 is measured linearly.
Adding 2mL of 0.1mg/mL MIP-6 into a 4mL fluorescence cuvette, adding pseudomonas fluorescens bacterial liquid with different concentrations into the cuvette, stirring for 6min, and measuring a fluorescence signal by using a fluorescence spectrophotometer (excitation wavelength 471nm and emission wavelength 520 nm) and marking as F; calculating to obtain a fluorescence response change value ((F)0-F)/F0). As shown in FIG. 7, MIP-6 is at 101-105Good linearity in the CFU/mL range.
Comparative example 11
The difference from example 14 is that Pseudomonas fluorescens as a template was not added.
The fluorescent nanoparticles prepared in this comparative example were designated NIP-6 with a yield of 23%
The gram-negative bacteria molecularly imprinted fluorescent nanoparticles prepared in example 14 were subjected to a selectivity evaluation test.
Respectively adding 2mL of MIP-6 and NIP-6 with 0.1mg/mL into a 4mL fluorescent cuvette, and adding pseudomonas fluorescens bacterial liquid into the cuvette to make the final concentration 102CFU/mL, stirring for 6min, measuring a fluorescence signal by using a fluorescence spectrophotometer (excitation wavelength 471nm, emission wavelength 520 nm), and recording as F; calculating to obtain a fluorescence response change value ((F)0-F)/F0). As shown in FIG. 6, the change value of the fluorescence response of MIP-6 to Pseudomonas fluorescens is significantly higher than that of the comparison fluorescent nanoparticle NIP-6. The molecular imprinting fluorescent nanoparticle (MIP-6) has good response selectivity on the pseudomonas fluorescens. Based on the change values of the fluorescence responses of MIP-6 and NIP-6 to Pseudomonas fluorescens, the imprinting factor of MIP-6 is calculated to be 1.6.
Comparative example 12-1
The difference from example 13 is that the template used is the gram-positive bacterium Staphylococcus aureus.
The molecularly imprinted fluorescent nanoparticle prepared in this example was named MIP-7. MIP-7 was prepared in 24% yield.
And (3) carrying out linear response measurement on the gram-positive bacterium molecularly imprinted fluorescent nanoparticles prepared in example 15.
Respectively adding 2mL of 0.1mg/mL MIP-7 into 4mL of fluorescence cuvettes, adding staphylococcus aureus bacteria liquid with different concentrations into the cuvettes, stirring for 6min, and measuring a fluorescence signal by using a fluorescence spectrophotometer (excitation wavelength 471nm and emission wavelength 520 nm) and marking as F; calculating to obtain a fluorescence response change value ((F)0-F)/F0). As shown in fig. 9, MIP-7 is at 101-106There was no significant fluorescence response in the CFU/mL range.
Comparative examples 12 to 2
The difference from comparative example 12-1 is that no template Staphylococcus aureus was added.
The fluorescent nanoparticles prepared in this comparative example were designated NIP-7 with a yield of 21%
And (3) carrying out a selectivity evaluation test on the gram-positive bacterium molecularly imprinted fluorescent nanoparticles prepared in the comparative example 12-1.
Respectively adding 2mL of 0.1mg/mL MIP-7 and NIP-7 into a 4mL fluorescent cuvette, and adding staphylococcus aureus liquid into the cuvette to make the final concentration 102CFU/mL, stirring for 6min, measuring a fluorescence signal by using a fluorescence spectrophotometer (excitation wavelength 471nm, emission wavelength 520 nm), and recording as F; calculating to obtain a fluorescence response change value ((F)0-F)/F0). As shown in fig. 8, MIP-7 andand NIP-7 has a low fluorescence response value to staphylococcus aureus, and the calculated imprinting factor is less than 1.5, which indicates that no imprinting effect exists, namely the method is not suitable for preparing the molecular imprinting fluorescent nanoparticles of gram-positive bacteria.
Example 15
The gram-negative bacteria molecularly imprinted fluorescent nanoparticles prepared in examples 4-12 were tested for selectivity evaluation.
2mL of 0.025mg/mL MIP and NIP were added to a 4mL fluorescent cuvette, respectively, and E.coli O157: H7 bacterial solution was added to the cuvette to give a final concentration of 102CFU/mL, stirring for 6min, measuring a fluorescence signal by using a fluorescence spectrophotometer (excitation wavelength 471nm, emission wavelength 513 nm), and recording as F; calculating to obtain a fluorescence response change value ((F)0-F)/F0）。
As shown in FIG. 3, the fluorescence response change values of MIP-1-3, MIP-2-2 and MIP-4-2 are all higher than those of the comparative fluorescent nanoparticles NIP-1-3, NIP-2-2 and NIP-4-2, and are significantly higher than those of other MIPs, which indicates that the three molecularly imprinted fluorescent nanoparticles have good response selectivity to E.coli O157: H7.
The imprinting factor is the ratio of the amount of the target substance adsorbed by the MIP or the change in the signal caused by the MIP to the amount of the target substance adsorbed by the NIP (blank control, no template molecule is used during synthesis) or the change in the signal caused by the MIP, and can be used for indicating the selectivity, and the higher the imprinting factor is, the higher the selectivity is. Generally, the imprinting factor is lower than 1.5, and the material synthesized under the condition has no selectivity or poor selectivity. Based on their fluorescence response change values, it can be calculated that the imprinting factors of MIP-1-1, MIP-1-2, MIP-1-3, MIP-2-1, MIP-2-2, MIP-3-1, MIP-3-2, MIP-4-1 and MIP-4-2 are 0.4, 1.5, 3.1, 1.2, 2.4, 1.5, 0.6, 1.8 and 1.5, respectively.
Example 16
The difference from example 13 is that rhodamine silylation agent RBITC-APTES is used as the fluorescent monomer.
The fluorescent nanoparticles prepared in this example were named MIP-8 with a yield of 21%
The response of the gram-negative bacteria molecularly imprinted fluorescent nanoparticle prepared in example 16 is linearly measured.
Respectively adding 2mL of 0.05mg/mL MIP-8 into 4mL of fluorescence cuvettes, adding E.coli O157: H7 bacterial liquids with different concentrations into the cuvettes, stirring for 6min, and measuring a fluorescence signal by using a fluorescence spectrophotometer (excitation wavelength of 545nm and emission wavelength of 584 nm) and marking as F; calculating to obtain a fluorescence response change value ((F)0-F)/F0). As shown in FIG. 11, MIP-8 is at 101-105The linear relation is shown in the CFU/mL range.
Comparative example 13
The difference from example 16 is that the template E.coli O157: H7 is not added.
The fluorescent nanoparticles prepared in this comparative example were designated NIP-8 with a yield of 22%
Respectively adding 2mL of 0.05mg/mL MIP-8 and NIP-8 into a 4mL fluorescent cuvette, and adding E.coli O157: H7 bacterial liquid into the cuvette to make the final concentration 102CFU/mL, stirring for 6min, measuring a fluorescence signal by using a fluorescence spectrophotometer (excitation wavelength is 545nm, emission wavelength is 584 nm), and recording as F; calculating to obtain a fluorescence response change value ((F)0-F)/F0). As shown in fig. 10, the change value of the fluorescence response of MIP-8 to e.coli O157: H7 is significantly higher than that of its comparative fluorescent nanoparticle NIP-8. The molecular imprinting fluorescent nanoparticles (MIP-8) are shown to have good response selectivity on E.coli O157: H7. Based on the fluorescence response change values of MIP-8 and NIP-8 to Pseudomonas fluorescens, the imprinting factor of MIP-8 is calculated to be 3.1.
The invention directly takes gram-negative bacteria as a template, overcomes the defect that biomolecules such as protein and the like are taken as the template, takes phenylboronic acid silanization reagent as a functional monomer, adopts a one-pot method to prepare the molecularly imprinted fluorescent nanoparticles of the gram-negative bacteria based on the sol-gel polymerization reaction of the silanization reagent, has simple preparation and low cost, and can carry out specific identification and detection analysis on target bacteria. The invention provides a stable molecular recognition material with low preparation cost for developing a rapid detection and analysis method of gram-negative bacteria, particularly pathogenic bacteria therein, and has good practical application value and prospect.
The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and any simple modifications or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention are within the scope of the present invention.

Claims (10)

1. A preparation method of molecularly imprinted fluorescent nanoparticles for detecting gram-negative bacteria is characterized by comprising the following steps:
step one, the concentration is 106-108 Adding CFU/mL template bacteria into the solution, and then adding a functional monomer with the concentration of 4.7-9.4 mu mol/mL and a fluorescent monomer with the concentration of 0.1-0.4 mu mol/mL for preassembling to obtain a molecular imprinting pre-aggregation solution, wherein the template bacteria are gram-negative bacteria;
step two, sequentially adding a cross-linking agent with the concentration of 100-1000 mM and an initiator with the mass concentration of 2-10% into the molecularly imprinted pre-polymerizing solution prepared in the step one, and stirring the polymerizing solution to generate molecularly imprinted fluorescent nanoparticles, wherein the nanoparticles are adsorbed on the surface of template bacteria;
and step three, removing the template bacteria in the step two by using an elution solution to obtain the molecularly imprinted fluorescent nanoparticles capable of specifically identifying the target bacteria.
2. The method according to claim 1, wherein the template bacteria in step one is Escherichia coli O157: H7 or Pseudomonas fluorescens, the solution is a mixture of phosphate buffer and ethanol, and the volume fraction of ethanol is 5-15%.
3. The preparation method of molecularly imprinted fluorescent nanoparticles for detecting gram-negative bacteria according to claim 1, characterized in that in the first step, the concentration of functional monomers is 4.7 μmol/mL, the concentration of fluorescent monomers is 0.2 μmol/mL, and the template is filled with a fluorescent solutionThe concentration of the Plateobacteria was 0.42 x 108CFU/mL, and the volume fraction of ethanol in the mixture of phosphate buffer solution and ethanol is 15%.
4. The method as claimed in claim 1, wherein the concentration of the cross-linking agent in step two is 849 mM, and the mass fraction of the initiator is 6%.
5. The method as claimed in claim 1, wherein the cross-linking agent is tetraethoxysilane or tetramethoxysilane, the initiator is ammonia water, and the stirring speed is 700-1500 rpm.
6. The preparation method of the molecularly imprinted fluorescent nanoparticle for detecting gram-negative bacteria as claimed in claim 1, wherein the functional monomer in the step one is phenylboronic acid silylation reagent, which is obtained by one-step reaction of aldehyde phenylboronic acid and amino silylation reagent in ethanol according to a molar ratio of 1: 1; the fluorescent monomer can be obtained by one-step reaction of fluorescent dye containing isocyanate or isothiocyanate and amino silanization reagent in ethanol according to the molar ratio of 1:1, and the obtained functional monomer and the fluorescent monomer do not need to be purified.
7. A method for preparing molecularly imprinted fluorescent nanoparticles for detecting gram-negative bacteria according to claim 6, wherein the aldehyde phenylboronic acid is any one of 3-formyl phenylboronic acid, 2-formyl phenylboronic acid, 4-formyl phenylboronic acid, 2-fluoro-3-aldehyde phenylboronic acid, 4-fluoro-3-aldehyde phenylboronic acid, or 3-fluoro-4-aldehyde phenylboronic acid.
8. The method according to claim 6, wherein the amino silanization reagent is 3-aminopropyl-triethoxysilane, 3-aminopropyl-trimethoxysilane, 3- (2-aminoethylamino) propyl-trimethoxysilane, or diethylenetriaminopropyl-trimethoxysilane.
9. A preparation method of molecularly imprinted fluorescent nanoparticles for detecting gram-negative bacteria according to claim 6, wherein the fluorescent dye is isocyanate fluorescein dye, isothiocyanate fluorescein dye or isothiocyanate rhodamine dye.
10. The molecularly imprinted fluorescent nanoparticle for detecting gram-negative bacteria prepared by the preparation method according to any one of claims 1 to 9.
CN202111622929.4A 2021-12-28 2021-12-28 Molecularly imprinted fluorescent nanoparticle for detecting gram-negative bacteria and preparation method thereof Pending CN114428070A (en)

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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004027084A1 (en) * 2002-09-20 2004-04-01 Queen's University At Kingston Detection of biological molecules by differential partitioning of enzyme substrates and products
CN104655695A (en) * 2015-02-11 2015-05-27 江南大学 Magnetic molecular imprinting sensor for detecting Gram-negative bacterium signal molecule
US20160146813A1 (en) * 2014-11-25 2016-05-26 The Johns Hopkins University Multifunctional nanoprobe-enabled capture and early detection of microbial pathogens
CN107271410A (en) * 2017-05-17 2017-10-20 武汉汉瑞隆德检测技术有限公司 Bacterium or the active quick determination method of fungi
CN108051419A (en) * 2018-01-15 2018-05-18 江南大学 A kind of fluorescence probe detected and quantify Gram-negative bacteria group signaling molecule
CN109810281A (en) * 2018-12-27 2019-05-28 江苏大学 It is a kind of based on polymethyl methacrylate nano ball-molecular engram fluorescent material preparation method and its usage
CN112321833A (en) * 2020-11-11 2021-02-05 南京师范大学 Preparation method and application of fluorescent molecularly imprinted silica gel nanoparticles
CN112898965A (en) * 2021-03-05 2021-06-04 河南理工大学 Preparation and use method of ratiometric fluorescent probe for visual differential detection

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004027084A1 (en) * 2002-09-20 2004-04-01 Queen's University At Kingston Detection of biological molecules by differential partitioning of enzyme substrates and products
US20160146813A1 (en) * 2014-11-25 2016-05-26 The Johns Hopkins University Multifunctional nanoprobe-enabled capture and early detection of microbial pathogens
CN104655695A (en) * 2015-02-11 2015-05-27 江南大学 Magnetic molecular imprinting sensor for detecting Gram-negative bacterium signal molecule
CN107271410A (en) * 2017-05-17 2017-10-20 武汉汉瑞隆德检测技术有限公司 Bacterium or the active quick determination method of fungi
CN108051419A (en) * 2018-01-15 2018-05-18 江南大学 A kind of fluorescence probe detected and quantify Gram-negative bacteria group signaling molecule
CN109810281A (en) * 2018-12-27 2019-05-28 江苏大学 It is a kind of based on polymethyl methacrylate nano ball-molecular engram fluorescent material preparation method and its usage
CN112321833A (en) * 2020-11-11 2021-02-05 南京师范大学 Preparation method and application of fluorescent molecularly imprinted silica gel nanoparticles
CN112898965A (en) * 2021-03-05 2021-06-04 河南理工大学 Preparation and use method of ratiometric fluorescent probe for visual differential detection

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JEAN DE DIEU HABIMANA 等: "A class-specific artificial receptor-based on molecularly imprinted polymer-coated quantum dot centers for the detection of signaling molecules, N-acyl-homoserine lactones present in gram-negative bacteria", 《ANALYTICA CHIMICA ACTA》, 8 May 2018 (2018-05-08), pages 134 - 144, XP085443586, DOI: 10.1016/j.aca.2018.05.018 *

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