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CN114236121A - pH-responsive color-changing nanoparticles based on bacitracin and thymolphthalein and their applications - Google Patents

pH-responsive color-changing nanoparticles based on bacitracin and thymolphthalein and their applications Download PDF

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CN114236121A
CN114236121A CN202111577397.7A CN202111577397A CN114236121A CN 114236121 A CN114236121 A CN 114236121A CN 202111577397 A CN202111577397 A CN 202111577397A CN 114236121 A CN114236121 A CN 114236121A
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CN114236121B (en
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林天然
来去平
蒋高艳
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Guangxi Normal University
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Abstract

The invention discloses a preparation method of pH response color-changing nanoparticles based on the co-assembly of bacitracin and thymolphthalein, and a method for visually detecting escherichia coli by using the nanoparticles. During preparation, bacitracin and thymolphthalein are assembled together, and the thymolphthalein and bacitracin are assembled together to form nano particles by utilizing the polarity change of a solution; then, a sandwich structure is constructed by utilizing magnetic beads modified by nano particles and aptamers and bacteria, finally, a NaOH solution is added to stimulate thymolphthalein to develop color, and the delta OD value is detected by an enzyme-linked immunosorbent assay (ELISA) instrument or the delta (R + G)/B value and the sum of the R + G)/B value detected by a smart phoneE.coliThe concentration change constructs a linear relation, thereby realizing the pairE.coliQuantitative visual detection of (3). The AMP/TP NPs material is verified to be used for structure characterization, experimental condition optimization, sensing performance analysis and other experimentsE.coliThe analysis and detection of (2). The detection method of the invention is not only convenient to operate, but also has the advantages of simple operation, convenient detection, and low costThe response speed is fast, and the popularization and the use are easy.

Description

PH response color-changing nano-particles based on bacitracin and thymolphthalein and application thereof
Technical Field
The invention relates to a nano material and application thereof, in particular to pH response color changing Nano Particles (NPs) assembled based on bacitracin (an apparent light yellow antibacterial peptide, AMP) and Thymolphthalein (TP), and a preparation method and application thereof.
Background
Escherichia coli (Escherichia coli，E. coli) The Escherichia coli is a pathogenic bacterium causing the disease of human and livestock, and has serious harm to the health of human and animals. Due to pathogenicityE. coliThe resulting animal infections and contamination of animal products are a serious problem in animal health and public health safety.E. coliThe serotype (b) can cause gastrointestinal infections of human or animals, mainly caused by infection with specific pilus antigens, pathogenic toxins and the like, and can cause urinary tract infection, arthritis, meningitis, sepsis type infection and the like besides gastrointestinal infections. Therefore, the temperature of the molten metal is controlled,E. colithe accurate detection is very critical, and the method has important significance for the fields of environmental science and life medicine.
AMP is a broad-spectrum polypeptide antibiotic synthesized by nonribosomal peptide synthetases and is a secondary metabolite produced by fermentation of Bacillus subtilis and Bacillus licheniformis. AMPs have a strong bactericidal effect on gram-positive bacteria and most gram-negative bacteria. AMP is stable in both acidic and neutral aqueous solutions, but degrades rapidly at room temperature with a pH >8.0 in a basic environment. Under the stimulation of pH value or temperature, the antibacterial peptide can form a nano self-assembly structure with a specific shape through spontaneous molecular arrangement of acting forces such as non-covalent bond, hydrophobic effect, nonspecific Van der Waals effect and the like, thereby improving the stability of the antibacterial peptide in vivo, increasing the capture sites of bacteria and improving the biocompatibility. Therefore, AMP has a good prospect in the field of Escherichia coli detection.
Bacterial cell culture techniques are commonly used for bacterial detection, but the method is time-consuming and labor-consuming. Detection methods such as quartz crystal microbalance, surface plasmon resonance, surface enhanced raman scattering, fluorescence spectroscopy, electrochemical sensors, polymerase chain reaction, DNA microarray analysis, microfluidic analytical devices, and flow cytometry have been subsequently developed. Although the method is time-saving compared with the traditional bacterial culture technology, the method needs professional personnel to operate a specific chemical instrument and is complex to operate, thus being not beneficial to application and popularization. In order to effectively prevent bacterial infection, the development of a new instant detection method has important significance for food detection, environmental detection and clinical diagnosis.
At present, antibodies, aptamers and phage are often used as molecular recognition reagents to design pathogen immune biosensing detection methods. However, the use of these recognition elements to design a sensor for detecting bacteria requires the use of signal amplification techniques to increase sensitivity, such as enzyme labeling or fluorescent molecular labeling. However, these labeling processes tend to inactivate antibodies or enzymes, leading to false negative signals, and also suffer from the high cost of antibodies and phages as molecular recognition elements.
In the published detection of E.coliE. coliCN113152091A a polysaccharide-based hydrogel-based fabric for visually detecting escherichia coli and pH response and a preparation method thereof. By preparing a modified chitosan which becomes a grafted chromogenic/fluorescent compound by an amide reactionE. coliThe secreted bacterial enzyme cleaves, releasing the chromophoric or fluorescent group within the fastest 30 minutes, the chromophoric being visible under natural light. By fluorescence intensity, solution color andE. colilinear relation of concentration, realization ofE. coliQuantitative monitoring of (3). However, the method has the disadvantages of complicated material synthesis, complex detection method, small detection range and low detection limit.
CN113201585A A quantitative detection method based on fluorescence Polymerase Chain Reaction (PCR) technologyE. coliThe method of (1). Putting genome DNA into a PCR Reaction tube, adding a forward primer, a reverse primer, a SYBR Green Reaction Mix, ROX and sterile water into the tube, and simultaneously carrying out PCR amplification and melting curve determination on a standard control solution and a sample to be detected to obtain the PCR probeE. coliThe Ct value of real-time quantitative PCR is obtained by fitting the linear relation between the Ct value of the standard control solution and the copy numberE. coliQuantitative monitoring of (3). However, the method has the disadvantages of complicated material synthesis and complex detection method. And the labeling process easily causes inactivation of antibodies or enzymes, leads to false negative signals, the required detection medicine is expensive, and the invention needs to be carried out in a sterile environment, which has high requirements on the detection environment.
Disclosure of Invention
The inventionAiming at the defects of the prior art, a pH response color change nanoparticle based on co-assembly of AMP and TP is provided, and the nanoparticle is used for visually detecting escherichia coli (C: (A)E. coli) The method of (1). The AMP/TP NPs prepared by the invention have the advantages of label-free, integrated target signal amplification and bacterial growth inhibition. The colorimetric signal can be combined with the smart phone, so that the on-site instant detection is facilitated.
The technical scheme for realizing the purpose of the invention is as follows:
a preparation method of pH response color-changing nanoparticles based on bacitracin and thymolphthalein comprises the following steps:
(1) weighing BSA (bovine serum albumin) and dissolving the BSA in water, and stirring and mixing the BSA and the water at a low temperature to obtain a BSA aqueous solution;
(2) weighing TP, dissolving the TP in DMSO, stirring, and uniformly mixing to obtain a DMSO mixed solution of the TP;
(3) dripping the DMSO mixed solution of TP prepared in the step (2) into the BSA aqueous solution prepared in the step (1) and stirring;
(4) and (3) weighing AMP to be dissolved in the mixed solution prepared in the step (3), stirring, centrifugally washing a product with ultrapure water, and dispersing the product into the ultrapure water to synthesize the AMP/TP NPs material, namely the pH response color-changing nano-particles based on bacitracin and thymolphthalein.
In the preparation method, in the step (1), the mass ratio of BSA to water is 1: 1-1000000; the low temperature is-20 ℃ to 10 ℃;
in the step (2), the mass ratio of TP to DMSO is 1: 1-100000;
in the step (3), the volume ratio of the BSA aqueous solution to the TP DMSO solution is 1: 1-1000;
in the step (4), the volume ratio of AMP to the mixed solution in the step (3) is 1: 1-10000000.
The invention also aims to use the prepared pH-response color-changing nano-particles for visually detecting the Escherichia coli.
The method for detecting escherichia coli by using the pH response color-changing nano-particles comprises the following steps:
s1 magnetic bead Apt-MB modified by synthetic aptamer
S1.1, centrifuging before uncovering an aptamer chain Apt, adding water after centrifuging, and mixing uniformly to prepare an aptamer chain solution;
s1.2, taking Streptavidin marked ferroferric oxide (Streptavidin-Fe) after ultrasonic treatment3O4) Magnetic bead stock solution is magnetically separated and washed by PBS solution, and water is added after washing to prepare Streptavidin-Fe3O4An aqueous solution of magnetic beads;
s1.3, Streptavidin-Fe formulated to S1.23O4Adding the Bio-Apt into the magnetic bead aqueous solution, uniformly mixing, and incubating;
s1.4, magnetically separating the S1.3 mixed solution to remove excessive Bio-Apt, magnetically separating and washing the mixture by using a PBS (phosphate buffer solution) with the pH of 7.4, and dispersing the washed mixture in the PBS to obtain aptamer-coupled magnetic beads Apt-MB;
s2 detection of Escherichia coli
S2.1, centrifugally washing the escherichia coli solution by using a PBS solution, and dispersing the escherichia coli precipitate in the PBS solution to obtain an escherichia coli stock solution;
s2.2, taking the Escherichia coli stock solution in the S2.1 to dilute the Escherichia coli stock solution into Escherichia coli standard solutions with different concentrations step by step;
s2.3, uniformly mixing the escherichia coli standard solutions with different concentrations in the S2.2 with Apt-MB and AMP/TP NPs materials in the S1.4, uniformly mixing, and incubating;
s2.4, magnetically separating the mixed solution of the S2.3, removing excessive AMP/TP NPs, and washing after magnetic separation;
s2.5, adding PBS and NaOH to the mixed solution after S2.4 washing to elute Apt-MB, carrying out magnetic separation, simultaneously observing the color of AMP/TP NPs @ escherichia coli solution, taking supernatant to a 96-well plate and measuring OD value;
if the actual sample of the escherichia coli is determined, replacing the escherichia coli standard solution with different concentrations in the S2.3 with the actual sample of the escherichia coli to be determined;
measuring OD value at 590 nm, increasing OD value of AMP/TP NPs @ E.coli solution at 37 deg.C with the increase of E.coli concentration, and detecting E.coli by linear relationship between OD value and E.coli content.
In the detection method S1.1, an aptamer chain Apt is centrifuged for 50-60S at 4000 rpm/min before being uncapped, and the volume ratio of the Apt to water is 1: 1-1000;
in S1.2, Streptavidin-Fe is taken for 2-3min of ultrasound3O4The stock solution of the magnetic beads is magnetically separated and washed for 2 to 3 times by PBS solution, and water is added to prepare Streptavidin-Fe3O4An aqueous solution of magnetic beads;
Streptavidin - Fe3O4the volume ratio of the stock solution of the magnetic beads to water is 1: 0.0001-100;
s1.3, Streptavidin-Fe prepared to S1.23O4Adding Bio-Apt into the magnetic bead water solution, mixing uniformly, and incubating at 35-37 ℃ for 50-60 min;
Streptavidin - Fe3O4the volume ratio of the magnetic bead aqueous solution to the Bio-Apt is 1: 0.001-100;
in S1.4, carrying out magnetic separation on the S1.3 mixed solution to remove excessive Bio-Apt, carrying out magnetic separation and washing for 2-3 times by using a PBS solution with pH of 7.4, and dispersing the washed solution in a 300 mu L PBS solution to obtain aptamer-coupled magnetic beads Apt-MB;
the volume ratio of the mixed solution to the PBS is 1: 0.001-100.
In the detection method S2.1, the Escherichia coli solution is centrifugally washed for 2-3 times by using a PBS solution; the activity of the Escherichia coli stock solution is (0-10000000000) CFU mL-1；
In S2.3, uniformly mixing the escherichia coli standard solutions with different concentrations with Apt-MB and AMP/TP NPs materials in S1.4, and incubating for 90-100 min at 35-37 ℃;
the volume ratio of the Escherichia coli standard solution, Apt-MB and AMP/TP NPs with different concentrations is 1 to (0.00001-100) to (0.0000001-1000);
in S2.4, magnetically separating the mixed solution in S2.3, removing excessive AMP/TP NPs, and washing for 2-3 times after magnetic separation;
in S2.5, adding PBS and NaOH to the mixed solution after S2.4 washing to elute Apt-MB, carrying out magnetic separation, simultaneously observing the color of AMP/TP NPs @ escherichia coli solution, taking 150 mu L of supernatant to a 96-well plate, and measuring OD value;
the volume ratio of the mixed solution to the PBS solution to the NaOH solution is 1 to (0.00001-1000) to (0.0000001-10000).
The detection method of the invention is a method which does not need to be marked and is simple and convenient to operate. AMP/TP integrated NPs with the functions of label-free, target signal amplification integration and bacterial growth inhibition are prepared by adopting a one-step co-assembly strategy, and pairs are constructedE. coliThe sensitive visual portable detection platform. Stimulation of AMP/TP NPs @ by addition of NaOH solutionE. coliThe indicator in (1) is developed by an OD valueE. coliLinear relation of contents realizes pairE. coliDetection of (3). In addition, the colorimetric signal is combined with the smart phone, so that the on-site instant detection is facilitated.
The AMP/TP NPs material has simple synthesis process and easy popularization and use. The AMP/TP NPs material prepared by the preparation method has higher sensitivity and higher response speed.
Drawings
FIG. 1 is a schematic diagram of the preparation of pH-responsive color-changing nanoparticles and a procedure for detecting Escherichia coli according to an embodiment;
FIG. 2 is a Fourier transform infrared spectrum of AMP, TP, BSA and AMP/TP NPs in the examples;
FIG. 3 shows the value of Δ OD 590 nm in the embodimentE. coliA linear plot of concentration;
FIG. 4 is a table of (R + G)/B values of the digital photos by analysis in the embodimentE. coliA linear plot of concentration;
FIG. 5 is a graph of the effect of different nanoparticles and bacteria on the AMP/TP NPs OD 590 nm in the examples.
Detailed Description
The present invention will be further described with reference to the following examples, but the present invention is not limited thereto.
Examples
Referring to fig. 1, the method for preparing pH-responsive color-changing nanoparticles based on bacitracin and thymolphthalein comprises the following steps:
(1) weighing 10 mg of BSA, placing the BSA in a glass bottle, dissolving the BSA in 10 mL of water, and stirring and mixing the BSA at the temperature of minus 20-10 ℃ to obtain a BSA aqueous solution;
(2) weighing 4 mg of TP, dissolving the TP in 1.6 mL of DMSO, stirring, and uniformly mixing to obtain a DMSO mixed solution of the TP;
(3) dripping the DMSO mixed solution of TP prepared in the step (2) into the BSA aqueous solution prepared in the step (1) and stirring;
(4) weighing 2 mg of AMP to dissolve in the mixed solution prepared in the step (3), stirring for 3 h, centrifugally washing the product with ultrapure water, and dispersing the product into the ultrapure water to synthesize an AMP/TP NPs material, namely the pH response color-changing nano-particles based on bacitracin and thymolphthalein, wherein a Fourier transform infrared spectrogram of the nano-particles is shown in figure 2: 1257 cm-1And 800 cm-1C-O bonds of the carboxyl group and C-H bonds of the pyrimidine ring on the AMP structural formula respectively; BSA and AMP were successfully immobilized on TP to form AMP/TP NPs.
Detection of Escherichia coli by using prepared AMP/TP NPs materialE. coliThe method of (1):
will be provided withE. coliThe solution was washed 3 times by centrifugation with PBS (5000 rpm, 5 min),E. colidispersing the precipitate into PBS, uniformly mixing, taking 200 mu L of bacterial liquid, and measuring the bacterial liquid by an enzyme-linked immunosorbent assay (OD = 0.32) with the concentration of 2.08 multiplied by 108 CFU mL-1Is thatE. coliStock solution;
to 500 mu LE. coliDiluting the stock solution into standard solutions with different concentrations, and storing for later use;
adding 500 muL of standard solution (the concentration of the standard solution is 2.08X 10)7 CFU mL-1) Incubating with 10 muL Apt-MB and 25 muL AMP/TP NPs at 37 ℃ for 90 min, magnetically separating to remove excessive AMP/TP NPs, and magnetically separating and washing for 2 times;
130 μ L PBS and 50 μ L NaOH (1 mol L) were added-1) Eluting Apt-MB, magnetically separating, and observing AMP/TP NPs @E. coliTaking 150 mu L of supernatant to a 96-well plate to measure the OD value according to the solution color;
through the data analysis, the data analysis shows that,E. colithe concentration is 2.08X 101 ~ 2.08 × 105 CFU mL-1Within a range of (D), a value of Δ ODE. coliNegative logarithm of concentration can be fitted to the linear equation (R)2= 0.997), the linear equation is y =0.031x-0.005 (x denotesE. coliNegative logarithm of concentration in lg CFU mL-1(ii) a y is AMP/TP NPs @E. coliSolution mixing systemAfter adding different concentrationsE. coliThe latter Δ OD value);
through the data analysis, the data analysis shows that,E. colithe concentration is 2.08X 101 ~ 2.08 × 105 CFU mL-1Δ (R + G)/B value within the range ofE. coliNegative logarithm of concentration can be fitted to the linear equation (R)2= 0.980). The linear equation is Δ (R + G)/B = 0.105 log (C) E. coli[] / CFU mL-1）- 0.114。
Referring to FIG. 3, AMP/TP NPs @ in the exampleE. coliIn a mixed systemE. coliThe linear relation between the concentration variation and the delta OD value; as can be taken from figure 3 of the drawings,E. coliat a concentration of 2.08X 101 ~ 2.08 × 105Within the range, Δ OD andE. colithe concentration is linear. According to the formula can implementE. coliAccurate detection of.
Referring to FIG. 4, AMP/TP NPs @ in the exampleE. coliIn a mixed systemE. coliA linear fit graph of the concentration and the value of (R + G)/B; as can be taken from figure 4 of the drawings,E. coliat a concentration of 2.08X 10-3 ~ 2.08 × 108Δ (R + G)/B value variable within the rangeE. coliThe concentration is linear. According to the formula can implementE. coliAccurate detection of.
The invention also explores the AMP/TP NPs @ from other bacteriaE. coliExtent of influence of Δ OD of the mixed system to verify AMP/TP NPs pairsE. coliSpecific recognition of (3). As can be seen from FIG. 5, 104 CFU mL-1 Is/are as followsE. coliAn intense signal is generated at 590 nm and 104 CFU mL-1The delta OD values of Staphylococcus aureus produced no significant signal, similar to the blank (phosphate buffer). Also synthesizes MP/TP NPs @ as TP NPsE. coliThe extent of the effect of Δ OD in the mixed system is similar to that of the blank control (phosphate buffer), as can be seen from FIG. 5, and the Δ OD value of TP NPs is similar to that of the blank control without a significant signal. Therefore, other bacteria, TP NPs, have negligible effect on the present invention in practical applications.
The invention is based on AMP/TP NPs material, and can not only detectE. coliThe content of (2) can also combine the colorimetric signal with the smart phone, thus being beneficial to on-site instant detection. Provides a new idea for realizing the instant detection of bacteria in the fields of food safety, clinical diagnosis, environmental monitoring and the like.

Claims (7)

1.基于杆菌肽与百里酚酞的pH响应变色纳米颗粒的制备方法，其特征在于，包括如下步骤：1. the preparation method of the pH-responsive color-changing nanoparticles based on bacitracin and thymolphthalein, is characterized in that, comprises the steps: （1）称取BSA溶解在水中，并在低温条件下搅拌混合，得到BSA水溶液；(1) Weigh BSA and dissolve it in water, and stir and mix at low temperature to obtain an aqueous BSA solution; （2）称取TP溶解在DMSO中，搅拌，混合均匀得到TP的DMSO混合溶液；(2) Weigh TP and dissolve it in DMSO, stir and mix evenly to obtain the DMSO mixed solution of TP; （3）将步骤（2）配制的TP的DMSO混合溶液滴入步骤（1）中配制的BSA水溶液中搅拌；(3) drop the DMSO mixed solution of TP prepared in step (2) into the BSA aqueous solution prepared in step (1) and stir; （4）称取AMP溶解于步骤（3）配制的混合溶液中，搅拌，产物用超纯水离心洗涤，并分散到超纯水中，即合成AMP/TP NPs材料，即为基于杆菌肽与百里酚酞的pH响应变色纳米颗粒。(4) Weigh AMP and dissolve it in the mixed solution prepared in step (3), stir, wash the product with ultrapure water centrifugation, and disperse it into ultrapure water, that is, to synthesize AMP/TP NPs material, which is based on bacitracin and pH-responsive color-changing nanoparticles of thymolphthalein. 2.根据权利要求1所述的pH响应变色纳米颗粒的制备方法，其特征在于：2. the preparation method of pH-responsive color-changing nanoparticles according to claim 1, is characterized in that: 步骤（1）中，BSA与水的质量比为1∶（1~1000000）；低温条件为-20℃~10℃；In step (1), the mass ratio of BSA and water is 1:(1~1000000); the low temperature condition is -20°C~10°C; 步骤（2）中，TP与DMSO的质量比为1∶（1~100000）；In step (2), the mass ratio of TP to DMSO is 1: (1~100000); 步骤（3）中，BSA水溶液与TP的DMSO溶液体积比为1∶（1~1000）；In step (3), the volume ratio of the BSA aqueous solution to the DMSO solution of TP is 1: (1~1000); 步骤（4）中，AMP与步骤（3）中混合溶液体积比为1∶（1~10000000）。In step (4), the volume ratio of AMP to the mixed solution in step (3) is 1:(1~10000000). 3.权利要求1-2任一项所述的制备方法制得的AMP/TP NPs材料。3. The AMP/TP NPs material prepared by the preparation method of any one of claims 1-2. 4.权利要求3所述的AMP/TP NPs材料在检测大肠杆菌中的应用。4. the application of the described AMP/TP NPs material of claim 3 in detecting Escherichia coli. 5.根据权利要求4所述的应用，其特征在于，检测大肠杆菌的方法包括以下步骤：5. application according to claim 4 is characterized in that, the method for detecting Escherichia coli comprises the following steps: S1、合成适配体修饰磁珠Apt-MBS1. Synthesis of aptamer-modified magnetic beads Apt-MB S1.1、适配体链Apt开盖前先离心，离心后加入水混匀，配置为适配体链溶液；S1.1. Centrifuge the aptamer chain Apt before opening the cap, add water after centrifugation and mix well, and configure it as an aptamer chain solution; S1.2、取超声后的Streptavidin-Fe3O4磁珠原液用PBS溶液磁分离洗涤，洗涤后加入水配置为Streptavidin - Fe3O4磁珠水溶液；S1.2, take the Streptavidin-Fe 3 O 4 magnetic bead stock solution after sonication and wash it by magnetic separation with PBS solution, and add water after washing to configure the Streptavidin-Fe 3 O 4 magnetic bead aqueous solution; S1.3、向S1.2配制的Streptavidin - Fe3O4磁珠水溶液中加入Bio-Apt混匀，混匀后孵育；S1.3. Add Bio-Apt to the Streptavidin - Fe 3 O 4 magnetic bead aqueous solution prepared in S1.2, mix well, and incubate after mixing; S1.4、将S1.3混合溶液磁分离除去过量的Bio-Apt，并用pH 7.4的PBS溶液磁分离洗涤，洗涤后分散在PBS溶液中即得到适配体偶联的磁珠Apt-MB；S1.4, magnetically separate the mixed solution of S1.3 to remove excess Bio-Apt, and magnetically separate and wash the mixed solution with a pH 7.4 PBS solution. After washing, disperse in the PBS solution to obtain the aptamer-conjugated magnetic beads Apt-MB; S2、检测大肠杆菌S2. Detection of Escherichia coli S2.1、将大肠杆菌溶液用PBS溶液离心洗涤，大肠杆菌沉淀在PBS溶液中分散得到大肠杆菌原液；S2.1. Centrifugally wash the Escherichia coli solution with a PBS solution, and disperse the Escherichia coli precipitation in the PBS solution to obtain an Escherichia coli stock solution; S2.2、取S2.1中大肠杆菌原液逐级稀释为不同浓度的大肠杆菌标准溶液；S2.2, take the Escherichia coli stock solution in S2.1 and dilute it stepwise to the Escherichia coli standard solution of different concentrations; S2.3、取S2.2中不同浓度的大肠杆菌标准溶液与S1.4中Apt-MB及AMP/TP NPs材料混匀，混匀后孵育；S2.3. Take the E. coli standard solutions of different concentrations in S2.2 and mix them with the Apt-MB and AMP/TP NPs materials in S1.4, and incubate after mixing; S2.4、磁分离S2.3的混合溶液，去除过量的AMP/TP NPs，磁分离后洗涤；S2.4, magnetically separate the mixed solution of S2.3, remove excess AMP/TP NPs, and wash after magnetic separation; S2.5、向S2.4洗涤后的混合溶液中，加入 PBS和 NaOH洗脱Apt-MB，磁分离，同时观察AMP/TP NPs＠大肠杆菌溶液颜色，取上清液至96孔板测量OD值；S2.5. To the mixed solution after washing in S2.4, add PBS and NaOH to elute Apt-MB, separate magnetically, and observe the color of AMP/TP NPs@Escherichia coli solution at the same time, take the supernatant to 96-well plate to measure OD value; 若测定大肠杆菌实际样，则将S2.3中不同浓度的大肠杆菌标准溶液替换为待测大肠杆菌的实际样；If the actual sample of Escherichia coli is determined, replace the standard solution of Escherichia coli with different concentrations in S2.3 with the actual sample of Escherichia coli to be tested; 在590 nm处测定其OD值，在37 ℃下AMP/TP NPs＠大肠杆菌溶液的OD值会随着大肠杆菌的浓度增加而增加，利用OD值与大肠杆菌含量的线性关系实现对大肠杆菌的检测。Its OD value was measured at 590 nm. At 37 ℃, the OD value of AMP/TP NPs@E. coli solution increased with the increase of E. coli concentration. The linear relationship between OD value and E. coli content was used to achieve the OD value of E. coli. detection. 6.根据权利要求5所述的应用，其特征在于：6. application according to claim 5, is characterized in that: S1.1中，适配体链Apt开盖前用4000 rpm/min离心50-60 s，Apt与水的体积比为1∶（1~1000）；In S1.1, the aptamer chain Apt was centrifuged at 4000 rpm/min for 50-60 s before opening the cap, and the volume ratio of Apt to water was 1:(1~1000); S1.2中，取超声2-3min的Streptavidin - Fe3O4磁珠原液用PBS溶液磁分离洗涤2-3次，加入水配置为Streptavidin - Fe3O4磁珠水溶液；In S1.2, take the Streptavidin-Fe 3 O 4 magnetic bead stock solution that has been sonicated for 2-3 min and wash it 2-3 times with PBS solution for magnetic separation, and add water to configure the Streptavidin-Fe 3 O 4 magnetic bead aqueous solution; Streptavidin - Fe3O4磁珠原液与水的体积比为1∶（0.0001~100）；The volume ratio of Streptavidin - Fe 3 O 4 magnetic bead stock solution and water is 1: (0.0001~100); S1.3中，向S1.2配制的Streptavidin - Fe3O4磁珠水溶液中加入Bio-Apt混匀，混匀后在35-37 ℃孵育50-60 min；In S1.3, add Bio-Apt to the Streptavidin - Fe 3 O 4 magnetic bead aqueous solution prepared in S1.2 and mix well, incubate at 35-37 °C for 50-60 min after mixing; Streptavidin - Fe3O4磁珠水溶液与Bio-Apt的体积比为1∶（0.001~100）；The volume ratio of Streptavidin - Fe 3 O 4 magnetic bead aqueous solution to Bio-Apt is 1: (0.001~100); S1.4中，将S1.3混合溶液磁分离除去过量的Bio-Apt，并用pH 7.4的PBS溶液磁分离洗涤2-3次，洗涤后分散在300 µL PBS溶液中即得到适配体偶联的磁珠Apt-MB； In S1.4, the mixed solution of S1.3 was magnetically separated to remove excess Bio-Apt, and washed 2-3 times with PBS solution of pH 7.4. After washing, it was dispersed in 300 µL of PBS solution to obtain aptamer coupling The magnetic beads Apt-MB; 所述混合溶液与PBS的体积比为1∶（0.001~100）。The volume ratio of the mixed solution to PBS is 1:(0.001-100). 7.根据权利要求5所述的应用，其特征在于：7. application according to claim 5, is characterized in that: S2.1中，将大肠杆菌溶液用PBS溶液离心洗涤2-3次；大肠杆菌原液活度为（0~10000000000）CFU mL-1；In S2.1, the E. coli solution was centrifuged and washed with PBS solution for 2-3 times; the activity of the E. coli stock solution was (0~10000000000) CFU mL -1 ; S2.3中，不同浓度的大肠杆菌标准溶液与S1.4中Apt-MB及AMP/TP NPs材料混匀，混匀后在35-37 ℃孵育90-100 min；In S2.3, different concentrations of E. coli standard solutions were mixed with Apt-MB and AMP/TP NPs materials in S1.4, and then incubated at 35-37 °C for 90-100 min; 不同浓度的大肠杆菌标准溶液、Apt-MB、AMP/TP NPs材料体积比为1∶（0.00001~100）∶（0.0000001~1000）；The volume ratio of Escherichia coli standard solution, Apt-MB and AMP/TP NPs materials with different concentrations is 1:(0.00001~100):(0.0000001~1000); S2.4中，磁分离S2.3中混合溶液，去除过量的AMP/TP NPs，磁分离后洗涤2-3次；In S2.4, the mixed solution in S2.3 was magnetically separated to remove excess AMP/TP NPs, and washed 2-3 times after magnetic separation; S2.5中，向S2.4洗涤后的混合溶液中，加入 PBS和 NaOH洗脱Apt-MB，磁分离，同时观察AMP/TP NPs＠大肠杆菌溶液颜色，取150 µL上清液至96孔板测量OD值；In S2.5, to the mixed solution after washing in S2.4, add PBS and NaOH to elute Apt-MB, magnetically separate it, and observe the color of AMP/TP NPs@E. coli solution, take 150 µL of supernatant to 96 wells Plate to measure OD value; 所述混合溶液、 PBS溶液、NaOH溶液的体积比为1∶（0.00001~1000）∶（0.0000001~10000）。The volume ratio of the mixed solution, the PBS solution, and the NaOH solution is 1:(0.00001~1000):(0.0000001~10000).
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