CN114236121A - PH response color-changing nano-particles based on bacitracin and thymolphthalein and application thereof - Google Patents
PH response color-changing nano-particles based on bacitracin and thymolphthalein and application thereof Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
- G01N21/80—Indicating pH value
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
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. The preparation method of the pH response color-changing nano-particles based on bacitracin and thymolphthalein is characterized by comprising 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.
2. The method for preparing pH-responsive color-changing nanoparticles according to claim 1, characterized in that:
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.
3. AMP/TP NPs material produced by the production method as set forth in any one of claims 1 to 2.
4. Use of the AMP/TP NPs material of claim 3 in the detection of E.
5. The use according to claim 4, wherein the method for detecting E.coli comprises the steps of:
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, obtaining Streptavidin-Fe after ultrasonic treatment3O4Magnetic 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.
6. Use according to claim 5, characterized in that:
s1.1, centrifuging an aptamer chain Apt for 50-60S at 4000 rpm/min before uncovering, wherein the volume ratio of 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.
7. Use according to claim 5, characterized in that:
s2.1, centrifugally washing the escherichia coli solution 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).
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN111796092A (en) * | 2020-08-17 | 2020-10-20 | 青岛农业大学 | pH response-based heterochromatic nanoparticles, pathogen detection kit containing nanoparticles and detection method |
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN111796092A (en) * | 2020-08-17 | 2020-10-20 | 青岛农业大学 | pH response-based heterochromatic nanoparticles, pathogen detection kit containing nanoparticles and detection method |
| AU2021100947A4 (en) * | 2020-08-17 | 2021-04-22 | Qingdao Agricultural University | Heterochromatic nanoparticle based on ph response, pathogenic bacteria detection kit containing the nanoparticle |
Non-Patent Citations (2)
| Title |
|---|
| SARA MALEKKHAIAT HÄFFNER: "Interplay between amphiphilic peptides and nanoparticles for selective membrane destabilization and antimicrobial effects", CURRENT OPINION IN COLLOID & INTERFACE SCIENCE, vol. 44, 31 December 2019 (2019-12-31), pages 59 * |
| 秦雨欣: "一种基于智能手机可视化多色荧光检测碱性磷酸酶活性的方法", 大学化学, vol. 35, no. 6, 29 February 2020 (2020-02-29), pages 110 * |
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