CN105424771B - A kind of method of quick detection gram negative pathogenic bacteria toxicity - Google Patents
A kind of method of quick detection gram negative pathogenic bacteria toxicity Download PDFInfo
- Publication number
- CN105424771B CN105424771B CN201510945115.2A CN201510945115A CN105424771B CN 105424771 B CN105424771 B CN 105424771B CN 201510945115 A CN201510945115 A CN 201510945115A CN 105424771 B CN105424771 B CN 105424771B
- Authority
- CN
- China
- Prior art keywords
- cell
- lps
- magnetic
- chitosan
- sensor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/308—Electrodes, e.g. test electrodes; Half-cells at least partially made of carbon
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Electrochemistry (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analyzing Materials By The Use Of Electric Means (AREA)
Abstract
The invention discloses a kind of gold nano-carbon nanotube-application of the chitosan complex film cell sensor in terms of food-borne pathogens toxicity detection, belong to gram negative pathogenic bacteria toxicity detection technical field.The present invention is chitosan-modified on magnetic glassy carbon electrode by nanogold-carbon nanotube-by self-assembling method, has the mouse macrophage Ana-1 of magnetic nano particle as sensor information using endocytosis, is directly adsorbed to modified electrode, completes the building of sensor；It is finally evaluated applied to the LPS toxicity of Gram-negative bacteria, in concentration range in 1~5 μ g mL‑1Interior to change with lipopolysaccharide concentration variation with apparent poisonous effect, detection is limited to 0.3 μ g mL‑1.The method of the present invention efficiently, it is convenient, rapidly realize toxicity detection to Gram-negative bacteria, the defect of traditional pathogenic bacteria detection method of toxicity is overcome, gained sensor production is simple, easy to operate, it is low in cost, there is good application prospect in terms of evaluating pathogenic mushroom toxin.
Description
Technical field
The present invention relates to a kind of gold nano-carbon nanotube-chitosan complex film cell sensors in food-borne pathogenic bacterium
The application of property context of detection, belongs to gram negative pathogenic bacteria toxicity detection technical field.
Background technique
In recent years, as economic globalization process is accelerated, food safety has become world today's property public health hot spot.Food
The harm of borne pathogen is an importance of food safety, does not reduce or disappears with economic development and technological progress
It loses, the microbial food safety malignant event of food-borne pathogenic occurs in succession in world wide, and food origin disease incidence occupies high
Under not.Therefore, have become an important topic of field of food safety for the toxicity assessment of food-borne pathogens.From leather
The lipopolysaccharides of gram-negative bacteria, which is found to be, causes one of numerous infection or most critical factor of disease, is to be confirmed as endangering
The human pathogen of danger.
Food is easy in production, transport, sales process by microbial contamination, therefore microorganism detection is in Food Inspection
An important content.The method for being usually used in detection and identification food-borne pathogens at present is mainly separately cultured identification method, life
Change identification, enzyme linked immunosorbent assay (ELISA) and automatic enzyme-linked fluorescence immunoassay detection method (VIDAS) combine etc. it is some simple
Molecular biology and immunological method.For these detection methods in addition to cumbersome, Check-Out Time is longer, expensive, has
Very big operating error.
Summary of the invention
To solve the above-mentioned problems, it is compound based on gold nano/Carbon Nanotubes/Chitosan that the object of the present invention is to provide one kind
The preparation method of theca cell sensor, and optimize the use condition of the invention material has been determined, use chitosan as gold nano with
The dispersion liquid of carbon nanotube takes and prepares composite membrane in the modification to carbon electrode of 20 μ l dispersion liquids, obtains good electric conductivity, and benefit
Cell sensor is prepared with the certain density cell of magnetic absorption, and the cell sensor is used to quickly to detect Gram-negative
Cause a disease mushroom toxin.The present invention is vivo immunization cell using macrophage, the virulence factor LPS's of gram-negative bacteria cell wall
Major receptors are the marker molecule CD of Macrophage Surface14, can quick activating macrophage, cause intracellular calcium concentration liter
Height generates a large amount of active oxygens (ROS) and the characteristics of a series of inflammatory factors cause inflammatory reaction, it is established that macrophages in vitro is commented
Valence model simultaneously constructs sensor, more direct than conventional detection means, faster.The present invention is more using gram negative pathogenic bacteria rouge
Sugar is used as test object.LPS is the Major Virulence Factors of Gram-negative bacteria, can activating macrophage generate inflammatory reaction,
Make cell that metamorphosis and apoptosis occur.Using this feature of LPS, it is detection medium with mouse macrophage Ana-1, passes through
Electrochemical signals detect influence of the LPS to cell growth condition, and carry out quantitative analysis, quickly detect pathogenic bacteria to reach
Purpose.Furthermore composite membrane is prepared as medium dispersing Nano carbon tubes and gold nano using the chitosan of bioactivity, improved thin
Born of the same parents' adhesion strength and vitality.
The cell biography that the first purpose of the invention is to provide a kind of for quickly detecting gram negative pathogenic bacteria toxicity
Sensor, preparation method are as follows: (1) cleaning carbon electrode magnetic glassy carbon electrode；(2) functionalized multi-wall carbonnanotubes are dispersed in shell
In glycan solution, ultrasound, centrifugation obtain carbon nanotube-chitosan dispersion, and then HAuCl is added in strong stirring at room temperature4,
80 DEG C of heating stirrings do not change to solution colour, obtain gold nano/Carbon Nanotubes/Chitosan dispersion liquid；(3) by Jenner
Rice/Carbon Nanotubes/Chitosan dispersant liquid drop is added to thorough clean magnetic glassy carbon electrode surface, dries under infrared lamp, obtains gold
Nanometer-carbon nanotube-chitosan complex film modifies carbon electrode；(4) magnetic cell is then directly adsorbed to modification magnetoelectricity pole surface,
Obtain gold nano-carbon nanotube-chitosan complex film cell sensor.
The magnetic cell the preparation method is as follows: the magnetic nanometer of synthesis is configured to RPMI 1640 culture medium dense
Degree is 0.05mg mL-1, by mouse macrophage with every hole 106mL-1It is inoculated in six orifice plates, every hole is added that 2mL is above-mentioned to be received containing magnetic
Rice grain culture solution is placed in 37 DEG C of 5%CO2It is cultivated in incubator.
The preparation of the magnetic nano particle: by 1.35g FeCl3·6H2O dissolves in 40mL ethylene glycol, and it is anhydrous that 3.6g is added
Sodium acetate and 1.0g polyethylene glycol stir 30min strongly, are sealed in autoclave, are heated to 200 DEG C of reaction 8h, are cooled to room
Temperature, product dehydrated alcohol and ultrapure water respectively wash 4 times, in a vacuum drying oven 80 DEG C be dried overnight it is spare.
The present invention also provides a kind of method for quickly detecting gram negative pathogenic bacteria toxicity using the cell sensor,
The method is incubated in the lipopolysaccharides (lipopolysaccharide, LPS) for be placed in cell sensor different known concentrations
It educates, inserts the sensors into electrolytic cell and measure after flushing, obtain the impedance value of various concentration LPS solution, it is bent to draw standard
Then the LPS of the bacterial strain to be detected extracted is configured to solution by line, detect impedance value, the impedance that will be obtained using sensor
Value, which brings standard curve into, can be obtained the LPS content of bacterial strain to be detected；For pathogenic bacteria of the same race, LPS content it is higher explanation to
The bacterial content of survey bacterial strain is more, toxicity is stronger, and for different types of pathogenic bacteria (for variety classes pathogenic bacteria, rouge
Polysaccharide structures are not also identical, and toxicity is also just different, and LPS content does not have comparativity with pathogenic), the difference of identical bacterial content
The LPS content that bacterium to be measured extracts is higher, toxicity is stronger；The cell sensor is the mouse that endocytosis is had to magnetic nano particle
Macrophage Ana-1, i.e. magnetic cell are directly adsorbed to construct on gold nano-carbon nanotube-chitosan complex film modification carbon electrode
's.
The method is: nanogold and the carbon nano-tube material modification for being dispersed chitosan by the method for self assembly to magnetic
Gold nano-carbon nanotube-chitosan complex film modification carbon electrode is obtained on electrode, using the mouse macrophage of endocytosis magnetic nano particle
Cell Ana-1 is directly adsorbed to the complex film modified magnetic of gold nano/Carbon Nanotubes/Chitosan as sensor information, and by magnetic cell
The cell sensor of preparation, is finally applied to the detection of LPS by the building that cell sensor is completed on electrode；Specific steps are as follows:
(1) nanogold-carbon nanotube-chitosan is self-assembled modified on carbon electrode；
(2) preparation of magnetic nano particle；
(3) preparation of the culture of mouse macrophage and magnetic cell；
(4) absorption of the magnetic cell on carbon electrode；
(5) detection of LPS.
Described as follows using sensor detection impedance value method: electrochemical workstation ATUO LAB is in initial amplitude 0.05V, frequency
It is 2.5mM Fe (CN) in reaction medium liquid under conditions of 1~100kHz6 3-/4-It is measured in solution, using EIS method, with
Working electrode adsorbs the impedance value of magnetic cell as blank AC impedance R after modifyinget(Ab), different from containing with this electrode
The post-stimulatory cell of concentration LPS surveyed impedance value in same electrolyte is Ret(Ab-Ag), the R in different LPS concentration is found outet
Changing value △ Ret:
△Ret=Ret(Ab-Ag)-Ret(Ab)
Ret(Ab): blank AC impedance；
Ret(Ab-Ag): transmitting resistance value with the electrodic electron of the post-stimulatory cell of LPS；
Δ Ret: the changing value of reaction front and back electrodic electron transmitting resistance；
It is mapped with the relationship of the changing value of electron transmission resistance and LPS solution concentration to get the detection of LPS cell sensor
Standard curve.
The cell sensor carries out cyclic voltammetric and ac impedance measurement；Wherein cyclic voltammetric condition are as follows: scanning model
It encloses: -0.2~0.6V, amplitude: 0.05V；AC impedance condition are as follows: initial potential: 0.2V, amplitude: 0.05V, frequency range 1~
100kHz。
Macrophage Cell electrochemical sensor detection Gram-negative bacteria lipopolysaccharides prepared by the present invention compares tradition side
Method has the advantage that
(1) inherently belong to immune system as the macrophage of sensor information, can occur when cell contacts LPS strong
Immune response, show as in form cell membrane surface microvillus and reduce, intracellular calcium increases, generate a large amount of active oxygens,
Inflammatory factor etc., therefore the spirit of detection will be greatly improved using macrophage as the medium of detection pathogenic bacteria to construct sensor
Sensitivity and reaction speed；
(2) complex film modified electrode is prepared using biologically active chitosan, improves cell to a certain extent
Adhesion strength and vigor.The present invention, as the signal of detection, not only increases the sensitive of detection using electrochemical gaging impedance
The speed for spending and accelerating detection, is just realizing the quick detection to pathogenic bacteria at the drawbacks of overcoming conventional method really.
Measured impedance value and LPS concentration are in 1~5 μ g mL-1It is in good linear relationship, equation of linear regression are as follows: y=in range
153.27x+120.47 r=0.9944, detection is limited to 0.3 μ g mL-1(S/N=3).The impedance value that will test brings standard curve into
The testing result of LPS can be obtained in equation, can determine that gram causes with the content of LPS is compared by detection data result
The pathogenic power of germ.
Detailed description of the invention
Fig. 1: the flow chart of electro-chemical cells sensor building；
Fig. 2: the adherent cell time (A) of electro-chemical cells sensor and adsorption concentration (B) optimization figure: (A) a 0min；b
1min；c 3min；d 5min；e 10min；f 15min；(B)a 6×102mL-1；b 6×103mL-1；c 6×104mL-1；d 6
×105mL-1；e 6×106mL-1；f 6×107mL-1；g 6×108mL-1；
Fig. 3: carbon electrode surface modification characterizes cyclic voltammogram (A) and differential pulse figure (B)；
A bare electrode；Carbon electrode after b carbon nanotube-is chitosan-modified；Magnetic after c gold nano-carbon nanotube-is chitosan-modified
Electrode, carbon electrode adsorbs magnetic nano particle after d gold nano/Carbon Nanotubes/Chitosan modification；E gold nano/carbon nanotube/shell is poly-
Carbon electrode adsorbs magnetic cell after sugar-modified
Fig. 4: modified electrode superficial cell SEM scanning electron microscope (SEM) photograph；
Fig. 5: LPS detection AC impedance figure and standard curve.
Specific embodiment
The preparation of 1 electro-chemical cells sensor of embodiment
The process of electro-chemical cells sensor building is as shown in Figure 1, be that will first be added in carboxylic carbon nano-tube dispersion liquid
HAuCl4Gold nano/Carbon Nanotubes/Chitosan dispersion liquid is prepared, modified electrode on electrode is added drop-wise to, then receives the magnetic of preparation
Cell absorption is fixed to the magnetic glassy carbon electrode surface of modification, preparation using magnetism into mouse macrophage by rice grain endocytosis
Obtain cell sensor.
1, nanogold-carbon nanotube-chitosan complex film modifies carbon electrode
It is the clean method of carbon electrode magnetic glassy carbon electrode is as follows: carbon electrode (Φ=5mm) is placed in Piranha solution
After impregnating 15min, respectively with 0.3 μm, 0.05 μm of Al2O3Powder is polished, electrode surface polishing is thrown into mirror surface, then use 1:1
(V:V) nitric acid, dehydrated alcohol, ultrapure water is successively cleaned by ultrasonic 5min, is dried with nitrogen, and 4 DEG C spare.By the carboxylic of 10mg after purification
Base multi-walled carbon nano-tube is dispersed in 20mL chitosan solution, and ultrasonic 2h is simultaneously centrifuged 10min, and strong stirring will at room temperature
1mL 25mM HAuCl4It is added in carbon nanotube-chitosan dispersion, 80 DEG C of heating stirrings do not change to solution colour.
20 μ l gold nanos/Carbon Nanotubes/Chitosan dispersant liquid drop is added to thorough clean magnetic glassy carbon electrode surface, is dried under infrared lamp
It is dry.
2, the preparation of magnetic nano particle
By 1.35g FeCl3·6H2O dissolves in 40mL ethylene glycol, and 3.6g anhydrous sodium acetate is added and 1.0g polyethylene glycol is strong
Strong stirring 30min, is sealed in autoclave, is heated to 200 DEG C of reaction 8h, is cooled to room temperature, product dehydrated alcohol and ultrapure
Water respectively washes 4 times, in a vacuum drying oven 80 DEG C be dried overnight it is spare.
3, the culture and fixation of cell
It is 0.05mg mL that the magnetic nano particle of synthesis, which is configured to concentration, with RPMI 1640 culture medium-1, by mouse macrophage
Cell is with every hole 106mL-1It is inoculated in six orifice plates, the above-mentioned culture solution containing magnetic nano particle of 2mL is added in every hole, is placed in 37 DEG C 5%
CO2It is cultivated in incubator.Magnetic cell is directly adsorbed to the magnetoelectricity pole surface modified.
4, cell sensor electrochemistry is identified
Cyclic voltammetric and ac impedance measurement are carried out to the cell electrochemical sensor prepared, SEM scanning electron microscope carries out
Characterization.Differential Pulse Voltammetry condition are as follows: scanning range: -0.2~0.6V, amplitude: 0.05V；AC impedence method condition are as follows: just
Beginning current potential: 0.2V, amplitude: 0.05V, 1~100kHz of frequency range.In the building process of cell sensor, to adherent cell
Concentration be optimized to obtain stable cell sensor, as a result as shown in Fig. 2, as seen from the figure, working as cell concentration
Reach 6 × 106mL-1When, R that electrode measuresetLasting variation no longer occurs, illustrates that electrode surface is at this time to reach adsorption saturation,
The optimal adsorption concentration of cell is 6 × 106mL-1.As shown in figure 3, the complex film modified magnetoelectricity of gold nano/Carbon Nanotubes/Chitosan
(than unmodified bare electrode, (curve a) is remarkably reinforced curve c) electric signal after extremely, illustrates that gold nano/carbon nanotube/shell is poly-
The sugared good electric conductivity of composite membrane, after cell is adsorbed onto modified electrode, due to the insulating properties of cell membrane, electric signal is substantially reduced
(curve e), as shown in figure 4, showing that cell has been adsorbed onto modified electrode surface, it was demonstrated that cell sensor is successfully prepared.
The application of 2 electro-chemical cells sensor of embodiment
1, the detection of LPS
It weighs 1mg LPS and is dissolved as 1mg mL with PBS-1Solution.Being diluted to concentration with RPMI 1640 culture medium is respectively 1,
1.5,2,2.5,3,5 μ g mL-1LPS solution.The cell sensing electrode that will have been modified is respectively placed in various concentration LPS solution 37
DEG C be incubated for 3h after, insert electrodes into electrolytic cell and measure after being rinsed with PBS.
2, analysis condition and method
Analysis method is as follows: electrochemical workstation ATUO LAB is in initial amplitude 0.05V, under conditions of frequency is 1~100kHz,
It is 2.5mM Fe (CN) in reaction medium liquid6 3-/4-It is measured in solution, using EIS method, and passes through best equivalence circuit meter
It calculates, adsorbs the impedance value of magnetic cell after modifying using working electrode as blank AC impedance Ret(Ab), with this electrode with contain
Having the post-stimulatory cell of various concentration LPS surveyed impedance value in same electrolyte is Ret(Ab-Ag), it finds out dense in different LPS
R in degreeetChanging value △ Ret:
△Ret=Ret(Ab-Ag)-Ret(Ab)
Ret(Ab): blank AC impedance；
Ret(Ab-Ag): transmitting resistance value with the electrodic electron of the post-stimulatory cell of LPS；
Δ Ret: the changing value of reaction front and back electrodic electron transmitting resistance；
It is mapped with the relationship of the changing value of electron transmission resistance and LPS solution concentration to get the detection of LPS cell sensor
Standard curve.
3, result judges
By preparing a series of LPS solution, measurement does standard song to obtain the impedance value of various concentration LPS solution
Line (as shown in Figure 4).Its measured impedance value and LPS concentration are in 1~5 μ g mL-1It is in good linear relationship in range, it is linear
Regression equation is y=153.27x+120.47, r=0.9944, and detection is limited to 0.3 μ g mL-1(S/N=3).
The impedance value that will test brings in calibration curve equation the testing result that LPS can be obtained into, can pass through testing number
The pathogenic power of gram pathogenic bacteria is determined with the content of LPS is compared according to result.
The LPS of micro rank can be quickly measured in 3h using this method, high sensitivity, accuracy are good, therefore can be with
It carries out providing technical support for gram negative pathogenic bacteria present in rapid quantitative detection and identification food.Cell of the invention
Sensor is that the cell of endocytosis magnetic nanometer is directly adsorbed on the carbon electrode containing detachable magnetic core by magnetism, and magnetic core is torn open
Except rear electrode loses magnetism, cell, which loses magnetism, to fall off from electrode surface, therefore the cell sensor can be by controlling magnetic
Property realize circulating repetition use.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Claims (3)
1. a kind of method of quickly detection gram negative pathogenic bacteria toxicity, which is characterized in that the method first prepares gold nano-
Sensor, is then placed in the LPS of different known concentrations and is incubated for by carbon nanotube-chitosan complex film cell sensor, punching
It inserts the sensors into electrolytic cell and measures after washing, obtain the impedance value of various concentration LPS solution, draw standard curve, so
The LPS of the bacterial strain to be detected extracted is configured to solution afterwards, impedance value is detected using sensor, obtained impedance value is brought into
The LPS content of bacterial strain to be detected can be obtained in standard curve；For pathogenic bacteria of the same race, LPS content is higher to illustrate strain to be tested
Bacterial content is more, toxicity is stronger, for different types of pathogenic bacteria, what the difference bacterium to be measured of identical bacterial content extracted
LPS content is higher, toxicity is stronger；The cell sensor is the mouse macrophage Ana-1 that endocytosis is had to magnetic nano particle, i.e.,
What magnetic cell was directly adsorbed to construct on gold nano-carbon nanotube-chitosan complex film modification carbon electrode；
The preparation method of the gold nano-carbon nanotube-chitosan complex film cell sensor is: (1) cleaning magnetic glass carbon electricity
Pole；(2) functionalized multi-wall carbonnanotubes are dispersed in chitosan solution, ultrasound, centrifugation obtain carbon nanotube-chitosan dispersion
Liquid, then HAuCl is added in strong stirring at room temperature4, 80 DEG C of heating stirrings do not change to solution colour, obtain Jenner
Rice/Carbon Nanotubes/Chitosan dispersion liquid；(3) gold nano/Carbon Nanotubes/Chitosan dispersant liquid drop is added to thorough clean magnetic
Property glassy carbon electrode surface, dry under infrared lamp, obtain gold nano-carbon nanotube-chitosan complex film modification carbon electrode；(4) so
Magnetic cell modification magnetoelectricity pole surface is directly adsorbed to afterwards to sense to get to gold nano-carbon nanotube-chitosan complex film cell
Device；
The incubation is in 37 DEG C of incubation 3-4h；
The magnetic cell the preparation method is as follows: the magnetic nano particle of synthesis be configured to concentration with RPMI 1640 culture medium being
0.05mg mL-1, culture solution containing magnetic nano particle is obtained, by mouse macrophage with every hole 106mL-1It is inoculated in six orifice plates, often
The above-mentioned culture solution containing magnetic nano particle of 2mL is added in hole, is placed in 37 DEG C of 5%CO2It is cultivated in incubator；
The preparation of the magnetic nano particle: by 1.35g FeCl3·6H2O dissolves in 40mL ethylene glycol, and 3.6g acetic anhydride is added
Sodium and 1.0g polyethylene glycol stir 30min strongly, are sealed in autoclave, are heated to 200 DEG C of reaction 8h, are cooled to room temperature, and produce
Object dehydrated alcohol and ultrapure water respectively wash 4 times, in a vacuum drying oven 80 DEG C be dried overnight it is spare.
2. the method according to claim 1, wherein the method is: by the method for self assembly by chitosan
Gold nano-carbon nanotube-chitosan complex film modification is obtained in nanogold and the carbon nano-tube material modification to carbon electrode of dispersion
Carbon electrode using the mouse macrophage Ana-1 of endocytosis magnetic nano particle as sensor information, and magnetic cell is directly adsorbed to
The building that cell sensor is completed on the complex film modified carbon electrode of gold nano/Carbon Nanotubes/Chitosan, finally by the cell of preparation
Sensor application is in the detection of LPS；Specific steps are as follows:
(1) nanogold-carbon nanotube-chitosan is self-assembled modified on carbon electrode；
(2) preparation of magnetic nano particle；
(3) preparation of the culture of mouse macrophage and magnetic cell；
(4) absorption of the magnetic cell on carbon electrode；
(5) detection of LPS.
3. the method according to claim 1, wherein described as follows using sensor detection impedance value method: electricity
Chem workstation ATUO LAB is in initial amplitude 0.05V, is 2.5mM Fe in reaction medium liquid under conditions of frequency is 1~100kHz
(CN)6 3-/4-It is measured in solution, using EIS method, adsorbs the impedance value of magnetic cell after modifying using working electrode as blank
AC impedance Ret(Ab), with this electrode with contain the surveyed impedance in same electrolyte of the post-stimulatory cell of various concentration LPS
Value is Ret(Ab-Ag), the R in different LPS concentration is found outetChanging value △ Ret:
△Ret=Ret(Ab-Ag)-Ret(Ab)
Ret(Ab): blank AC impedance；
Ret(Ab-Ag): transmitting resistance value with the electrodic electron of the post-stimulatory cell of LPS；
Δ Ret: the changing value of reaction front and back electrodic electron transmitting resistance；
It is mapped with the relationship of the changing value of electron transmission resistance and LPS solution concentration to get the examination criteria of LPS cell sensor
Curve.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510945115.2A CN105424771B (en) | 2015-12-16 | 2015-12-16 | A kind of method of quick detection gram negative pathogenic bacteria toxicity |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510945115.2A CN105424771B (en) | 2015-12-16 | 2015-12-16 | A kind of method of quick detection gram negative pathogenic bacteria toxicity |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105424771A CN105424771A (en) | 2016-03-23 |
| CN105424771B true CN105424771B (en) | 2018-12-04 |
Family
ID=55503117
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510945115.2A Active CN105424771B (en) | 2015-12-16 | 2015-12-16 | A kind of method of quick detection gram negative pathogenic bacteria toxicity |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105424771B (en) |
Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107219274B (en) * | 2017-05-27 | 2019-05-17 | 江南大学 | A kind of cell electrochemical sensor for analyzing mycotoxin joint toxicity |
| CN107449821B (en) * | 2017-08-14 | 2019-05-28 | 南京农业大学 | A method of oxidation resistance is evaluated using cell sensor |
| US20210231637A1 (en) * | 2018-04-27 | 2021-07-29 | Ion Chat Research Corporate | Method for measuring intracellular potential with a capacitance type potential measurement device |
| CN108802148B (en) * | 2018-07-21 | 2020-07-03 | 福建医科大学 | Micro-fluidic paper chip for on-line monitoring of intracellular dopamine based on nano-gold modified silk-screen printing electrode |
| CN112969780A (en) * | 2018-10-29 | 2021-06-15 | 离子通道与转运研究公司 | Electrode with nano structure at front end |
| CN110470721A (en) * | 2019-07-12 | 2019-11-19 | 佛山职业技术学院 | A kind of acrylamide electrochemical fast detecting method |
| CN110632160B (en) * | 2019-09-23 | 2022-02-01 | 南京市食品药品监督检验院 | Three-dimensional cell paper chip sensor and application thereof in bacterial lipopolysaccharide detection |
| CN111812167A (en) * | 2020-07-15 | 2020-10-23 | 哈尔滨工业大学（深圳） | Chemical indirect toxicity detection platform and application thereof |
| CN114019004B (en) * | 2021-11-05 | 2022-09-02 | 核工业四一六医院 | Electrochemical detection method of gram-negative bacteria, sensor and preparation method of sensor |
| CN114199975B (en) * | 2021-11-15 | 2023-07-18 | 江苏大学 | Method for evaluating pesticide immunotoxicity based on three-dimensional cell electrochemical sensor |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102608189A (en) * | 2012-03-30 | 2012-07-25 | 山东理工大学 | Method for manufacturing nanometer magnetic ferroferric oxide modified immunosensor |
| CN204203160U (en) * | 2014-10-24 | 2015-03-11 | 济南大学 | The colibacillary electrochemical sensor of a kind of detection |
| CN104655695A (en) * | 2015-02-11 | 2015-05-27 | 江南大学 | Magnetic molecular imprinting sensor for detecting Gram-negative bacterium signal molecule |
-
2015
- 2015-12-16 CN CN201510945115.2A patent/CN105424771B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102608189A (en) * | 2012-03-30 | 2012-07-25 | 山东理工大学 | Method for manufacturing nanometer magnetic ferroferric oxide modified immunosensor |
| CN204203160U (en) * | 2014-10-24 | 2015-03-11 | 济南大学 | The colibacillary electrochemical sensor of a kind of detection |
| CN104655695A (en) * | 2015-02-11 | 2015-05-27 | 江南大学 | Magnetic molecular imprinting sensor for detecting Gram-negative bacterium signal molecule |
Non-Patent Citations (6)
| Title |
|---|
| A label-free electrochemical impedance immunosensor based on AuNPs/PAMAM-MWCNT-Chi nanocomposite modified glassy carbon electrode for detection of Salmonella typhimurium in milk;Jing Dong等;《Food Chemistry》;20130510;第141卷;第1981-1982页 * |
| Attenuation of cellular toxicity by calpain inhibitor induced by bacterial endotoxin: a mechanistic study using muscle precursor cells as a model system;Ke Shang等;《Mol Biol Rep》;20150327;第42卷;第1286页 * |
| Development of highly sensitive electrochemical genosensor based on multiwalled carbon nanotubes–chitosan–bismuth and lead sulfide nanoparticles for the detection of pathogenic Aeromonas;António Maximiano Fernandes等;《Biosensors and Bioelectronics》;20140728;第63卷;第399–406页 * |
| Enzymatic amplification detection of peanut allergen Ara h1 using a stem-loop DNA biosensor modified with a chitosan-mutiwalled carbon nanotube nanocomposite and spongy gold film;Xiulan Sun等;《Talanta》;20140814;第131卷;第521–527页 * |
| Fluorescent magnetic bead-based mast cell biosensor for electrochemical detection of allergens in foodstuffs;Donglei Jiang等;《Biosensors and Bioelectronics》;20150328;第70卷;摘要 * |
| Synthesis and electrochemical properties of single-walled carbon nanotube–gold nanoparticle composites;Hui-Jun Jiang等;《Materials Chemistry and Physics》;20081024;第114卷;第879–883页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105424771A (en) | 2016-03-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105424771B (en) | A kind of method of quick detection gram negative pathogenic bacteria toxicity | |
| Huang et al. | Silver nanoparticles coated graphene electrochemical sensor for the ultrasensitive analysis of avian influenza virus H7 | |
| CN104459124B (en) | A kind of preparation method of the electrochemical immunosensor based on HS-β-CD-Ag-GOD conjugate and application | |
| Zhang et al. | Optimized dendrimer-encapsulated gold nanoparticles and enhanced carbon nanotube nanoprobes for amplified electrochemical immunoassay of E. coli in dairy product based on enzymatically induced deposition of polyaniline | |
| Jain et al. | Development of an antibody functionalized carbon nanotube biosensor for foodborne bacterial pathogens | |
| Han et al. | Electrochemical immunosensor based on self-assembled gold nanorods for label-free and sensitive determination of Staphylococcus aureus | |
| CN108020587A (en) | The detection method of the staphylococcus aureus in milk of dual signal amplification | |
| CN106442994B (en) | A kind of preparation method and application of the electrochemical immunosensor based on Ag@Au nano composite materials | |
| Wang et al. | Electrochemical immunosensor for α-fetoprotein detection using ferroferric oxide and horseradish peroxidase as signal amplification labels | |
| Wang et al. | Rapid and ultrasensitive detection of Salmonella typhimurium using a novel impedance biosensor based on SiO2@ MnO2 nanocomposites and interdigitated array microelectrodes | |
| Wang et al. | Immunosensor based on electrodeposition of gold-nanoparticles and ionic liquid composite for detection of Salmonella pullorum | |
| Tabrizi et al. | Flow injection amperometric sandwich-type aptasensor for the determination of human leukemic lymphoblast cancer cells using MWCNTs-Pdnano/PTCA/aptamer as labeled aptamer for the signal amplification | |
| Feng et al. | A novel electrochemical immunosensor based on Fe3O4@ graphene nanocomposite modified glassy carbon electrode for rapid detection of Salmonella in milk | |
| Zhao et al. | Sensitive and selective dopamine determination in human serum with inkjet printed Nafion/MWCNT chips | |
| Zhang et al. | An ultrasensitive multi-walled carbon nanotube–platinum–luminol nanocomposite-based electrochemiluminescence immunosensor | |
| Su et al. | Chemical pre-reduction and electro-reduction guided preparation of a porous graphene bionanocomposite for indole-3-acetic acid detection | |
| Wang et al. | A Sensitive and simple macrophage-based electrochemical biosensor for evaluating lipopolysaccharide cytotoxicity of pathogenic bacteria | |
| Zhu et al. | Cooperation mode of outer surface and inner space of nanochannel: separation-detection system based on integrated nanochannel electrode for rapid and facile detection of Salmonella | |
| Gao et al. | A novel electrochemical immunosensor using β-cyclodextrins functionalized silver supported adamantine-modified glucose oxidase as labels for ultrasensitive detection of alpha-fetoprotein | |
| Wei et al. | Molecularly imprinted electrochemical sensor for the determination of ampicillin based on a gold nanoparticle and multiwalled carbon nanotube‐coated pt electrode | |
| CN102520168B (en) | Immunosensor for detecting aspergillus parasiticus used for producing aflatoxin and preparing method thereof | |
| CN103822949A (en) | Preparation method and application of electrochemical immunosensor for detecting escherichia coli | |
| CN106442666B (en) | Ion liquid functionalization azotized carbon nano sheet modified electrode and its preparation and the application for detecting chlorophenol | |
| CN109239343A (en) | A kind of fast quantitative measurement method for detecting of Lactobacillus rhamnosus bacteriophage | |
| CN109507409A (en) | A kind of test strips and its preparation method and application based on the amplification of molybdenum disulfide composite nano materials signal |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |