CN106290326B - Detect colorimetric sensor, preparation method and the application of lipopolysaccharides - Google Patents
Detect colorimetric sensor, preparation method and the application of lipopolysaccharides Download PDFInfo
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- CN106290326B CN106290326B CN201610577032.7A CN201610577032A CN106290326B CN 106290326 B CN106290326 B CN 106290326B CN 201610577032 A CN201610577032 A CN 201610577032A CN 106290326 B CN106290326 B CN 106290326B
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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
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
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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
- G01N2021/751—Comparing reactive/non reactive substances
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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
- G01N2021/7756—Sensor type
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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
- G01N2021/7769—Measurement method of reaction-produced change in sensor
Abstract
The invention discloses a kind of colorimetric sensor, preparation method and applications for detecting lipopolysaccharides, sensor is detected by ultraviolet-visible light spectral technology, it is characterised in that the inhibition that ion is transported in identification of the magnetic nano particle to lipopolysaccharides and its catalytic oxidation activity and liopopolysaccharides double-layer of lipoid similar to peroxidase that aminobenzene boric acid modified is utilized.The inhibition that present invention utilizes 3- amino phenyl boric acids in conjunction with the high specific of lipopolysaccharides and liopopolysaccharides double-layer of lipoid transports ion, it ensure that the high degree of specificity of detection, it is similar to the catalytic oxidation activity of peroxidase by magnetic nano particle, improves the sensitivity of detection.Magnetic nano-catalytic H is captured by ultraviolet-visible light spectral technology2O2The color change that ABTS is generated is aoxidized, the analysis detection of lipopolysaccharides is realized.The method of the present invention is simple, quickly, without signal marks；High sensitivity, can within the scope of 0.00001 μ g/mL to 180 μ g/mL linearity test lipopolysaccharides；High specificity can efficiently differentiate lipopolysaccharides and other control substance of plant drug.
Description
Technical field
The present invention relates to a kind of food hygiene quality characterization processes equipment, methods and applications, more particularly to a kind of bacterium
Instrument for testing endotoxin device, methods and applications are applied to food quality control and health care management analysis technical field.
Background technique
Lipopolysaccharides (LPS) is one of gram-negative bacterial cell wall ingredient, when bacterial death dissolution or manually
Method releases after destroying bacterium cell, therefore claims endotoxin.Endotoxin consists of three parts: lipoid A, core oligosaccharide and special
Property O- o antigen polysaccharide o side chain, toxic component is mainly lipoids A.Lipid A is the glycolipid for constituting endotoxin toxicity, with covalent bond
It is connected to heteroglycan chain.Endotoxin is located at the outermost layer of cell wall, is covered on the glutinous peptide of cell wall, can cause fever, micro- follow
Ring obstacle, endotoxin shock and disseminated intravascular coagulation (DIC) etc..After food-pasteurization, microorganism level tends to meet health
Standard requirements, therefore microorganism level can not reflect the hygienic quality of food processing and production process.But level of endotoxin is sterilized
It then will not change afterwards, therefore it can show well the health level of food processing and production, to serve food hygiene and safety
The control of quality.Therefore, the lipopolysaccharides level for detecting food is of great significance.
Currently, the method for detection lipopolysaccharides has limulus reagent test, Enzyme-Linked Immunospot, rapid silver staining etc..Reagents inspection
Operation is simple for survey method, checks endotoxin high specificity, high sensitivity, but its false positive reaction is more.Enzyme-linked immunization ratio
Compared with simple economy, semiquantitative determination can be carried out within the scope of turbidity appropriate, defect is that specificity is not strong.Rapid silver staining tool
Have time-consuming, cumbersome, precision and it is quantitative poor the features such as.These methods be difficult to meet it is daily quickly analyze want
It asks.It can be seen that establishing endotoxic analyzing novel methods and research and development Mycotoxin identification device becomes technical problem urgently to be resolved.
Summary of the invention
In order to solve prior art problem, it is an object of the present invention to overcome the deficiencies of the prior art, and to provide one kind
Colorimetric sensor, preparation method and the application of lipopolysaccharides are detected, energy is efficient, sensitive, quickly detects lipopolysaccharides, and application prospect is excellent
Different, data measured is accurate and reliable.The present invention is double-deck by the lipoids of the peroxidase activity of magnetic nano particle and lipopolysaccharides
The ion transport that structure is adjusted combines, using the recognition reaction of 3- amino phenyl boric acid and lipopolysaccharides, using UV-vis spectroscopy
Spectrum realizes highly sensitive, high specific analysis detection the purpose to lipopolysaccharides as detection means.
Purpose is created to reach foregoing invention, the present invention uses following inventive concept:
The ion transport that the present invention is adjusted using the lipoids double-layer structure of lipopolysaccharides is with 3- amino phenyl boric acid to lipopolysaccharides
Recognition reaction realize the detection to the specificity and sensitivity of lipopolysaccharides.Mechanism of the present invention is as follows: selecting 3-
Recognition component of the amino phenyl boric acid as lipopolysaccharides passes through the carboxylic of amino group and magnetic nano particle surface in 3- amino phenyl boric acid
Base group, which reacts the amido bond to be formed, on small molecule 3- aminobenzene boric acid modified to magnetic nano particle, will obtain surface covering boric acid
The magnetic nano particle of recognition group.Since magnetic nano particle has the property of similar catalase, in acid condition and peroxide
Change hydrogen (H2O2) it is existing under the conditions of, catalysis 2,2- connection (3- ethyl-benzothiazole -6- sulfonic acid) di-ammonium salts (ABTS) of nitrogen-two oxidation
For 2'- hydrazine-bis- -3- ethyl benzo thiazole phenanthroline -6- sulfonic acid free radical (ABTS.-), occur high absorption peak in certain wave strong point,
Supernatant shows deeper green.Under the conditions of having existing for lipopolysaccharides, the boric acid base group and rouge on magnetic nano particle surface are more
Dihydroxy of taking advantage of a situation in sugar on sugar chain carries out specific recognition and reacts to form boric acid ester bond, and lipopolysaccharides is on magnetic nano particle surface
It is double-deck to form lipoids, hinders hydrogen ion (H+) contacted with magnetic nano particle surface, inhibit have catalytic activity ferrous iron from
Son (Fe2+) formation, while hindering Fe2+It is entered in solution from magnetic bead surfaces, H can not be catalyzed2O2ABTS is aoxidized, in certain wave
There is low absorption peak in strong point, while shallower green is presented in supernatant.In this detection architecture, magnetic nano particle surface boron
The specific binding of acid groups and lipopolysaccharides and liopopolysaccharides double-layer of lipoid hinder H+Transport and inhibition Fe2+Form the Gao Te of transport
Anisotropic and magnetic nano particle is similar to the highly sensitive feature of the catalytic activity of peroxidase, makes highly sensitive high special
Property analysis detection lipopolysaccharides is possibly realized.
Conceived according to foregoing invention, the present invention adopts the following technical solutions:
A kind of colorimetric sensor detecting lipopolysaccharides, mainly by kit, equipment for separating liquid from solid, ultraviolet-visible spectrum point
Analysis apparatus, analysis system and control system composition, the control system control the supply and detection data of the reagent in kit
Output and input；By on 3- aminobenzene boric acid modified to magnetic nano particle, using equipment for separating liquid from solid, magnetic nano particle is obtained
Surface covers the MNPs magnetic nano particle of boric acid recognition group, and the MNPs magnetic nano particle for preparing covering boric acid recognition group is molten
Liquid, as the first identification agent, storage is set in the first kit；In acid condition, join (the 3- second of nitrogen-two according to 2,2-
Base-benzothiazole -6- sulfonic acid) di-ammonium salts and hydrogen peroxide setting mixed proportion, by 2,2- join (the 3- ethyl-benzo thiophene of nitrogen-two
Azoles -6- sulfonic acid) di-ammonium salts are mixed with hydrogen peroxide, ABTS-H is made2O2Acidic solution system, as the second identification agent,
Storage is set in the second kit；According to setting mixed proportion, using the MNPs magnetic nano particle in the first identification agent as
Catalyst, using hydrogen peroxide as oxidant, using first identification agent and second identification agent as reactant into
Row mixing, is made MNPs-ABTS-H2O2Reactant system, the product system solution formed after reaction are utilized as referring to reagent
Ultraviolet-visible spectrum analytical equipment, the spectrum of detection reference reagent, and using the reference reagent as the ginseng with characteristic spectrum
Examine element；First identification agent and second identification agent are in addition taken according to setting mixed proportion, it first will be described
First identification agent is mixed with lipopolysaccharides sample solution to be measured first, obtains the magnetic nanometer of liopopolysaccharides double-layer of lipoid covering
Grain solution, as the MNPs-LPS system solution of preliminary target detection sample, then by MNPs-LPS system solution and described
The mixing of second identification agent, forms the MNPs-LPS-ABTS-H of the target detection sample of optimization after at least 5 min2O2Reagent
System makes MNPs-LPS-ABTS-H2O2Reagent system utilizes ultraviolet-visible spectrum as final target detection sample reagent
Analytical equipment detects MNPs-LPS-ABTS-H2O2The spectrum of reagent system passes through analysis system and the feature referring to reagent
Spectrum compares, and the contents level of lipopolysaccharides detected is determined by spectrum comparing result.
3- ammonia as currently preferred technical solution, in the MNPs magnetic nano particle solution described in the first identification agent
The mass fraction of the magnetic nano particle of base phenyl boric acid modification is not less than 2%(w/v).
The technical solution further preferred as above-mentioned technical proposal, in the second identification agent ABTS-H2O2Acid solution body
In system, 2,2- connection (3- ethyl-benzothiazole -6- sulfonic acid) di-ammonium salts of nitrogen-two and hydrogen peroxide mixing mol ratio are 1:(0.7
~0.9).
The technical solution further preferred as above-mentioned technical proposal, according to MNPs magnetic nano particle quality and ABTS-H2O2
The molal quantity of ABTS in acid solution is mixed for the ratio of 1:0.005~1:0.02(g/mol), prepares MNPs- respectively
ABTS-H2O2Reaction system and MNPs-LPS-ABTS-H2O2Reagent system.
The rouge of lipopolysaccharides sample solution to be measured is established in the technical solution further preferred as above-mentioned technical proposal, setting
Polysaccharide concentration and certain wave strong point MNPs-LPS-ABTS-H2O2The calculating mould of curved line relation between the absorbance value of reagent system
Block carries out quantitative calculating to the lipopolysaccharide concentration of lipopolysaccharides sample solution to be measured, and passes through output device for lipopolysaccharide concentration
Testing result is exported.
A kind of preparation method of the colorimetric sensor of present invention detection lipopolysaccharides, includes the following steps:
A. magnetic nano particle surface covering boric acid identification base will on 3- aminobenzene boric acid modified to magnetic nano particle, be obtained
The MNPs magnetic nano particle solution of group, as the first identification agent, the 3- amino phenyl boric acid in MNPs magnetic nano particle solution is repaired
The mass fraction of the magnetic nano particle of decorations is not less than 2%(w/v)；
B. in acid condition, join (3- ethyl-benzothiazole -6- sulfonic acid) di-ammonium salts of nitrogen-two and peroxidating according to 2,2-
Hydrogen mix mol ratio be 1:(0.7~0.9) mixed proportion, by 2,2- join nitrogen-two (3- ethyl-benzothiazole -6- sulfonic acid)
Di-ammonium salts are mixed with hydrogen peroxide, and ABTS-H is made2O2Acidic solution system, as the second identification agent；
C. according to MNPs magnetic nano particle quality and ABTS-H2O2The molal quantity of ABTS in acid solution is 1:0.005
~1:0.02(g/mol) ratio, take the first identification agent prepared in the step a and obtained in the b
First identification agent is mixed with lipopolysaccharides sample solution to be measured first first, obtains liopopolysaccharides by two identification agents
Then the magnetic nano particle solution of double-layer of lipoid covering will as the MNPs-LPS system solution of preliminary target detection sample
MNPs-LPS system solution and second identification agent mixing, form the target detection sample of optimization after at least 5 min
MNPs-LPS-ABTS-H2O2Reagent system makes MNPs-LPS-ABTS-H2O2Reagent system is as final target detection sample
Product reagent detects MNPs-LPS-ABTS-H using ultraviolet-visible light spectral technology analytical equipment2O2The spectrum of reagent system, with institute
The characteristic spectrum stated referring to reagent compares, and the content water of lipopolysaccharides detected is determined by spectrum comparing result
It is flat.
As currently preferred technical solution, the step a include it is following step by step:
1. by a certain amount of FeCl3﹒ 6H2O is added in ethylene glycol, is stirred to after being completely dissolved, and sodium citrate two is added
Water stirs at least 30 min, adds 1.200 g sodium acetates and continues 30 min of strong stirring, is then transferred into reaction kettle,
At least 10 h are reacted in 210 DEG C of baking ovens, obtain reaction product；
2. will the step 1. obtained in reaction product take out and be down to room temperature, then respectively with ethyl alcohol and go from
At least three times, Magneto separate obtains magnetic nano particle to sub- water washing, adds deionized water, obtains mass fraction not less than 1%(w/
V) magnetic nano particle solution；
3. by 0.22 M 1- ethyl-(3- dimethylaminopropyl) carbodiimide hydrochloride and 0.22 M N- hydroxyl amber
Amber acid imide is added in 1 mL MNPs, ultrasonic vibration at least 30 min, and after then at least washing three times, the 4 of 0.2 mL are added
MM 3- amino phenyl boric acid ethanol solution, mixing concussion at least 3 hours, then wash at least three times, deionized water is added, obtains
MNPs magnetic nano particle solution.
The technical solution further preferred as above-mentioned technical proposal, the step c include it is following step by step:
The MNPs magnetic nano particle solution prepared in the step a and lipopolysaccharides sample solution to be measured are mixed in by I
In Tris-HCl reaction buffer of the pH value no more than 5.4, cultivates at room temperature at least 30 minutes, obtain reaction mixture；
II to the reaction mixture obtained in the step I using Magneto separate after remove supernatant, then use pH value
Tris-HCl reaction buffer washing no more than 5.4 obtains the magnetic nanometer of liopopolysaccharides double-layer of lipoid covering at least three times
Grain solution.
A kind of application of the colorimetric sensor of present invention detection lipopolysaccharides, the lipopolysaccharides in lipopolysaccharides sample solution to be measured
Concentration be 0.00001~180 μ g/mL.
The technical solution further preferred as above-mentioned technical proposal, be suitable for detection drinking water, milk, fruit drink or
Lipopolysaccharide concentration in tea beverage sample.
The present invention compared with prior art, has following obvious prominent substantive distinguishing features and remarkable advantage:
1. present invention measurement lipopolysaccharides colorimetric sensor is selectively good, high sensitivity, detection range are wide；
2. lipopolysaccharides is fixed to the surface of magnetic nano particle by covalent bond by the present invention, so that the fixation of substrate is very
Stablize, to improve the stability of sensor；
3. the present invention utilizes the double grading of lipopolysaccharides, that is, pass through identification and liopopolysaccharides lipid of the boric acid to lipopolysaccharides
Dual layer barrier H+Transport and inhibition Fe2+The detection that lipopolysaccharides is realized in transport is formed, so that sensor strong antijamming capability, specificity
It is high；
4. the present invention utilizes magnetic nano particle Catalyzed Synthesis By Peroxidase H2O2The activity of ABTS is aoxidized to detect lipopolysaccharides
Activity, so that sensor has high sensitivity；
5. the present invention is based on ultraviolet spectral analysis method, with highly sensitive, the response time is short, required instrument and equipment valence
The advantages that lattice are cheap can be widely used in the chemical detection of field of food, and it is active to reach efficient, sensitive, quick detection lipopolysaccharides
Purpose.
Detailed description of the invention
Fig. 1 is the schematic illustration for the colorimetric sensor that the embodiment of the present invention one detects lipopolysaccharides.
Fig. 2 is that the magnetic nano particle of the 3- aminobenzene boric acid modified of the embodiment of the present invention one is catalyzed H2O2Aoxidize ABTS reaction
Later and the magnetic nano particle of 3- aminobenzene boric acid modified is in conjunction with lipopolysaccharides specific recognition and liopopolysaccharides double-layer of lipoid hinders
Magnetic nano particle is hindered to be catalyzed H2O2After aoxidizing ABTS reaction, the H of comparative example one2O2Aoxidize ABTS reaction after and lipopolysaccharides and
H2O2After aoxidizing ABTS reaction, the spectrogram comparison diagram of generation in 380nm ~ 500nm wave-length coverage.
Fig. 3 be the embodiment of the present invention two in the case that various concentration lipopolysaccharides there are, the magnetic of 3- aminobenzene boric acid modified
Nano particle is in conjunction with lipopolysaccharides specific recognition and liopopolysaccharides double-layer of lipoid hinders magnetic nano particle to be catalyzed H2O2Aoxidize ABTS
After reaction, the spectrogram of generation in the nm of 380 nm ~ 500 wave-length coverage.
Fig. 4 is the linear relationship chart at the lipopolysaccharide concentration and 414nm of the embodiment of the present invention two between absorbance value.
Fig. 5 is the lipopolysaccharides (LPS) of the embodiment of the present invention one and bovine serum albumin(BSA) (BSA), the egg white egg of comparative example two
White (OVA), pectin (Pectin) respectively in conjunction with the magnetic nano particle of 3- aminobenzene boric acid modified after obtain in 414nm wavelength
Locate light absorption value comparison diagram.
Specific embodiment
Details are as follows for the preferred embodiment of the present invention:
Embodiment one:
In the present embodiment, referring to Fig. 1, Fig. 2 and Fig. 5, a kind of colorimetric sensor detecting lipopolysaccharides, mainly by reagent
Box, equipment for separating liquid from solid, ultraviolet-visible spectrum analytical equipment, analysis system and control system composition, the control system control
The supply of reagent in kit and outputting and inputting for detection data；By on 3- aminobenzene boric acid modified to magnetic nano particle,
Using equipment for separating liquid from solid, the MNPs magnetic nano particle of magnetic nano particle surface covering boric acid recognition group is obtained, and prepares and covers
The MNPs magnetic nano particle solution of lid boric acid recognition group, as the first identification agent, storage is set in the first kit；?
Under acid condition, mixed according to the setting that 2,2- joins (3- ethyl-benzothiazole -6- sulfonic acid) di-ammonium salts of nitrogen-two and hydrogen peroxide
2,2- connection nitrogen-two (3- ethyl-benzothiazole -6- sulfonic acid) di-ammonium salts are mixed with hydrogen peroxide, ABTS- are made by ratio
H2O2Acidic solution system, as the second identification agent, storage is set in the second kit；According to setting mixed proportion, with the
MNPs magnetic nano particle in one identification agent is as catalyst, and using hydrogen peroxide as oxidant, first identification is tried
Agent and second identification agent are mixed as reactant, and MNPs-ABTS-H is made2O2Reactant system is formed after reaction
Product system solution, as referring to reagent, using ultraviolet-visible spectrum analytical equipment, detection referring to reagent spectrum, and with
The reference reagent is as the reference element with characteristic spectrum；The first identification examination is in addition taken according to setting mixed proportion
First identification agent is mixed with lipopolysaccharides sample solution to be measured first first, is obtained by agent and second identification agent
The magnetic nano particle solution covered to liopopolysaccharides double-layer of lipoid, the MNPs-LPS system as preliminary target detection sample are molten
Then MNPs-LPS system solution and second identification agent are mixed, form the target of optimization after at least 5 min by liquid
The MNPs-LPS-ABTS-H of test sample2O2Reagent system makes MNPs-LPS-ABTS-H2O2Reagent system is as final mesh
Test sample reagent is marked, using ultraviolet-visible spectrum analytical equipment, detects MNPs-LPS-ABTS-H2O2The spectrum of reagent system,
It is compared, and is determined by spectrum comparing result detected by analysis system and the characteristic spectrum referring to reagent
The contents level of lipopolysaccharides.
In the present embodiment, the lipopolysaccharide concentration and certain wave strong point of lipopolysaccharides sample solution to be measured are established in setting
MNPs-LPS-ABTS-H2O2The computing module of curved line relation between the absorbance value of reagent system, to lipopolysaccharides sample to be measured
The lipopolysaccharide concentration of product solution carries out quantitative calculating, and is exported lipopolysaccharide concentration testing result by output device.
In the present embodiment, a kind of preparation method for the colorimetric sensor detecting lipopolysaccharides, includes the following steps:
Step 1: the preparation flow of the MNPs magnetic nano particle of aminobenzene boric acid modified:
1. by the FeCl of 1.080 g3﹒ 6H2O is added in 20 mL ethylene glycol, is stirred to after being completely dissolved, and is added 0.200
Two water of g sodium citrate stirs 30 min, adds 1.200 g sodium acetates and continues 30 min of strong stirring, is then transferred into reaction
In kettle, 10 h are reacted in 210 DEG C of baking ovens, obtain reaction product；
2. will the step 1. obtained in reaction product take out and be down to room temperature, then respectively with ethyl alcohol and go from
Three times, Magneto separate obtains magnetic nano particle to sub- water washing, adds 100 ml deionized waters, and obtaining mass fraction is 1%(w/v)
Magnetic nano particle solution；
3. by the N- hydroxyl of 1- ethyl-(3- dimethylaminopropyl) carbodiimide hydrochloride and 0.22 M of 0.22 M
Base succinimide is added in the MNPs of 1 mL, 30 min of ultrasonic vibration, and after being washed out three times, 4 mM of 0.2 mL are added
3- amino phenyl boric acid ethanol solution, mixing concussion 3 hours, then wash three times is added 20 mL deionized waters, obtains quality point
Count the MNPs magnetic nano particle solution of the 3- aminobenzene boric acid modified for 2%(w/v).
Step 2: 3- amino phenyl boric acid and lipopolysaccharides specific recognition and the reaction process for hindering ion to transport:
I by the process of the step 2 3. in prepare 5 μ L concentration be 2 mg/mL MNPs magnetic nano particle
The lipopolysaccharide concentration of solution and 95 μ L are 50 mM that the lipopolysaccharides sample solution to be measured of 10 μ g/mL is mixed in that pH value is 5.4
Tris-HCl reaction buffer in, at room temperature cultivate 30 minutes, obtain reaction mixture；
II to the reaction mixture obtained in the step I using Magneto separate after remove supernatant, then use pH value
The magnetic nanometer for obtaining liopopolysaccharides double-layer of lipoid three times and covering is washed for the Tris-HCl reaction buffer of 5.4 50 mM
Grain solution, the nano particle can hinder Fe2+、H+Deng ion transport.
Step 3: uv-visible absorption spectra analysis detection lipopolysaccharides process:
100 mM of 50 μ L are added in the magnetic nano particle covered to the liopopolysaccharides double-layer of lipoid prepared in step 2
2- (N- morpholine) ethanesulfonic acid, the H of 50 μ L22, the 2- of O, 5 mM of 20 μ L join (the 3- ethyl-benzothiazole-of nitrogen-two
6- sulfonic acid) di-ammonium salts, the hydrogen peroxide of 4 mM of 20 μ L forms the MNPs- of the target detection sample of optimization after 5 min
LPS-ABTS-H2O2Reagent system makes MNPs-LPS-ABTS-H2O2Reagent system as final target detection sample reagent,
Using ultraviolet-visible light spectral technology analytical equipment, MNPs-LPS-ABTS-H is detected2O2The spectrum of reagent system, and LPS is not added
MNPs-ABTS-H2O2Reagent system characteristic spectrum compares, and determines that rouge detected is more by spectrum comparing result
The contents level of sugar.
As shown in Figure 1, under the conditions of lipopolysaccharides is existing, in magnetic nano particle surface boric acid base group and lipopolysaccharides on sugar chain
Cis-form dihydroxy carry out specific recognition and react to form boric acid ester bond, the structure of liopopolysaccharides double-layer of lipoid hinders H+It is close
Magnetic nano particle surface and Fe2+Formation and transport, H can not be catalyzed2O2Aoxidize ABTS.The preparation of the present embodiment colorimetric sensor
Method and application, the ion transport detection lipopolysaccharides adjusted by pH sensitive kinds double-layer of lipoid, can efficient, sensitive, quick detection
Lipopolysaccharides in milk, tea beverage, fruit drink and drinking water is horizontal, is applied to food and health care analysis technical field.
Comparative example one:
This comparative example is substantially the same as in the previous example, and is particular in that:
In comparison Shi Lizhong, referring to fig. 2, the system of the colorimetric sensor of the detection lipopolysaccharides of MNPs magnetic nano particle is not added
Preparation Method includes the following steps:
2- (N- morpholine) ethanesulfonic acid of 100 mM of 50 μ L, 50 μ L are added into lipopolysaccharides sample solution to be measured
H22, the 2- of O, 5 mM of 20 μ L join nitrogen-two (3- ethyl-benzothiazole -6- sulfonic acid) di-ammonium salts, 4 mM's of 20 μ L
Hydrogen peroxide forms the LPS-ABTS-H of the target detection sample of optimization after 5 min2O2Reagent system makes LPS-ABTS-
H2O2Reagent system detects LPS- using ultraviolet-visible light spectral technology analytical equipment as final target detection sample reagent
ABTS-H2O2The spectrum of reagent system, with the ABTS-H that LPS is not added2O2Reagent system characteristic spectrum compares, and passes through
Spectrum comparing result determines the contents level of lipopolysaccharides detected.As in Fig. 2 it is found that H2O2Aoxidize ABTS reaction after and
Lipopolysaccharides and H2O2After aoxidizing ABTS reaction, the spectrogram comparison diagram that generates in 380nm ~ 500nm wave-length coverage it is found that
H2O2Aoxidize ABTS reaction after and lipopolysaccharides and H2O2The curve of spectrum essentially coincides after oxidation ABTS reaction, without apparent
Spectrum distinguishing characteristics.
According to Fig. 1, in the absence of lipopolysaccharides, magnetic nano particle has the property of similar catalase, in acid
Under the conditions of property, Fe2+It can freely be free in containing H2O2In the solution of ABTS, it is catalyzed H2O2ABTS is aoxidized, at 414nm
High absorbance value is obtained, solution shows deeper green.Under the conditions of existing for the lipopolysaccharides, magnetic nano particle surface boric acid
Group carries out specific recognition with the cis-form dihydroxy on sugar chain in lipopolysaccharides and reacts to form boric acid ester bond, liopopolysaccharides lipid
Double-deck structure hinders H+Close to magnetic nano particle surface and Fe2+Formation and transport, H can not be catalyzed2O2ABTS is aoxidized,
Low absorbance value is obtained at 414nm, solution liquid shows shallower green.As shown in Fig. 2, when there is no lipopolysaccharides to deposit in system
When, the catalytic activity of magnetic nano particle will not be hindered, and can be catalyzed ABTS2-Oxidation generates color change, and light absorption value is bright
Aobvious higher, solution colour is deep.In the presence of having lipopolysaccharides, the catalytic activity of magnetic nano particle is hindered, and ultraviolet absorption value is bright
Aobvious to reduce, solution colour is shallow.
Comparative example two:
This comparative example is particular in that:
The special Journal of Sex Research of biosensor is carried out referring to Fig. 5 in comparison Shi Lizhong:
In order to verify the specificity of present invention detection lipopolysaccharides, we have used different albumen such as bovine serum albumin(BSA)
(BSA), ovalbumin (OVA) and pectin (Pectin) verify the biosensor according to the operating process of embodiment two processing
Specificity.As shown in figure 5, still cannot even if the concentration of BSA, OVA albumen and Pectin are 2000 times higher than lipopolysaccharide concentration
Solution light absorption value is caused significantly to change.This compares the 3- amino phenyl boric acid of reaction descriptions above-described embodiment in conjunction with lipopolysaccharides
The inhibition transported with lipopolysaccharides double-layer of lipoid to ion has the specificity of height, ensure that the height of biosensor detection
Selectivity.
By the comparative analysis of embodiment one and above-mentioned comparative example it is found that embodiment one is double-deck using the lipoids of lipopolysaccharides
The ion transport and 3- amino phenyl boric acid that structure is adjusted realize specificity and spirit to lipopolysaccharides to the recognition reaction of lipopolysaccharides
The detection of quick property.Mechanism used by embodiment one is as follows: selecting recognition component of the 3- amino phenyl boric acid as lipopolysaccharides, passes through
Amino group reacts the amido bond to be formed with the carboxylic group on magnetic nano particle surface for small molecule 3- ammonia in 3- amino phenyl boric acid
In the modification to magnetic nano particle of base phenyl boric acid, the magnetic nano particle of surface covering boric acid recognition group is obtained.Due to magnetic nanometer
Grain has the property of similar catalase, can be in acid condition and hydrogen peroxide (H2O2) it is existing under the conditions of, be catalyzed 2'-
Bis- -3- ethyl benzo thiazole phenanthroline -6- the sulfonic acid (ABTS) of hydrazine-are oxidized to 2'- hydrazine-bis- -3- ethyl benzo thiazole phenanthroline -6- sulfonic acid
Free radical (ABTS.-), occur high absorption peak at 414 nm, supernatant shows deeper green.When with the presence of lipopolysaccharides
Under conditions of, the dihydroxy of taking advantage of a situation in the boric acid base group and lipopolysaccharides on magnetic nano particle surface on sugar chain carries out specific recognition simultaneously
Reaction forms boric acid ester bond, and lipopolysaccharides forms lipoids bilayer on magnetic nano particle surface, hinders hydrogen ion (H+) and magnetic nanometer
Particle surface contact, inhibits the ferrous ion (Fe with catalytic activity2+) formation, while hindering Fe2+From magnetic bead surfaces into
Enter into solution, H can not be catalyzed2O2Oxidation ABTS, the low absorption peak of appearance at 414 nm, while supernatant present shallower
Green.In this detection architecture, the specific binding of magnetic nano particle surface boric acid base group and lipopolysaccharides and lipopolysaccharides lipoid
Matter dual layer barrier H+Transport and inhibition Fe2+The high specific and magnetic nano particle for forming transport are similar to peroxidase
The highly sensitive feature of catalytic activity, makes it possible highly sensitive high specific analysis detection lipopolysaccharides.
Embodiment two:
The present embodiment is basically the same as the first embodiment, and is particular in that:
In the present embodiment, referring to Fig. 3 and Fig. 4, the detection of the lipopolysaccharides of various concentration is carried out.
By lipopolysaccharides (0.00001 μ g/mL, 0.00005 of the magnetic nano particle of 3- aminobenzene boric acid modified and various concentration
μg /mL、0.0001 μg/mL、0.0005 μg/mL、0.001 μg/mL、0.005μg/mL、0.01μg/mL、0.05μg/mL、
0.1μg/mL、0.5μg/mL、1μg/mL、5μg/mL、10μg/mL、20μg/mL、40μg/mL、80μg/mL、180μg/mL、280μ
G/mL, 380 μ g/mL) reaction, after specific recognition combination and oxidation reaction, with the inspection of UV-vis spectroscopy spectroscopic methodology
It surveys.
As shown in figure 3, light absorption value of the reaction product solution at 414 nm wavelength is increased with the concentration of lipopolysaccharides and is reduced.
Under the conditions of Fig. 3 is existing for the various concentration lipopolysaccharides, the spectrogram in 380nm ~ 500nm wave-length coverage.Fig. 4 is 414 nm suction
Linear relationship chart between shading value and lipopolysaccharide concentration, in 0.00001 μ g/mL to the lipopolysaccharide concentration range of 180 μ g/mL
Interior, absorbance increase is linear with lipopolysaccharide concentration.
Above-described embodiment sensor is detected by ultraviolet-visible light spectral technology, and aminobenzene boric acid modified is utilized
Magnetic nano particle is to the identification of lipopolysaccharides and its similar to the catalytic oxidation activity and liopopolysaccharides double-layer of lipoid of peroxidase
To the inhibition of ion transport.Embodiments described above utilize 3- amino phenyl boric acid is in conjunction with the high specific of lipopolysaccharides and rouge is more
The inhibition that carbohydrate double-layer of lipoid transports ion ensure that the high degree of specificity of detection, be similar to by magnetic nano particle
The catalytic oxidation activity of peroxidase improves the sensitivity of detection.Magnetic nanometer is captured by ultraviolet-visible light spectral technology to urge
Change H2O2The color change that ABTS is generated is aoxidized, the analysis detection of lipopolysaccharides is realized.Above-described embodiment method is simple, quickly, nothing
Signal is needed to mark；High sensitivity, can within the scope of 0.00001 μ g/mL to 180 μ g/mL linearity test lipopolysaccharides；Specificity
By force, lipopolysaccharides and other control substance of plant drug can be efficiently differentiated.
Actual sample experiment detection research:
Using sample-adding absorption method, drinking water, milk, fruit drink, tea-drinking are determined with the present embodiment colorimetric sensor
The concentration for expecting lipopolysaccharides in (green tea), referring to following table.It is found that the present embodiment electrochemical sensor being capable of Accurate Determining actual sample
Middle lipopolysaccharide concentration.
Colorimetric sensing of the above-described embodiment using the magnetic nano particle detection lipopolysaccharides of 3- aminobenzene boric acid modified, feature
It is the contact that the specific binding based on 3- amino phenyl boric acid and lipopolysaccharides can hinder magnetic nano particle with extraneous solution, and
Due to the special construction of liopopolysaccharides double-layer of lipoid, so that Fe2+、H+Plasma can not be carried out by liopopolysaccharides double-layer of lipoid from
Son transport, and combine magnetic nano particle that can aoxidize 2,2- with catalyzing hydrogen peroxide and join (the 3- ethyl-benzothiazole -6- sulphur of nitrogen-two
Acid) di-ammonium salts this features, it is analyzed by ultraviolet-visible light spectral technology and detects lipopolysaccharides.
The embodiment of the present invention is illustrated above in conjunction with attached drawing, but the present invention is not limited to the above embodiments, it can be with
The purpose of innovation and creation according to the present invention makes a variety of variations, under the Spirit Essence and principle of all technical solutions according to the present invention
Change, modification, substitution, combination or the simplification made, should be equivalent substitute mode, as long as meeting goal of the invention of the invention,
Colorimetric sensor, preparation method and the technical principle of application and inventive concept of lipopolysaccharides are detected without departing from the present invention,
Belong to protection scope of the present invention.
Claims (10)
1. it is a kind of detect lipopolysaccharides colorimetric sensor, it is characterised in that: mainly by kit, equipment for separating liquid from solid, it is ultraviolet-can
See spectral analysis device, analysis system and control system composition, the supply of the reagent in control system control kit and
Detection data is output and input；
By on 3- aminobenzene boric acid modified to magnetic nano particle, using equipment for separating liquid from solid, the covering of magnetic nano particle surface is obtained
The MNPs magnetic nano particle of boric acid recognition group, and the MNPs magnetic nano particle solution of covering boric acid recognition group is prepared, as
First identification agent, storage are set in the first kit；
In acid condition, join setting for (3- ethyl-benzothiazole -6- sulfonic acid) di-ammonium salts of nitrogen-two and hydrogen peroxide according to 2,2-
Determine mixed proportion, 2,2- connection nitrogen-two (3- ethyl-benzothiazole -6- sulfonic acid) di-ammonium salts are mixed with hydrogen peroxide, are made
ABTS-H2O2Acidic solution system, as the second identification agent, storage is set in the second kit；
Made according to setting mixed proportion using the MNPs magnetic nano particle in the first identification agent as catalyst with hydrogen peroxide
For oxidant, is mixed using first identification agent and second identification agent as reactant, MNPs- is made
ABTS-H2O2Reactant system, the product system solution formed after reaction utilize ultraviolet-visible spectrum point as referring to reagent
Analysis apparatus, the spectrum of detection reference reagent, and using the reference reagent as the reference element with characteristic spectrum；
First identification agent and second identification agent are in addition taken according to setting mixed proportion, first by described first
Identification agent is mixed with lipopolysaccharides sample solution to be measured first, and the magnetic nano particle for obtaining the covering of liopopolysaccharides double-layer of lipoid is molten
Liquid, as the MNPs-LPS system solution of preliminary target detection sample, then by MNPs-LPS system solution and described second
Identification agent mixing, forms the MNPs-LPS-ABTS-H of the target detection sample of optimization after at least 5min2O2Reagent system,
Make MNPs-LPS-ABTS-H2O2Reagent system is filled as final target detection sample reagent using ultraviolet-visible light spectrum analysis
It sets, detects MNPs-LPS-ABTS-H2O2The spectrum of reagent system, by analysis system and the characteristic spectrum referring to reagent into
Row compares, and the contents level of lipopolysaccharides detected is determined by spectrum comparing result.
2. detecting the colorimetric sensor of lipopolysaccharides according to claim 1, it is characterised in that: as the first identification agent,
The mass fraction of the magnetic nano particle of 3- aminobenzene boric acid modified in the MNPs magnetic nano particle solution is not less than 2%w/v.
3. detecting the colorimetric sensor of lipopolysaccharides according to claim 1, it is characterised in that: as the second identification agent,
ABTS-H2O2In acidic solution system, 2,2- connection (3- ethyl-benzothiazole -6- sulfonic acid) di-ammonium salts of nitrogen-two and hydrogen peroxide are mixed
Conjunction mol ratio is 1:(0.7~0.9).
4. detecting the colorimetric sensor of lipopolysaccharides according to claim 1, it is characterised in that: according to MNPs magnetic nano particle matter
Amount and ABTS-H2O2The molal quantity of ABTS in acid solution is that the ratio of 1:0.005~1:0.02g/mol is mixed, respectively
Prepare MNPs-ABTS-H2O2Reaction system and MNPs-LPS-ABTS-H2O2Reagent system.
5. detecting the colorimetric sensor of lipopolysaccharides described according to claim 1~any one of 4, it is characterised in that: setting is built
Found the lipopolysaccharide concentration and certain wave strong point MNPs-LPS-ABTS-H of lipopolysaccharides sample solution to be measured2O2The extinction of reagent system
The computing module of curved line relation between angle value carries out quantitative calculating to the lipopolysaccharide concentration of lipopolysaccharides sample solution to be measured,
And lipopolysaccharide concentration testing result is exported by output device.
6. detecting the preparation method of the colorimetric sensor of lipopolysaccharides described in a kind of claim 1, which is characterized in that including walking as follows
It is rapid:
A. magnetic nano particle surface covering boric acid recognition group will on 3- aminobenzene boric acid modified to magnetic nano particle, be obtained
MNPs magnetic nano particle solution, as the first identification agent, 3- aminobenzene boric acid modified in MNPs magnetic nano particle solution
The mass fraction of magnetic nano particle is not less than 2%w/v；
B. in acid condition, join (3- ethyl-benzothiazole -6- sulfonic acid) di-ammonium salts of nitrogen-two according to 2,2- and hydrogen peroxide is mixed
Conjunction mol ratio be 1:(0.7~0.9) mixed proportion, by 2,2- join nitrogen-two (3- ethyl-benzothiazole -6- sulfonic acid) diammonium
Salt is mixed with hydrogen peroxide, and ABTS-H is made2O2Acidic solution system, as the second identification agent；
C. according to MNPs magnetic nano particle quality and ABTS-H2O2The molal quantity of ABTS in acid solution is 1:0.005~1:
The ratio of 0.02g/mol takes the first identification agent prepared in the step a and the second identification obtained in the b
Reagent first mixes first identification agent with lipopolysaccharides sample solution to be measured first, and it is double to obtain liopopolysaccharides lipid
The magnetic nano particle solution of layer covering, as the MNPs-LPS system solution of preliminary target detection sample, then by MNPs-
LPS system solution and second identification agent mixing, form the MNPs- of the target detection sample of optimization after at least 5min
LPS-ABTS-H2O2Reagent system makes MNPs-LPS-ABTS-H2O2Reagent system as final target detection sample reagent,
Using ultraviolet-visible light spectral technology analytical equipment, MNPs-LPS-ABTS-H is detected2O2The spectrum of reagent system, with the reference
The characteristic spectrum of reagent compares, and the contents level of lipopolysaccharides detected is determined by spectrum comparing result.
7. detecting the preparation method of the colorimetric sensor of lipopolysaccharides according to claim 6, which is characterized in that the step a
Including as follows step by step:
1. by a certain amount of FeCl3﹒ 6H2O is added in ethylene glycol, is stirred to after being completely dissolved, and two water of sodium citrate is added, stirs
At least 30min is mixed, 1.200g sodium acetate is added and continues strong stirring 30min, be then transferred into reaction kettle, dried at 210 DEG C
At least 10h is reacted in case, obtains reaction product；
2. will the step 1. obtained in reaction product take out and be down to room temperature, then washed respectively with ethyl alcohol and deionization
It washs at least three times, Magneto separate obtains magnetic nano particle, adds deionized water, obtains magnetic of the mass fraction not less than 1%w/v and receives
Rice grain solution；
3. by 0.22M 1- ethyl-(3- dimethylaminopropyl) carbodiimide hydrochloride and 0.22M n-hydroxysuccinimide
It is added in 1mL MNPs, after then at least washing three times, the 4mM 3- aminobenzene boron of 0.2mL is added in ultrasonic vibration at least 30min
Sour ethanol solution, mixing concussion at least 3 hours, then wash at least three times, deionized water is added, it is molten to obtain MNPs magnetic nano particle
Liquid.
8. detecting the preparation method of the colorimetric sensor of lipopolysaccharides according to claim 6, which is characterized in that the step c
Including as follows step by step:
The MNPs magnetic nano particle solution prepared in the step a and lipopolysaccharides sample solution to be measured are mixed in pH value not by I
In Tris-HCl reaction buffer greater than 5.4, cultivates at room temperature at least 30 minutes, obtain reaction mixture；
II to the reaction mixture obtained in the step I using Magneto separate after remove supernatant, be then not more than using pH value
5.4 Tris-HCl reaction buffer washing at least three times, obtains the magnetic nano particle solution of liopopolysaccharides double-layer of lipoid covering.
9. detecting the application of the colorimetric sensor of lipopolysaccharides described in a kind of claim 1, it is characterised in that: lipopolysaccharides sample to be measured
The concentration of lipopolysaccharides in product solution is 0.00001~180 μ g/mL.
10. detecting the application of the colorimetric sensor of lipopolysaccharides according to claim 9, it is characterised in that: be suitable for detection drink
With the lipopolysaccharide concentration in water, milk, fruit drink or tea beverage sample.
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