CN109900757A - A kind of aptamer sensor and preparation method thereof - Google Patents
A kind of aptamer sensor and preparation method thereof Download PDFInfo
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- CN109900757A CN109900757A CN201910106931.2A CN201910106931A CN109900757A CN 109900757 A CN109900757 A CN 109900757A CN 201910106931 A CN201910106931 A CN 201910106931A CN 109900757 A CN109900757 A CN 109900757A
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- 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
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- 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
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
Abstract
The present invention relates to a kind of property using the specific recognition between target molecule and its aptamer, the listeria monocytogenes fast super sensitivity detection devices based on nanochannel confinement characteristic of building.The invention further relates to the multiaperture pelluminas that will be modified through the DNA aptamer of listeria monocytogenes as electrode, assembling self-control electrolytic cell；Potassium ferricyanide ion is as probe ion, the method detected to listeria monocytogenes.When LM exists and concentration is lower, electric current lift-off value is remarkably decreased with the increase of its concentration；With the increase of LM concentration, current variation value decline；It is in a linear relationship between electric current lift-off value when LM concentration is within the scope of 100-1250CFU/mL；When its concentration is greater than 1500CFU/mL, current variation value tends towards stability.Therefore, which can complete detection to the minimum detection limit of LM up to 100CFU/mL, range of linearity 100-1250CFU/mL in 10 minutes.Escherichia coli and staphylococcus aureus control experiment through 108CFU/mL show that it has high selectivity to Listeria.
Description
The application is, application number 201610333218.8, entitled " is based on aptamer on May 2016 applying date 19
Modify multiaperture pellumina the highly sensitive new detecting method of Listeria " divisional application.
Technical field
The present invention relates to a kind of property using the specific recognition between target molecule and its aptamer, building based on nanometer
The listeria monocytogenes super sensitivity detection device of channel confinement characteristic.The invention further relates to will be through listeria monocytogenes
The multiaperture pellumina of DNA aptamer modification is as electrode, assembling self-control electrolytic cell；Potassium ferricyanide ion is right as probe ion
Listeria monocytogenes carry out quick, Sensitive Detection method.
Background technique
Listeria monocytogenes (scientific name: Listeria monocytogenes), are a kind of facultative anaerobic bacterias,
For the pathogen of Listeria disease；It is gram-positive bacteria, belongs to Firmicutes.Listeria monocytogenes (following letter
Claim LM) be a kind of zoonosis pathogen.It is with Escherichia coli O 157: H7, staphylococcus aureus, together with salmonella
Four big food-borne pathogens are classified as by the World Health Organization.Listeriosis is exactly to be caused by Listeria Monocytogenes
The rare serious disease of one kind.This pathogen can be killed under normal cooking temp, also can be low in refrigerated storage temperature
It is slowly grown under conditions of to 0 DEG C.The people of most of health, which may occur without, shows clinical symptoms but infected Li Si
The case where special bacterium, but some Susceptible population, such as people (such as AIDS patient, glycosuria of pregnant woman, the elderly and hypoimmunity
Patient etc.), extremely serious health risk can be faced.The presence of LM in food may be (especially former derived from its ingredient
Material) or from surrounding environment, including equipment, the cross contamination between finished product and raw material.If in rings such as temperature
Under border condition, the especially permission of acidity and moisture content, LM can be grown by the process mass propagation stored for a long time.LM's
Growth is for the of less demanding of environment, regardless of aerobic or anoxic environment is ok；Its wider model of temperature range grown,
But its optimum growth temperature is 37 DEG C, at 4 DEG C, just relatively slowly, -18 DEG C of whens, can survive for growth；It is resistance to high salt and acidproof
Property it is strong: 20% sodium chloride, pH value range be 4.4~9.6 under conditions of can grow；Partial pressure of oxygen is slightly lower, titanium dioxide
Well-grown under conditions of carbon pressure power is slightly higher.It is nearly ubiquitous in the natural environment that these properties result in LM.Because LM is
One of most fatal foodborne bacterial pathogens can cause 20~30% clinical infection person dead, so, have very to the detection of LM
Big necessity.
Currently, the detection method of LM has very much, it can be divided into following several: the 1. method of traditional separation and identification；②
The Rapid Identification of chromogenic culture medium；3. the method for full automatic microorganism analysis system 4. molecular biology；5. immunology side
Method etc..The traditional detection method of Listeria in food has 3 key links: increasing bacterium, separation, identification, has in whole process
A series of biochemical reaction and other experiments.This method is not only time-consuming also especially cumbersome.The Rapid identification skill of chromogenic culture medium
Art is then the glucuroide and virulence factor for LM.Full automatic microorganism analysis system is the developing direction of microorganism detection
One of, its process is then fairly simple, convenient, and accuracy rate is relatively high.However in the detection process of LM, if tested test sample
The small number of target bacteria in product, this makes detection have certain difficulty.By the effort of people, based on nucleic acid basis
Molecular biology for detection occur.This method is completed to examine by expanding the specific gene signal of a small amount of target bacteria
It surveys, can achieve the detection in the level of listeria or kind.Application of the polymerase chain reaction (PCR) in food inspection
So that it not only can use DNA molecular, RNA molecule can also be used.The detection speed of these probe reagent boxes is fast and detection is tied
Fruit is accurate.Immunologic detection method is based on antibody for the natural affinity between antigen, but needed for this detection method
The sample wanted is to have to pass through purifying ability accurate detection to go out LM.However it is compared with PCR detection, polymerase chain reaction
Testing result is more easier to be influenced by sample.This detection method is that very fast and accurately, and operating process is fairly simple.
But, its deficiency is exactly that sensitivity is low, poor specificity.Therefore, it is intended that it is more accurate, more to develop a kind of detection for LM
Add sensitive and more convenient and fast method.
Emerging nano material is used due to its special nature is concerned.Zhang Guangteng et al. utilizes gold nano grain
The fixed mercapto-modified DNA probe of glass-carbon electrode is prepared for the electrochemica biological sensor of detection LM in its surface.This method
Minimum detectability reached 1.0 × 10-12Mol/L (DNA concentration meter).Zhong Ziqing et al. is exempted from using the nanometer of surface modification
Epidemic disease magnetic bead is enriched with the LM in sample, is adsorbed separation under magnetic fields to eliminate disturbing factor；To improve inspection
Sensitivity is surveyed, reaches high detection rate and shortens detection time.The present invention receives using through the aptamer modified multiaperture pellumina of LM
Rice material as electrode carries out Electrochemical Detection to LM.
Metallic aluminium chemical property is active, is easily oxidized and forms one layer of fine and close aluminum oxide film layer in aluminium surface.Aluminium oxide
Film layer is more stable, can prevent internal aluminium from continuing to be oxidized, play a protective role to aluminium.Through studying, aluminum oxide film layer is nanometer
Porous structure is able to maintain fixed shape and structure feature.Aluminium is added in some acid mediums and carries out electrification as anode
Reaction is learned, multiaperture pellumina, also known as porous anodic alumina films (porous aluminium oxide film, abbreviation are generated
PAA film).PAA film has the micropore of hexagonal, forms the nano array structure of special, self-organizing high-sequential；These micropores
Perpendicular to aluminum substrate.The diameter of micropore changes with the variation of anodic oxidation condition.It is micro- according to different oxidizing condition
The diameter in hole can change between 10~500nm.Understanding and understanding with people to the microstructure and properties of PAA film,
PAA film be gradually widely used as sensor material, separation membrane material, antimicrobial film material, catalysis material, optics and photoelectric cell,
Magnetic pipe recording material etc..The present invention makees the sensor base element in Electrochemical Detection using PAA film.
Aptamer sensor is one kind of biosensor.Biosensor covers biology, chemistry and signal processing etc.
Technology, specificity, accuracy and precision with height.It is divided into many types: aptamer sensor, microbial sensitive
Device, immunosensor and enzyme sensor etc..In terms of the safety detection of food, the application of aptamer sensor is increasingly by people
Pay close attention to.The present invention is tested using aptamer sensor.Aptamer is one section of single stranded DNA or RNA oligonucleotide chain,
The target cell with high-affinity can be specifically bound.Compared with antibody, aptamer has great potentiality.High-affinity resists
The production of body is costly and time-consuming.It therefore, is all very difficult for the preparation of the antibody of any one target molecule.But parent
With power height, specificity is high, and stability is high, simple to mark, and PCR directed expansion nucleic acid and low cost production etc. are all that aptamer is opposite
In terms of the advantage of antibody.To target molecule, during identification, aptamer has the specific three-dimensional structure of comparison.
After since nineteen ninety, first aptamer is found, different types of aptamer is assessed in vitro, there is protein, poison
Element, ATP, or even virus and cell surface marker.Therefore, because the selectivity and sensitivity of its height, are based on nearly all class
The biosensor and nano biological sensor of the aptamer of type have been widely studied.Aptamer used in the present invention is DNA suitable
Body.It is section of DNA mononucleotide chain, there is 47 amino, wherein " 5 ' " terminal modified aldehyde radical (5 '-CHO-ATC CAT
GGG GCG GAG ATG AGG GGG AGG AGG GCG GGT ACC CGG TTG AT-3 ') it is more in order to be fixed in
The nanochannel inner surface of porous aluminum oxide film.This aptamer can by it is external it is artificial synthesized, method is simple；It can carry out targeting knowledge
Not；There is the affinity of height with LM；There is high selectivity.This is also one important advantage of aptamer sensor.Therefore, originally
Inventive method can be accurate, and simply, height suitably detects LM.
Summary of the invention
The present invention relates to the listeria monocytogenes super sensitivity detection devices based on nanochannel confinement characteristic, prepare the inspection
The purposes that the method and the detector for surveying device detect single listeria monocytogenes.
On the one hand, the present invention provides through listeria monocytogenes DNA aptamer modification porous aluminas membrane electrode,
Wherein the multiaperture pellumina electrode is the multiaperture pellumina that surface is modified through the DNA aptamer of listeria monocytogenes,
And it is gold-plated in its another side.Specifically, the aperture of the multiaperture pellumina is 20nm.More specifically, the DNA aptamer is
DNA mononucleotide chain has 47 amino, wherein " 5 ' " terminal modified aldehyde radical (5 '-CHO-ATC CAT GGG GCG GAG
ATG AGG GGG AGG AGG GCG GGT ACC CGG TTGAT-3′).In addition, more specifically, it is described it is gold-plated be by making
It is realized with vacuum ion sputtering instrument.
On the other hand, the present invention provides the methods for preparing above-mentioned porous aluminas membrane electrode, which comprises (1)
Multiaperture pellumina is gradually cleaned, boil, is impregnated to modify functional group；(2) with vacuum ion sputtering instrument in porous aluminas
Film one side is gold-plated, crosses modeling is made membrane electrode；(3) it will be specifically bound with listeria monocytogenes aptamer modified in porous oxygen
On the channel inner surface for changing aluminium film.Specifically, the aperture of the multiaperture pellumina is 20nm.More specifically, in step (1)
In, hydroxyl is modified using 30% hydrogen peroxide；Amino is modified using 3- aminopropyl triethoxysilane.In step (3)
In, used aptamer is DNA mononucleotide chain, has 47 amino, wherein " 5 ' " terminal modified aldehyde radical (5 '-CHO-ATC
CAT GGG GCG GAG ATG AGG GGG AGG AGG GCG GGT ACC CGG TTG AT-3′)。
On the other hand, the present invention provides the listeria monocytogenes super sensitivity detections based on nanochannel confinement characteristic
Device comprising above-mentioned porous aluminas membrane electrode and electrolytic cell, wherein the electrolytic cell contains sodium dihydrogen phosphate and phosphoric acid hydrogen two
The PBS buffer solution that the pH value that sodium is prepared is 7, and the potassium ferricyanide ion as probe ion.Specifically, the PBS is slow
The concentration for rushing solution is 1mM；The concentration of potassium ferricyanide ion is more preferably 1mM.
On the other hand, monokaryon hyperplasia Lee that the present invention provides above-mentioned porous aluminas membrane electrodes in qualitative detection sample
The purposes of this special bacterium.Specifically, optionally also with staphylococcus aureus (being lower than 108CFU/mL) and large intestine bar in the sample
Bacterium (is lower than 108CFU/mL).
On the other hand, listeria monocytogenes the present invention also provides above-mentioned detector in quantitative detection sample
Purposes.Specifically, (it is lower than in the sample optionally also with staphylococcus aureus (being lower than 108CFU/mL) and Escherichia coli
108CFU/mL)。
The present invention is by Listeria detection technique of the research based on nanochannel confinement characteristic, using aptamer modified through LM
Multiaperture pellumina make electrode assembling self-control electrolytic cell to listeria monocytogenes carry out quantitative detection.It is obtained from experiment,
The listeria monocytogenes that electrochemical detection method based on nanometer confinement space is used to detect in sample liquid are feasible.
The concentration of staphylococcus aureus, Escherichia coli and LM is 108CFU/mL.As shown in the picture, high concentration is detected
There were significant differences for staphylococcus aureus and Escherichia coli and the current variation value of current variation value and LM generated, can define
Ground distinguishes.As above, the LM aptamer of the present invention has very high selectivity, when there are a large amount of Staphylococcus aureus in sample
In the case where bacterium, Escherichia coli, still the detection of LM is not also influenced.
Detailed description of the invention
Fig. 1 is the schematic illustration of aptamer modified multiaperture pellumina detection LM.
Fig. 2 is the aptamer modified schematic diagram in porous aluminas film surface of LM.
Fig. 3 is the scanning electron microscope SEM figure of multiaperture pellumina (PAA).
Fig. 4 is the experimental result of aptamer modified multiaperture pellumina detection LM.(a) current-responsive of various concentration LM is bent
Line；(b) electric current lift-off value is obtained with LM concentration curve figure by the current-responsive curve arrangement in Fig. 4 (a).
Fig. 5 is the control Current experiments figure of staphylococcus aureus (SA), Escherichia coli (E.coli) and LM.(a) different
The current-responsive curve of type bacterium；(b) variation diagram of different type bacterium electric current lift-off value, by the current-responsive curve in Fig. 5 (a)
Arrangement obtains.
Fig. 6 is the control SEM figure of staphylococcus aureus (SA), Escherichia coli (E.coli) and LM.(a) control experiment；
(b)103CFU/mL LM；(c)108CFU/mL E.coli；(d)108CFU/mL SA.
Specific embodiment
In the following, the attached drawing in the embodiment of the present invention will be combined, technical solution in the embodiment of the present invention carries out detailed
Description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on this hair
Embodiment in bright, those of ordinary skill in the art's every other reality obtained without making creative work
Example is applied, shall fall within the protection scope of the present invention.
It should be noted that, in this document, the terms "include", "comprise" or its any other variant are intended to non-row
His property includes, so that the process, method, article or equipment for including a series of elements not only includes those elements, and
And further include other elements that are not explicitly listed, or further include for this process, method, article or equipment institute it is intrinsic
Element.
1 materials and methods
1.1 materials and instrument
Table 1
Table 2
Gold plaque, electric furnace, PET film, the hand held punch of 2mm, high-pressure sterilizing pot, (Varioskan Flash) are full-automatic
Microplate reader etc..Experimental water is ultrapure water (> 18.2M Ω, Millipore France).LM aptamer has " 5 ' " are terminal modified
One aldehyde functions.Buffer solution used is the PBS buffer solution that sodium dihydrogen phosphate and disodium hydrogen phosphate are prepared in electrolytic cell,
PH value is 7.0.
1.2 experimental method
The specific binding and Electrochemical Detection of aptamer and listeria monocytogenes have been used in the present invention.Firstly, with
PAA film preparation gold electrode.The multiaperture pellumina that aperture is 20nm is gradually cleaned, boil, is impregnated to modify functional group.So
Afterwards, with vacuum ion sputtering instrument multiaperture pellumina one side it is gold-plated, cross modeling membrane electrode is made；LM aptamer is modified again in nano-pore
Diameter inner surface.The bacteria suspension of the listeria monocytogenes prepared is added anti-with mounted membrane electrode in self-control electrolytic cell
It answers, is detected.
Fig. 1 show the principle of aptamer modified multiaperture pellumina detection LM.In self-control electrolytic cell, in sample inlet pool
Potassium ferricyanide ion can be reached in the golden film in this face of detection cell by nanochannel, so that high price iron is reduced, be generated
Electric current.Listeria there are when, Listeria and aptamer specifically bind and are attached in PAA film surface, hinder iron cyaniding
Potassium ion is changed by nanochannel, this size for allowing for the electric current generated.Because the aperture of nanochannel is 20nm,
And the size of listeria monocytogenes is 0.5~2.0 μm of 0.4~0.5 μ m；LM is then attached to PAA film surface, causes LM itself
Blocking nano pore decline potassium ferricyanide ion can by the quantity of nanochannel.Secondly, the negative electric field formed around LM
(there are the groups such as phosphoric acid and carboxylic acid on the surface LM, lead to the surface charge of LM band -14.2mV) and potassium ferricyanide ionic interaction
So that the amount of the nanochannel transmission potassium ferricyanide ion near it is also reduced.We can be based on receiving by utilizing as a result,
The electrochemical aptamer sensor of rice material to carry out quantitative detection to listeria monocytogenes.
1.2.1 the preparation of porous aluminas membrane electrode
The preparation of LM shown in Fig. 2 aptamer modified porous aluminas membrane electrode, the specific steps of which are as follows:
(1) it cleans: taking a piece of PAA film in 10mL small beaker.PAA film is cleaned 3-4 times with acetone in draught cupboard, the used time
About 5min, after cleaned with ultrapure water.
(2) it modifies hydroxyl: 30% hydrogenperoxide steam generator of 10mL being added in beaker, is boiled on electric furnace.Keep micro-
Boiling state 30min, outwells remaining hydrogenperoxide steam generator in beaker, cleans PAA film with ultrapure water.
(3) it modifies amino: being separately added into the 3- aminopropyl triethoxysilane (APTES) of 0.5mL in the beaker in (2)
With the acetone of 9.5mL.Preservative film sealing, puts into drier and stands for 24 hours.
(4) dry: to embathe PAA film 4-5 times with acetone in draught cupboard, about 5min, remove the unbonded of remnants
APTES.Then it is cleaned with ultrapure water, by the PAA film cleaned up with being dried with nitrogen.
(5) gold-plated: the PAA film of drying is gold-plated at a surface thereof with vacuum ion sputtering instrument.Gold-plated condition: electric current is
L0mA, time 200s.It should be noted that distinguishing gilding and non-gilding.
(6) envelope modeling: the circular hole of a diameter 2mm is made a call on a pet film with punch.PAA film is clipped among PET film, is made
Circular hole is located at PAA film middle position.Elongated golden conducting wire is placed on the gilding side of PAA film, Chong Die with film.It is sealed with laminator
Modeling, cuts off redundance.PAA membrane electrode is made for use.
1.2.2 the preparation and modification of LM aptamer solutions
LM aptamer is using preceding needing to be centrifuged, centrifugal condition: revolving speed 5000r/min, time 5min.Then suitable in the LM of 1OD
0.1M PBS (pH=7.0) solution of 200 μ L is added in body, is configured to 10 μM of solution.Electrolytic cell is assembled into (gilding court
Detection cell, non-gilding be sample inlet pool).It is separately added into the PBS buffer solution of 2.5mL in sample inlet pool and detection cell, stands
Half an hour.Buffer solution is sucked, electrolytic cell is erected, sample inlet pool is above.Then, it is contacted in sample inlet pool and PAA film
A few drop DNA aptamer solution (air that attention exhausts contact surface) are dripped on face.It places it in confined space 24 hours, side puts half
Cup water is to prevent the volatilization of LM aptamer solutions.The PAA film for having modified LM aptamer is stored in 4 DEG C of PBS solution.
1.2.3 the preparation of LM, Escherichia coli and staphylococcus aureus bacteria suspension
Listeria, Escherichia coli and the culture of staphylococcus aureus.With trypticase soy yeast extract broth (TSB-
YE LM culture) is carried out, culture solution is prepared are as follows: 3.6g (TSB-YE)/100mL；And Escherichia coli and staphylococcus aureus are used
Nutrient broth medium is cultivated, and culture solution is prepared are as follows: 1.9g/100mL.It is cultivated for 24 hours in biochemical cultivation case after inoculation.It will
Cultured bacterium solution respectively takes 1mL to be diluted (0.85 sterile saline) respectively, is diluted to experiment bacterial concentration, to
With.Then the OD600 value of bacterium solution is surveyed with microplate reader, while carrying out colony counting method number clump count with corresponding bacterium solution, is obtained
The relational graph of OD600 value and bacterial concentration, for use next time.
1.2.4 detection method
(1) blank assay: the PAA film for being modified with LM aptamer is examined in self-control electrolytic cell by electrochemical workstation
It surveys.Potential added by electrolytic cell both sides is 0V.
(2) control experiment: the Escherichia coli bacteria suspension made is added in sample inlet pool, detection cell is empty.Half an hour is stood,
Pay attention to excluding the gas of bacterium solution and PAA film contact surface.Electrolytic cell is cleaned with ultrapure water, is detected.Staphylococcus aureus
It detects identical as Escherichia coli step.
(3) detection of Listeria: sample inlet pool is added in the Listeria bacteria suspension prepared, from low concentration to high concentration
Successively detected.The bacteria suspension of each concentration will stand half an hour with sample inlet pool is equally added in (2), clean up, then
Detection.The concentration of each bacteria suspension is measured with the method for plate culture count.
2 results and discussion
The structure of 2.1 multiaperture pelluminas
Fig. 3 is scanning electron microscope (SEM) picture of PAA film, is the porous structure of high-sequential, and aperture size is about
20nm。
The detection of 2.2 listeria monocytogenes
Under optimal conditions, the PBS solution and potassium ferricyanide solution of 1mM concentration are prepared.Listeria bacteria suspension is by dense
Degree gradient is successively detected from low to high.It is added into sample inlet pool, stands 10min at room temperature.It cleans up, is examined
It surveys, as a result as shown in Figure 4.It concludes that electric current first quickly increases at any time by Fig. 4 (a), arrives reach to peak value, then drop to and tend to
Stablize.Current variation value becomes smaller with the increase of LM concentration.The potassium ferricyanide is reached in golden film by the nanochannel of PAA film, iron
Cyaniding potassium ion is restored by gold and generates electric current；And potassium ferricyanide ion first increases to peak by the speed that nanochannel reaches golden film
Then value slowly drops to stabilization.LM concentration becomes larger, and the LM adhered on PAA film is more, nanochannel transmission the potassium ferricyanide from
Son is fewer.Fig. 4 (b) is electric current lift-off value with LM concentration curve figure, is obtained by the current-responsive figure arrangement in Fig. 4 (a).When
When LM exists and concentration is lower, electric current lift-off value is remarkably decreased with the increase of its concentration；With the increase of LM concentration, current variation value
Decline；It is in a linear relationship between electric current lift-off value when LM concentration is within the scope of 100-1250CFU/mL；When its concentration is greater than
When 1500CFU/mL, current variation value tends towards stability.Therefore, the detection method to the minimum detection limit of LM up to 100CFU/mL,
Range of linearity 100-1250CFU/mL can complete detection in 10 minutes.
2.3 control experiment
Under the experiment condition of optimization, the PBS solution and potassium ferricyanide solution of respective concentration are prepared.Successively in sample inlet pool
The Escherichia coli bacteria suspension and staphylococcus aureus bacteria suspension made is added.Half an hour is stood at room temperature.It cleans up, then
It is detected.Testing result is as shown in the figure: Fig. 5 is the control of staphylococcus aureus (SA), Escherichia coli (E.coli) and LM
Lab diagram.The concentration of staphylococcus aureus, Escherichia coli and LM is 108CFU/mL.By scheming, the golden yellow of high concentration is detected
There were significant differences for staphylococcus and Escherichia coli and the current variation value of current variation value and LM generated, can clearly differentiate
Out.This illustrates that the LM aptamer of this experiment has very high selectivity, when there are a large amount of staphylococcus aureuses, big in sample
Enterobacteria does not still also influence the detection of LM.
Fig. 6 is the SEM figure after the aptamer modified PAA film of LM impregnates in different bacterium solutions.Fig. 6 (a) is blank control, Fig. 6
(b), Fig. 6 (c) and Fig. 6 (d) is through LM aptamer modified PAA film respectively in 103CFU/mL Listeria, 108CFU/mL large intestine
SEM figure after being impregnated in bacillus and 108CFU/mL staphylococcus aureus solution.Fig. 6 (a), Fig. 6 (b), Fig. 6 (c), Fig. 6 (d)
It is corresponding with Fig. 5, to demonstrate Fig. 5 conclusion.
3 conclusions
The present invention is by single Listeria detection technique of the research based on nanochannel confinement characteristic, using through LM aptamer
The multiaperture pellumina of modification makees electrode assembling self-control electrolytic cell and carries out qualitative detection to listeria monocytogenes.From testing
Out, the single listeria monocytogenes that the electrochemical detection method based on nanometer confinement space is used to detect in sample liquid are feasible
's.This method not only has the affinity and selectivity of height to the detection of LM, and there are also very high sensitivity.When LM exist and it is dense
When spending lower, electric current lift-off value is remarkably decreased with the increase of its concentration；With the increase of LM concentration, current variation value decline；LM is dense
It is in a linear relationship between electric current lift-off value when degree is within the scope of 100-1250CFU/mL；When its concentration is greater than 1500CFU/mL
When, current variation value tends towards stability.Therefore, the detection method is to the minimum detection limit of LM up to 100CFU/mL, the range of linearity
100-1250CFU/mL can complete detection in 10 minutes.This illustrates method that this experiment uses to the significant excellent of the detection of LM
Point is highly sensitive and selectivity, followed by simple to operation, time saving, low in cost.
Claims (8)
1. a kind of aptamer sensor comprising porous aluminas membrane electrode and prepared containing sodium dihydrogen phosphate and disodium hydrogen phosphate
PH value be 7 PBS buffer solution electrolytic cell, the porous aluminas membrane electrode be surface through listeria monocytogenes
The multiaperture pellumina of DNA aptamer modification, and it is gold-plated in the lateral surface of its electrode.
2. adapter sensor according to claim 1, wherein the aperture of the multiaperture pellumina is 20nm.
3. adapter sensor according to claim 1 or 2 has 47 ammonia wherein the DNA aptamer is DNA mononucleotide chain
Base, wherein " 5 ' " terminal modified aldehyde radical (5 '-CHO-ATC CAT GGG GCG GAG ATG AGG GGG AGG AGG
GCG GGT ACC CGG TTG AT-3′)。
4. adapter sensor as claimed in one of claims 1-3, it is described it is gold-plated be by using vacuum ion sputtering instrument
It realizes.
5. the method for preparing the adapter sensor of claim 1, which comprises
Step (1): multiaperture pellumina is gradually cleaned, boil, is impregnated to modify functional group；
Step (2): with vacuum ion sputtering instrument multiaperture pellumina one side it is gold-plated, cross modeling membrane electrode is made；
Step (3): the aptamer modified nanochannel in multiaperture pellumina that will be specifically bound with listeria monocytogenes
On surface.
6. method according to claim 5, the aperture of the multiaperture pellumina is 20nm.
7. according to the method for claim 5 or 6, wherein in step (1), modify hydroxyl using 30% hydrogen peroxide；Make
Amino is modified with 3- aminopropyl triethoxysilane.
8. according to the method for any one of claim 5-7, in step (3), used aptamer is DNA mononucleotide chain,
There are 47 amino, wherein " 5 ' " terminal modified aldehyde radical (5 '-CHO-ATC CAT GGG GCG GAG ATG AGG GGG
AGG AGG GCG GGT ACC CGG TTG AT-3′)。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910106931.2A CN109900757A (en) | 2016-05-19 | 2016-05-19 | A kind of aptamer sensor and preparation method thereof |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910106931.2A CN109900757A (en) | 2016-05-19 | 2016-05-19 | A kind of aptamer sensor and preparation method thereof |
| CN201610333218.8A CN106018508A (en) | 2016-05-19 | 2016-05-19 | Novel high-sensitivity LM (listeria monocytogene) detection method based on aptamer modified porous alumina membrane |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
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| CN107741446A (en) * | 2016-11-18 | 2018-02-27 | 广东海洋大学 | Mercury ion electrochemical sensor based on nanochannel confinement effect and application thereof |
| CN108982613A (en) * | 2018-07-02 | 2018-12-11 | 浙江大学 | Based on nanochannel arrays surface spraying plating carbon/platinum integrative electrochemical electrode system |
| CN108982614A (en) * | 2018-07-02 | 2018-12-11 | 浙江大学 | Integrative electrochemical electrode system based on nanochannel arrays surface spraying plating platinum |
| CN108982615B (en) * | 2018-07-02 | 2020-08-11 | 浙江大学 | Integrated electrochemical electrode system based on nanochannel array surface gold/platinum spraying |
| CN108982612B (en) * | 2018-07-02 | 2020-08-11 | 浙江大学 | Integrated electrochemical electrode system based on nanochannel array surface gold spraying |
| CN110879243A (en) * | 2019-12-04 | 2020-03-13 | 江南大学 | Preparation method and application of functional biological inorganic nano composite membrane |
| CN112098487A (en) * | 2020-08-08 | 2020-12-18 | 青岛科技大学 | Nano-pore photoelectric chemical DNA sensor and preparation method and application thereof |
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| CN111812177A (en) * | 2020-07-22 | 2020-10-23 | 吉林大学 | Porous anodic alumina-Cas/dCas family protein composite sensing membrane and preparation method and application method thereof |
| CN111812177B (en) * | 2020-07-22 | 2022-12-27 | 吉林大学 | Porous anodic alumina-Cas/dCas family protein composite sensing membrane and preparation method and application method thereof |
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