CN106018508A - Novel high-sensitivity LM (listeria monocytogene) detection method based on aptamer modified porous alumina membrane - Google Patents
Novel high-sensitivity LM (listeria monocytogene) detection method based on aptamer modified porous alumina membrane Download PDFInfo
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- CN106018508A CN106018508A CN201610333218.8A CN201610333218A CN106018508A CN 106018508 A CN106018508 A CN 106018508A CN 201610333218 A CN201610333218 A CN 201610333218A CN 106018508 A CN106018508 A CN 106018508A
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- 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
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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
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Abstract
The invention relates to a fast ultra-sensitive LM (listeria monocytogene) detector based on nanochannel confinement characteristics and constructed according to the nature of specific identification between a target molecule and an aptamer of the target molecules. The invention further relates to a method for detecting the LM by taking potassium ferricyanide ions as probe ions and taking an LM DNA modified porous alumina membrane as an electrode for assembling a self-made electrolytic tank. When LM is present and the concentration is lower, a current increasing value is remarkably reduced with the increase of the concentration; a current change value is reduced with increase of the LM concentration; when the LM concentration is in a range of 100-1,250 CFU/mL, a linear relation is formed between the LM concentration and the current increasing value; when the LM concentration is higher than 1,500 CFU/mL, the current change value becomes stable. Therefore, the lowest detection limit of the detection method for the LM can reach 100 CFU/mL, the linear range is 100-1,250 CFU/mL, and the detection can be completed within 10 min; a 108 CFU/mL of escherichia coli and staphylococcus aureus control experiment indicates that the method has high selectivity on the LM.
Description
Technical field
The present invention relates to a kind of utilize target molecule and its fit between the character of specific recognition,
The listeria monocytogenes super sensitivity detection device based on nanochannel confinement characteristic built.This
The multiaperture pellumina that invention further relates to the DNA aptamer through listeria monocytogenes is modified is made
For electrode, assemble self-control electrolyzer；Potassium ferricyanide ion is as probe ion, to monokaryon hypertrophy
Listerella is carried out quickly, the method for Sensitive Detection.
Background technology
Listeria monoeytogenes (formal name used at school: Listeria monocytogenes), is one
Plant facultative anaerobic bacteria, for the pathogen of Listerella disease；It is gram positive bacteria, belongs to heavy wall
Bacterium door.Listeria monoeytogenes (being the most all called for short LM) is the disease of a kind of zoonosis
Former bacterium.By the world together with it and Escherichia coli O 157: H7, staphylococcus aureus, Salmonella
Health organization is classified as four big food-borne pathogens.Listeriosis is exactly by monocyte hyperplasia Lee
The microbial a kind of the most serious rare disease of this Te Shi.This pathogen can be in normal culinary art
At a temperature of be killed, also slowly can grow under conditions of refrigerated storage temperature as little as 0 DEG C.Great majority
Healthy people it may happen that do not show clinical symptoms but infected listerial situation,
But the people of some Susceptible population, such as anemia of pregnant woman, old people and hypoimmunity is (such as AIDS sufferer
Person, diabetics etc.), extremely serious health risk can be faced.LM in food deposits
Come from its composition (particularly raw material) being probably or come from the environment of surrounding, including setting
Standby, between finished product and raw material cross-contamination.If in environmental conditions such as temperature, especially
Under the permission of acidity and moisture, LM can be grown by the process amount reproduction of long-term storage.
Less demanding for environment of growth of LM, no matter aerobic or the environment of anoxia can；Its
The wider model of temperature range of growth, but its optimum growth temperature is 37 DEG C, at 4 DEG C
Shi Shengchang the most slowly, can be survived when-18 DEG C；Resistance to high salt and acid resistance are strong: the chlorine of 20%
Change sodium, pH value can grow in the range of under conditions of 4.4～9.6；Lower slightly at partial pressure of oxygen,
Well-grown under conditions of pressure carbon dioxide is slightly higher.These character result in LM at natural environment
In nearly ubiquitous.Because LM is one of the most fatal foodborne pathogens, can cause
20～30% clinical infection person dead, so, the detection to LM has the biggest necessity.
At present, the detection method of LM has a lot, can be divided into following several: the most traditional separation
With the method identified；2. the Rapid Identification of chromogenic culture medium；3. full automatic microorganism analysis
The method of system 4. molecular biology；5. immunological method etc..Listerial biography in food
System detection method has 3 key links: increases bacterium, separate, identify, whole during have and one be
The biochemical reaction of row and other experiment.This method is not only the most loaded down with trivial details.Colour developing is cultivated
The Rapid Identification of base is then the glucosidase for LM and virulence factor.Full-automatic micro-life
Thing analysis system is one of developing direction of microorganism detection, and its process is the most fairly simple, just
Victory, and accuracy rate is higher.But during the detection of LM, if in detected sample
The quantity of target bacteria is fewer, and this makes to detect certain difficulty.By the effort of people,
Molecular biology for detection based on nucleic acid basis occurs in that.This method is a small amount of by amplification
The specific gene signal of target bacteria and complete detection, the water of listeria or kind can be reached
Detection on Ping.The polymerase chain reaction (PCR) application in food inspection makes it not only may be used
To utilize DNA molecular, it is also possible to use RNA molecule.The detection speed of these probe reagent boxes is fast
And testing result is accurate.Immunologic detection method is for the nature between antigen based on antibody
Affinity, but the sample required for this detection method is to have to pass through purification just can accurately examine
Measure LM.But it is compared with PCR detection, the testing result of polymerase chain reaction is more held
Easily affected by sample.This detection method is the most fast and accurately, and operating process is compared
Simply.But, its deficiency is exactly that sensitivity is low, poor specificity.Therefore, it is intended that work out
A kind of detection for LM more accurately, sensitiveer and method more easily.
Emerging nano material is used because its special nature is concerned.Zhang Guangteng et al. profit
Fix the DNA probe of sulfydryl modification in its surface with the glass-carbon electrode of gold nano grain, be prepared for
The electrochemica biological sensor of detection LM.The minimum detectability of this method has reached 1.0 × 10-12
Mol/L (DNA concentration meter).Zhong Ziqing et al. utilizes the nano immune magnetic bead of surface modification to inspection
LM in sample is enriched with, and under the action of a magnetic field, adsorbed separation is to eliminate interference factor；Thus
Improve detection sensitivity, reach high recall rate and shorten the detection time.The present invention uses warp
Multiaperture pellumina nano material aptamer modified for LM makees electrode, and LM is carried out Electrochemical Detection.
Metallic aluminium chemical property is active, the most oxidized and form one layer of fine and close oxidation on aluminum surface
Aluminum membranous layer.Pellumina layer is more stable, can prevent the aluminum of inside from continuing oxidized, play aluminum
Protective effect.After deliberation, pellumina layer is nano-porous structure, it is possible to keep fixing shape
Shape and architectural feature.Aluminum is added in some acid mediums and is electrochemically reacted as anode,
Generate multiaperture pellumina, also known as porous anodic alumina films (porous aluminium oxide
Film, is called for short PAA film).PAA film has the micropore of hexagonal, is formed special, self-organizing
The nano array structure of high-sequential；These micropores are perpendicular to aluminum substrate.The diameter of micropore along with
The change of anodic oxidation condition changes.According to different oxidizing condition, the diameter of micropore
Can change between 10～500nm.Along with people are to the microstructure and properties of PAA film
Understanding and understanding, PAA film is gradually widely used as sensor material, separation membrane material, antibacterial film
Material, catalysis material, optics and photoelectric cell, magnetic pipe recording material etc..The present invention uses
PAA film makees the sensor base element in Electrochemical Detection.
Aptamer sensor is the one of biosensor.Biosensor covers biology, change
The technology such as and signal processing, have specificity, accuracy and the precision of height.It is divided into
Many types: aptamer sensor, microbiological sensor, immunosensor and enzyme sensor etc.
Deng.In terms of the safety detection of food, the application of aptamer sensor is increasingly paid close attention to by people
?.The present invention uses aptamer sensor to test.Fit is one section of single stranded DNA or RNA
Oligonucleotide chain, it is possible to the specific binding target cell with high-affinity.Compared with antibody,
Fit have great potentiality.The production of the antibody of high-affinity is costly and time-consuming.Therefore,
Preparation for the antibody of any one target molecule is all very difficult.But affinity is high, special
The opposite sex is high, and stability is high, and simple labelling, PCR directed expansion nucleic acid and low-cost production etc. are all
It it is the fit advantage aspect relative to antibody.During target molecule is identified,
Aptamer has the three dimensional structure that comparison is clear and definite.Since nineteen ninety first is fit be found after,
Different types of fit assessed the most in vitro, had protein, toxin, ATP, even
Virus and cell surface marker.Accordingly, because the selectivity of its height and sensitivity, based on several
All types of fit biosensors and nano biological sensor are widely studied.This
Fit used in invention is DNA aptamer.It is section of 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 ') so that being fixed
Nanochannel inner surface at multiaperture pellumina.This is fit can pass through external synthetic,
Method is simple；Targets identification can be carried out；With the affinity that LM has height；There is high selection
Property.This is also one important advantage of aptamer sensor.Therefore, the inventive method can be accurate,
Simply, LM is detected the most aptly.
Summary of the invention
The present invention relates to listeria monocytogenes hypersensitive based on nanochannel confinement characteristic inspection
Survey device, the method preparing this detector, and this detector is to single listeria monocytogenes
Carry out the purposes detected.
On the one hand, the invention provides the DNA aptamer through listeria monocytogenes and modify many
Porous aluminum oxide membrane electrode, wherein said Woelm Alumina membrane electrode is that surface is through monokaryon hypertrophy Li Si
The multiaperture pellumina that the DNA aptamer of special bacterium is modified, and gold-plated at its another side.Specifically,
The aperture of described multiaperture pellumina is 20nm.More specifically, described DNA aptamer is DNA
Mononucleotide chain, have 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 addition, it is more specifically, described gold-plated by using vacuum ion sputtering instrument to realize.
On the other hand, the invention provides the method preparing above-mentioned Woelm Alumina membrane electrode, institute
The method of stating includes: multiaperture pellumina is progressively cleaned, boils, soaks to modify functional group by (1)；
(2), mistake the most gold-plated in multiaperture pellumina one side with vacuum ion sputtering instrument is moulded and is made membrane electrode；(3)
By the aptamer modified passage at multiaperture pellumina specific binding with listeria monocytogenes
On inner surface.Specifically, the aperture of described multiaperture pellumina is 20nm.More specifically,
In step (1), the hydrogen peroxide of 30% is used to modify hydroxyl；Use 3-aminopropyl-triethoxy
Amino modified by silane.In step (3), used fit be DNA mononucleotide chain, have
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 invention provides monokaryon hypertrophy Li Si based on nanochannel confinement characteristic
Special bacterium super sensitivity detection device, it includes above-mentioned Woelm Alumina membrane electrode and electrolyzer, Qi Zhongsuo
Stating electrolyzer, to contain the PBS that pH value is 7 buffering that sodium dihydrogen phosphate and disodium hydrogen phosphate prepare molten
Liquid, and the potassium ferricyanide ion as probe ion.Specifically, described PBS buffer solution
Concentration be 1mM；The concentration of potassium ferricyanide ion is more preferably 1mM.
On the other hand, the invention provides above-mentioned Woelm Alumina membrane electrode at qualitative detection sample
In the purposes of listeria monocytogenes.Specifically, optionally also with golden yellow in this sample
Color staphylococcus (less than 108CFU/mL) and escherichia coli (less than 108CFU/mL).
On the other hand, present invention also offers above-mentioned detector monokaryon in detection by quantitative sample
The purposes of hyperplasia Listeria.Specifically, optionally also with Staphylococcus aureus in this sample
Bacterium (less than 108CFU/mL) and escherichia coli (less than 108CFU/mL).
The present invention is by studying Listerella detection technique based on nanochannel confinement characteristic, sharp
Electrode assembling self-control electrolyzer is made to monokaryon hypertrophy Lee with the multiaperture pellumina aptamer modified through LM
This special bacterium carries out detection by quantitative.Draw from experiment, Electrochemical Detection based on nanometer confinement space
It is feasible that method is used for examining the listeria monocytogenes in sample measuring liquid.
The concentration of staphylococcus aureus, escherichia coli and LM is 108CFU/mL.Such as accompanying drawing institute
Show, the detection staphylococcus aureus of high concentration and escherichia coli and the current variation value that produces with
There were significant differences for the current variation value of LM, it is possible to distinguishes clearly.As above, the present invention uses
LM fit have the highest selectivity, when sample exists substantial amounts of staphylococcus aureus, big
In the case of enterobacteria, the still detection also no impact on LM.
Accompanying drawing explanation
Fig. 1 is the principle schematic of aptamer modified multiaperture pellumina detection LM.
Fig. 2 is the aptamer modified schematic diagram on multiaperture pellumina 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 () is different dense
The current-responsive curve of degree LM；B () electric current lift-off value is with LM concentration curve figure, by Fig. 4 (a)
In current-responsive curve arrange draw.
Fig. 5 is staphylococcus aureus (SA), escherichia coli (E.coli) and the comparison electric current of LM
Lab diagram.The current-responsive curve of (a) dissimilar bacterium；(b) dissimilar bacterium electric current lift-off value
Variation diagram, by Fig. 5 (a) current-responsive curve arrange draw.
Fig. 6 is staphylococcus aureus (SA), escherichia coli (E.coli) and the comparison SEM of LM
Figure.(a) control experiment；(b)103CFU/mL LM；(c)108CFU/mL E.coli；(d)108
CFU/mL SA。
Detailed description of the invention
Below, the accompanying drawing in the embodiment of the present invention will be combined, to the technology in the embodiment of the present invention
Scheme is described in detail, it is clear that described embodiment is only that a part of the present invention is real
Execute example rather than whole embodiments.Based on the embodiment in the present invention, the common skill in this area
The every other embodiment that art personnel are obtained on the premise of not making creative work, all
Belong to the scope of protection of the invention.
It should be noted that in this article, term " include ", " comprising " or its any its
His variant is intended to comprising of nonexcludability so that include the process of a series of key element,
Method, article or equipment not only include those key elements, but also include being not expressly set out
Other key elements, or also include intrinsic for this process, method, article or equipment
Key element.
1 materials and methods
1.1 materials and instrument
Table 1
Table 2
Gold plaque, electric furnace, PET film, the hand held card punch of 2mm, high-pressure sterilizing pot, (Varioskan
Flash) full-automatic microplate reader etc..Experimental water is ultra-pure water (＞ 18.2M Ω, Millipore
France).LM is fit has an aldehyde functions " 5 ' " are terminal modified.Used by electrolyzer
Buffer solution is the PBS buffer solution of sodium dihydrogen phosphate and disodium hydrogen phosphate preparation, and pH value is
7.0。
1.2 experimental technique
The present invention has used fit and listeria monocytogenes specific binding and electrochemistry
Detection.First, with PAA film preparation gold electrode.It is the multiaperture pellumina of 20nm by aperture
Progressively clean, boil, soak to modify functional group.Then, with vacuum ion sputtering instrument many
Porous aluminum oxide film one side is the most gold-plated, mistake is moulded and made membrane electrode；Modify LM more fit in nano aperture
Surface.The bacteria suspension of listeria monocytogenes prepared is added in self-control electrolyzer with peace
The membrane electrode reaction installed, detects.
Fig. 1 show the principle of aptamer modified multiaperture pellumina detection LM.In self-control electrolysis
Chi Zhong, the potassium ferricyanide ion in sample inlet pool can by nanochannel thus arrive detection cell this
On the golden film in face so that ferrum is reduced at high price, produce electric current.In the presence of listerial, Lee
This special bacterium, with fit specific binding and be attached on PAA film surface, hinders potassium ferricyanide ion
By nanochannel, the size of this electric current allowing for generation changes.Because nanochannel
Aperture be 20nm, and the size of listeria monocytogenes is 0.4～0.5 μ m 0.5～2.0
μm；LM is then attached to PAA film surface, cause LM itself blocking nano pore make the potassium ferricyanide from
Son can be declined by the quantity of nanochannel.Secondly, negative electric field (the LM table formed around LM
There is the group such as phosphoric acid and carboxylic acid in face, causes the surface charge of LM band-14.2mV) and ferrum cyaniding
Potassium ion interacts and the amount of its neighbouring nanochannel transmission potassium ferricyanide ion is also subtracted
Few.Thus, we just can be by utilizing electrochemical aptamer sensor based on nano material
Listeria monocytogenes is carried out detection by quantitative.
1.2.1 the preparation of Woelm Alumina membrane electrode
The preparation of the Woelm Alumina membrane electrode aptamer modified for LM shown in Fig. 2, its concrete steps
As follows:
(1) clean: take a piece of PAA film in 10mL small beaker.In fume hood clear with acetone
Wash PAA film 3-4 time, used time about 5min, cleans with ultra-pure water after terminating.
(2) modify hydroxyl: add in beaker 10mL 30% hydrogenperoxide steam generator, at electricity
Boil on stove.Holding slight boiling condition 30min, remaining hydrogenperoxide steam generator in outwelling beaker,
PAA film is cleaned with ultra-pure water.
(3) amino is modified: in the beaker in (2), be separately added into 3-aminopropyl three second of 0.5mL
TMOS (APTES) and the acetone of 9.5mL.Preservative film seals, and puts in exsiccator and stands
24h。
(4) it is dried: in fume hood, embathe PAA film 4-5 time with acetone, about 5min, removes
Remaining unconjugated APTES.Then clean with ultra-pure water, the PAA film nitrogen that will clean up
Air-blowing is done.
(5) gold-plated: the PAA film vacuum ion sputtering instrument dried up is the most gold-plated.Gold-plated
Condition: electric current is 10mA, time 200s.It should be noted that differentiation gilding and do not plate
Gold face.
(6) envelope is moulded: make a call to the circular hole of a diameter 2mm on a pet film with card punch.By PAA
Film is clipped in the middle of PET film, makes circular hole be positioned at PAA film centre position.Elongated golden wire is placed on
On the gilding limit of PAA film, overlapping with film.Mould by laminator envelope, cut off redundance.PAA
Membrane electrode is made stand-by.
1.2.2 the preparation of LM aptamer solutions and modification
Need to be centrifuged before use that LM is fit, centrifugal condition: rotating speed 5000r/min, time 5min.
Then at 0.1M PBS (pH=7.0) solution of the fit middle addition 200 μ L of LM of 1OD,
It is configured to the solution of 10 μMs.Electrolyzer is assembled that (gilding is towards detection cell, the most gold-plated
Face is sample inlet pool).The PBS buffer solution of 2.5mL it is separately added in sample inlet pool and detection cell,
Stand half an hour.Sucking buffer solution, erected by electrolyzer, sample inlet pool is above.Connect
, (attention is drained contact to drip several DNA aptamer solution with PAA film on the face that sample inlet pool contacts
The air in face).Placing it in confined space 24 hours, half cup water is put on side in case LM is fit
Solution evaporation.Modify PAA film fit for LM to be stored in the PBS solution of 4 DEG C.
1.2.3 LM, escherichia coli and the preparation of staphylococcus aureus bacteria suspension
The cultivation of Listerella, escherichia coli and staphylococcus aureus.With trypticase Semen sojae atricolor ferment
Female extractum meat soup (TSB-YE) carries out LM cultivation, and culture fluid is formulated as: 3.6g (TSB-YE)/100
mL；And escherichia coli and staphylococcus aureus are all cultivated with nutrient broth medium, training
Nutrient solution is formulated as: 1.9g/100mL.In biochemical cultivation case, 24h is cultivated after inoculation.Will training
The bacterium solution supported respectively takes 1mL and is diluted (sterile saline of 0.85) respectively, is diluted to reality
Test with bacterial concentration, stand-by.Then the OD600 value of bacterium solution is surveyed by microplate reader, simultaneously with corresponding
Bacterium solution carry out colony counting method number clump count, draw the graph of a relation of OD600 value and bacterial concentration,
So that next time uses.
1.2.4 detection method
(1) blank experiment: be modified with PAA film fit for LM in self-control electrolyzer by electrification
Learn work station to detect.Electromotive force added by electrolyzer both sides is 0V.
(2) control experiment: adding in sample inlet pool by the escherichia coli bacteria suspension made, detection cell is empty
?.Stand half an hour, note the gas getting rid of bacterium solution with PAA film contact surface.Clear with ultra-pure water
Wash electrolyzer, detect.The detection of staphylococcus aureus is identical with escherichia coli step.
(3) listerial detection: the Listerella bacteria suspension prepared is added sample inlet pool, from
Low concentration detects successively to high concentration.The bacteria suspension of each concentration equally will add with in (2)
Enter sample inlet pool and stand half an hour, clean up, then detect.The concentration flat board of each bacteria suspension
Colony counting method is measured.
2 results and discussion
The structure of 2.1 multiaperture pelluminas
Fig. 3 is scanning electron microscope (SEM) picture of PAA film, and the porous for high-sequential is tied
Structure, aperture size is about 20nm.
The detection of 2.2 listeria monocytogenes
Under optimal conditions, PBS solution and the potassium ferricyanide solution of 1mM concentration are prepared.Lee
This special bacterium bacteria suspension is detected the most successively by Concentraton gradient.It is added in sample inlet pool,
Left at room temperature 10min.Cleaning up, detect, result is as shown in Figure 4.
Concluded that electric current the most quickly raises in time by Fig. 4 (a), to peaking, then drop to
In stable.Current variation value diminishes with the increase of LM concentration.The potassium ferricyanide is by PAA film
Nanochannel arrives on gold film, and potassium ferricyanide ion is produced electric current by gold reduction；And the potassium ferricyanide
Ion first increases to peak value by the speed of nanochannel arrival gold film and the most slowly drops to surely
Fixed.LM concentration becomes big, and on PAA film, the LM of attachment is the most, the potassium ferricyanide of nanochannel transmission
Ion is the fewest.Fig. 4 (b) be electric current lift-off value with LM concentration curve figure, by Fig. 4 (a)
Current-responsive figure arrange draw.When LM exists and concentration is relatively low, electric current lift-off value is dense with it
The increase of degree is remarkably decreased；With the increase of LM concentration, current variation value declines；LM concentration exists
Time in the range of 100-1250CFU/mL and linear between electric current lift-off value；When its concentration
During more than 1500CFU/mL, current variation value tends towards stability.Therefore, this detection method is to LM
Lowest detectable limit up to 100CFU/mL, range of linearity 100-1250CFU/mL, can be 10
Detection is completed in minute.
2.3 control experiment
Under the experiment condition optimized, the PBS solution of preparation respective concentration and potassium ferricyanide solution.
In sample inlet pool, add the escherichia coli bacteria suspension made successively and staphylococcus aureus bacterium is hanged
Liquid.Left at room temperature half an hour.Clean up, then detect.Testing result such as schemes institute
Show: Fig. 5 is staphylococcus aureus (SA), escherichia coli (E.coli) and the control experiment of LM
Figure.The concentration of staphylococcus aureus, escherichia coli and LM is 108CFU/mL.By scheming,
The detection staphylococcus aureus of high concentration and escherichia coli and the current variation value that produces are with LM's
There were significant differences for current variation value, it is possible to distinguishes clearly.This illustrates the LM of this experiment
Fit have the highest selectivity, when there is substantial amounts of staphylococcus aureus, large intestine bar in sample
Bacterium, still the detection also no impact on LM.
Fig. 6 is the SEM figure after PAA film aptamer modified for LM soaks in different bacterium solution.Fig. 6 (a)
It is to divide through PAA film aptamer modified for LM for blank, Fig. 6 (b), Fig. 6 (c) and Fig. 6 (d)
Not in 103CFU/mL Listerella, 108CFU/mL escherichia coli and 108CFU/mL golden yellow
SEM figure after soaking in color staphylococcus solution.Fig. 6 (a), Fig. 6 (b), Fig. 6 (c), figure
6 (d) is corresponding with Fig. 5, thus demonstrates Fig. 5 conclusion.
3 conclusions
The present invention is by studying single Listerella based on nanochannel confinement characteristic detection skill
Art, utilizes and makees electrode assembling self-control electrolyzer to monokaryon through the multiaperture pellumina that LM is aptamer modified
Hyperplasia Listeria carries out qualitative detection.Draw from experiment, electrification based on nanometer confinement space
It is feasible for learning detection method for the single listeria monocytogenes examined in sample measuring liquid.This
Method does not only have affinity and the selectivity of height to the detection of LM, also has the highest sensitivity.
When LM exists and concentration is relatively low, electric current lift-off value is remarkably decreased with the increase of its concentration；With LM
The increase of concentration, current variation value declines；When LM concentration is in the range of 100-1250CFU/mL,
And it is linear between electric current lift-off value；When its concentration is more than 1500CFU/mL, electric current
Changing value tends towards stability.Therefore, this detection method to the lowest detectable limit of LM up to 100
CFU/mL, range of linearity 100-1250CFU/mL, detection can be completed in 10 minutes.This says
The method of bright experiment employing is high sensitivity and selectivity to the remarkable advantage of the detection of LM, its
Secondary is simple to operation, saves time, with low cost.
Claims (14)
1. the Woelm Alumina membrane electrode modified through the DNA aptamer of listeria monocytogenes, its
Described in Woelm Alumina membrane electrode to be surface modify through the DNA aptamer of listeria monocytogenes
Multiaperture pellumina, and gold-plated at the lateral surface of its electrode.
Woelm Alumina membrane electrode the most according to claim 1, wherein said multiaperture pellumina
Aperture be 20nm.
3. according to the Woelm Alumina membrane electrode of claim 1 or 2, wherein said DNA aptamer
It 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′)。
Woelm Alumina membrane electrode the most as claimed in one of claims 1-3, described gold-plated be
By using vacuum ion sputtering instrument to realize.
5. the method for the Woelm Alumina membrane electrode of preparation claim 1, described method includes:
Step (1): multiaperture pellumina is progressively cleaned, boil, soak to modify functional group；
Step (2):, mistake the most gold-plated in multiaperture pellumina one side with vacuum ion sputtering instrument is moulded and made
Membrane electrode；
Step (3): by specific binding with listeria monocytogenes aptamer modified at porous oxygen
Change on the nanochannel surface of aluminum film.
Method the most according to claim 5, the aperture of described multiaperture pellumina is 20nm.
7., according to the method for claim 5 or 6, wherein, in step (1), use 30%
Hydrogen peroxide modify hydroxyl；3-aminopropyl triethoxysilane is used to modify amino.
8., according to the method any one of claim 5-7, in step (3), used
Fit is DNA mononucleotide chain, have 47 amino, wherein " 5 ' " terminal modified aldehyde
Base (5 '-CHO-ATC CAT GGG GCG GAG ATG AGG GGG AGG AGG GCG GGT ACC
CGG TTG AT-3′)。
9. listeria monocytogenes super sensitivity detection device based on nanochannel confinement characteristic, its
Including the Woelm Alumina membrane electrode any one of claim 1-4 and electrolyzer, wherein said
Electrolyzer contains the PBS buffer solution that pH value is 7 of sodium dihydrogen phosphate and disodium hydrogen phosphate preparation,
And the potassium ferricyanide ion as probe ion.
Detector the most according to claim 9, the concentration of described PBS buffer solution is 1mM；
The concentration of potassium ferricyanide ion is 1mM.
11. according to the Woelm Alumina membrane electrode of claim 1-4 in detection by quantitative sample
The purposes of listeria monocytogenes.
12. purposes according to claim 11, optionally also with less than 10 in this sample8
The staphylococcus aureus of CFU/mL and less than 108The escherichia coli of CFU/mL.
13. detectors according to claim 9 or 10 monokaryon hypertrophy in detection by quantitative sample
Listerial purposes.
14. purposes according to claim 13, optionally also with less than 10 in this sample8
The staphylococcus aureus of CFU/mL and less than 108The escherichia coli of CFU/mL.
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