CN104076025B - A kind of antibacterial peptide electrochemical luminous sensor and preparation method thereof and detection method - Google Patents
A kind of antibacterial peptide electrochemical luminous sensor and preparation method thereof and detection method Download PDFInfo
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Abstract
The invention provides a kind of antibacterial peptide electrochemical luminous sensor, the antibacterial peptide electrochemical luminous sensor includes naked gold electrode and is cascading in the L cysteine layers on the naked gold electrode surface, nano gold layer and antibacterial peptide antibody layer that the antibacterial peptide antibody layer is combined together by electrostatic adsorption with the nano gold layer.The antibacterial peptide electrochemical luminous sensor small volume, low cost, can be with sensitive, rapidly on-line checking antibacterial peptide；The invention provides a kind of preparation method of antibacterial peptide electrochemical luminous sensor, the preparation method of the antibacterial peptide electrochemical luminous sensor is simple to operation.Present invention also offers the application of the antibacterial peptide electrochemical luminous sensor, electrochemical luminous sensor stability, repeatability and the specificity are good.
Description
Technical field
The present invention relates to biosensor technique and technical field of electrochemistry, more particularly to a kind of antibacterial peptide electrochemiluminescence biography
Sensor and preparation method thereof and detection method.
Background technology
Antibacterial peptide be it is a kind of be widely present in the biological internal micromolecule polypeptide for possessing multiple biological activities, it has heat
The advantages of stability, broad-spectrum antiseptic and potential antitumor properties, thus it is widely used in clinical medicine, medicine, biological feeding
The fields such as feed additives, antiseptics for natural food and animal and plant disease resisting genetic engineering.The activity of antibacterial peptide, mechanism of action, antibacterial
Peptide is produced and the quality control of antibacterial peptide product has become the hot issue that antibacterial peptide is studied, and the research of these problems is all
It is badly in need of accurate, sensitive, easy antibacterial peptide quantitative detecting method, is especially that by the detection method of on-line analyses.
The detection method of antibacterial peptide mainly has liquid chromatography, LC/MS and high performance capillary electrophoresis at present.But
During using these methods, sample pretreatment is loaded down with trivial details, required expensive equipment and volume is larger, and time-consuming for detection, and to operator
Member has high demands, it is difficult to realize on-line analyses.
Therefore, how to realize inexpensive, sensitive and rapidly on-line checking antibacterial peptide becomes the emphasis of current research.
The content of the invention
To solve the above problems, the invention provides a kind of antibacterial peptide electrochemical luminous sensor and preparation method thereof and should
With, the antibacterial peptide electrochemical luminous sensor low cost, can be with sensitive, rapidly on-line checking antibacterial peptide；The antibacterial peptide
The preparation method of electrochemical luminous sensor is simple to operation.
In a first aspect, the invention provides a kind of antibacterial peptide electrochemical luminous sensor, the antibacterial peptide electrochemiluminescence
Sensor includes naked gold electrode and is cascading in L-Cysteine layer, the nano gold layer on the naked gold electrode surface
With antibacterial peptide antibody layer, the antibacterial peptide antibody layer is combined together by electrostatic adsorption with the nano gold layer.
Preferably, the naked gold electrode and L-Cysteine layer are combined by Au-S bonds.
The antibacterial peptide that the antibacterial peptide antibody layer is adopted is moved from insecticide, plant, mammal, Amphibian, ocean
Thing, birds, antibacterial and virus etc..
Preferably, the antibacterial peptide antibody that the antibacterial peptide antibody layer is adopted is for cecropin B antibody.
L-Cysteine (Cys) is a kind of aminoacid containing sulfydryl, and the naked gold electrode and L-Cysteine layer pass through
With reference to can be very strong Au-S it is bonded, define stable L-Cysteine layer on naked gold electrode surface；L-Cysteine itself
With good electro-chemical activity, electron transfer can be promoted, increase naked gold electrode area, hence it is evident that strengthen the antibacterial peptide electricity
The electrochemiluminescence effect of chemiluminescence sensor.Meanwhile, L-Cysteine has good biocompatibility.
Nanometer gold has high-specific surface area, biocompatibility and high connductivity activity, not only can reduce electrochemiluminescence
Initial potential, moreover it is possible to accelerate the electron transmission between electrode and immune molecule and strengthen electrochemiluminescence intensity, improves electrochemistry and sends out
The sensitivity of light detection and stability.
Preferably, the L-Cysteine layer and nano gold layer are combined together by electrostatic adsorption, nano gold layer
The fixed amount of antibacterial peptide antibody can be increased, improve biomolecule microenvironment, biomolecule activity and work are kept with maximum
The advantages of property molecular orientation is fixed.
The nano gold layer and antibacterial peptide antibody layer are combined together by electrostatic interaction, and the introducing of nanometer gold is not only firm
Secure antibacterial peptide antibody, accelerate electron transfer rate.Reach fixed antibacterial peptide antibody simultaneously align, be orientated
The purpose of rule, further improves the activity of biomolecule.It is electrostatic adsorption between the nanometer gold and antibacterial peptide, therefore
Various antibacterial peptide antibodies can be combined together to form electrochemiluminescence immunosensor by electrostatic interaction with nanometer gold, can
To prepare different types of electrochemiluminescence immunosensor with the different types of antibacterial peptide of detection by quantitative.
Preferably, the remaining site in nano gold layer is closed using the bovine serum albumin.
The bovine serum albumin and antibacterial peptide antibody do not have special requirement, can be through being commercially available.
The antibacterial peptide electrochemical luminous sensor low cost, high specificity, strong antijamming capability, before sample being simplified
Processing procedure, can be with sensitive, rapidly on-line checking antibacterial peptide content.
Second aspect, the invention provides a kind of preparation method of antibacterial peptide electrochemical luminous sensor, including following step
Suddenly：
(1) naked gold electrode is provided, the naked gold electrode is placed in into the L-Cysteine solution that concentration is 15～25mmol/L
In, using cyclic voltammetry in the naked gold electrode electroplating surface L-Cysteine layer, obtain the electricity of L-Cysteine layer modification
Pole；The voltage range of the cyclic voltammetry be -0.5～1.0V, scanning speed be 5～15mV/s, electroplating time be 20～
30min；
(2) the L-Cysteine layer modification electrode surface Deca nanometer gold dispersion liquid, at 4 DEG C stand 20～
24h, prepares nano gold layer in the L-Cysteine layer surface, obtains the electrode of nano gold layer and the modification of L-Cysteine layer；
(3) the electrode surface Deca concentration modified in the nano gold layer and L-Cysteine layer is the anti-of 4～8 μ g/mL
Bacterium peptide antibody solution, and 12～24h is reacted at 4 DEG C, antibacterial peptide antibody layer, the antibacterial are prepared on the nano gold layer surface
Peptide antibody layer is combined together by electrostatic adsorption with the nano gold layer, obtains the antibacterial peptide electrochemiluminescence sensing
Device.
Preferably, the naked gold electrode that step (1) is provided is carried out into pretreatment, the naked gold electrode preprocess method is：Will
Naked gold electrode is polished on the abrasive paper for metallograph of 3000 mesh, then with 0.5 μm of Al2O3Slurry is polished to minute surface on chamois leather, then spend from
After sub- water is rinsed well, fully it is cleaned by ultrasonic in deionized water and dehydrated alcohol respectively；In the H that concentration is 0.5mol/L2SO4
In solution, 1.5V～-0.2V potential ranges interior circulation scanning about 20 is enclosed, and until obtaining stable cyclic voltammogram, takes out electrode
Deionized water is rinsed well standby.
Preferably, in step (1), the naked gold electrode is placed in into the L-Cysteine that 5mL concentration is 15～25mmol/L
In solution.
Preferably, in step (1), the hydrochloric acid containing 0.1mol/L in the L-Cysteine solution.
Preferably, after step (1) plating terminates, it is soaked in water, cleans the electrode that the L-Cysteine layer is modified, to remove
Remove the unreacted L-Cysteine molecule of electrode surface.
Preferably, disperse in the nanometer gold of the 40 μ L of electrode surface Deca of L-Cysteine layer modification in step (2)
Liquid, the particle diameter of the nanogold particle in the nanometer gold dispersion liquid is 7～10nm.
Preferably, nanometer gold dispersion liquid ultrasound before Deca is mixed.
Preferably, in step (2), the preparation method of the nanometer gold dispersion liquid is：
Take 1～2mL concentration to be added in 45～49mL distilled water for 0.02～0.03mol/L chlorauric acid solutions, be heated to
Boiling, then rapidly joins the citric acid three sodium solution that 3～6mL concentration is 0.025～0.035mol/L under agitation, keeps micro-
Boiling, and continuous stirring, when solution gradually becomes colourless, Lycoperdon polymorphum Vitt by light yellow, until when becoming claret, stopping heating, cooling
To room temperature, 50mL is settled to, obtains the nanometer gold dispersion liquid.
Preferably, in step (3), in the electrode surface Deca 40～60 that the nano gold layer and L-Cysteine layer are modified
The antibacterial peptide antibody-solutions of μ L.
It is highly preferred that in step (3), the electrode surface Deca modified in the nano gold layer and L-Cysteine layer 40～
60 μ L concentration are 5 μ g/mL antibacterial peptide antibody-solutions.
Preferably, in step (3), the antibacterial peptide antibody-solutions delay for the phosphate that antibacterial peptide antibody is dissolved in pH=7.4
Rush in solution and obtain.
Preferably, cleaned after the nano gold layer surface prepares antibacterial peptide antibody layer in step (3), cleaned
Operate and be：Electrode containing antibacterial peptide antibody layer is placed in the phosphate buffered solution of the pH=7.4 that concentration is 0.01mol/L
5～8min of immersion, then with the phosphate buffered solution cleaning electrode 2～3 times, to remove antibacterial peptide antibody not to be adsorbed.
Preferably, the antibacterial peptide electrochemical luminous sensor for step (3) being obtained adopts the bovine serum albumin
Remaining site in closing nano gold layer, specifically includes following steps：
The antibacterial peptide electrochemical luminous sensor that step (3) is obtained is placed in bovine serum albumin solution, room temperature
After 30～60min of lower reaction, the antibacterial peptide electrochemical luminous sensor is taken out, then the phosphate-buffered for being placed in pH=7.4
5～8min is soaked in solution, is then cleaned 2～3 times with the phosphate buffered solution, to remove unnecessary bovine serum albumin point
The antibacterial peptide electrochemical luminous sensor is finally preserved stand-by in 4 DEG C of refrigerators by son.
It is highly preferred that the mass concentration of the bovine serum albumin solution is 1%.
It is highly preferred that the concentration of the phosphate buffered solution is 0.01mol/L.
The naked gold electrode and L-Cysteine layer by combine can be very strong Au-S it is bonded, in naked gold electrode surface shape
Into stable electrochemical assembling film；Cys itself has good electro-chemical activity, can promote electron transfer, increase naked
Gold electrode area, hence it is evident that strengthen the electrochemiluminescence effect of the antibacterial peptide electrochemical luminous sensor.The presence of Cys can be with
Avoid the directly contact of antibacterial peptide antibody and naked gold electrode, it is to avoid the decrease to antibacterial peptide antibody activity.
Nanometer gold has high-specific surface area, biocompatibility and high connductivity activity, not only can reduce electrochemiluminescence
Initial potential, moreover it is possible to accelerate the electron transmission between electrode and immune molecule and strengthen electrochemiluminescence intensity, improves electrochemistry and sends out
The sensitivity of light detection and stability.
The Cys layers are connected by electrostatic adsorption with nano gold layer, and nano gold layer can increase antibacterial peptide antibody
Fixed amount, improves molecule microenvironment, realizes the advantages of maximum keeps its activity and bioactive molecule directional at-tachment.
The nanometer gold and antibacterial peptide antibody are combined together by electrostatic interaction, and the introducing of nanometer gold is not only firmly solid
Determine antibacterial peptide antibody, accelerate electron transfer rate.Reach fixed antibacterial peptide antibody simultaneously align, be orientated rule
Purpose, further improve biomolecule activity.It is adsorption between the nanometer gold and antibacterial peptide, therefore polytype
Antibacterial peptide antibody electrochemiluminescence immunosensor can be combined together to form by electrostatic interaction with nanometer gold, this
The preparation method versatility of bright electrochemiluminescence immunosensor is good, can prepare according to preparation method of the present invention not of the same race
The electrochemiluminescence immunosensor of class is with the different types of antibacterial peptide of detection by quantitative.
Antibacterial peptide electrochemical luminous sensor is prepared using the inventive method, method is simple, and the antibacterial peptide for preparing
Electrochemical luminous sensor small volume, low cost, strong antijamming capability can simplify the pretreatment process of sample, it is adaptable to corpusculum
In product biotic environment, the detection of antibacterial peptide, is capable of achieving the on-line analyses to antibacterial peptide.
The third aspect, above-described antibacterial peptide electrochemical luminous sensor are applied to the quantitative detecting method of antibacterial peptide,
Comprise the following steps：
The antibacterial peptide electrochemical luminous sensor is placed in antibacterial peptide sample, 1.5～4h is reacted at 36～38 DEG C,
Take out the antibacterial peptide electrochemical luminous sensor and clean, by antibacterial peptide electrochemical luminous sensor and electrochemistry after cleaning
After light-emitting appearance is connected, the antibacterial peptide electrochemical luminous sensor is placed in into the potassium peroxydisulfate that concentration is 0.08～0.12mol/L
In solution, using cyclic voltammetry, test obtains the electrochemiluminescence intensity of potassium peroxydisulfate, the voltage model of the cyclic voltammetry
Enclose for -2.0～0.0V, scanning speed is 80～120mV/s, it is bent according to the standard of electrochemiluminescence intensity and antibacterial peptide concentration
Line, obtains the antibacterial peptide content in antibacterial peptide sample.
Preferably, the manufacture method of the standard curve is：
The antibacterial peptide electrochemical luminous sensor is placed in the antibacterial peptide standard solution of series concentration, at 36～38 DEG C
1.5～4h of lower reaction, takes out the antibacterial peptide electrochemical luminous sensor and cleans, by the antibacterial peptide electrochemiluminescence after cleaning
After sensor is connected with electrochemiluminescence instrument, by the antibacterial peptide electrochemical luminous sensor be placed in concentration for 0.08～
In the potassium persulfate solution of 0.12mol/L, using cyclic voltammetry, test obtains the electrochemiluminescence intensity of potassium peroxydisulfate, institute
The voltage range for stating cyclic voltammetry is -2.0～0.0V, and scanning speed is 80～120mV/s, according to electrochemiluminescence intensity and
The linear relationship of antibacterial peptide concentration of standard solution logarithm, draws standard curve.
Preferably, the antibacterial peptide standard solution is obtained during the phosphate buffered solution of pH=7.4 is dissolved in for antibacterial peptide.
Preferably, the pH value of the potassium persulfate solution is 7.4.
It is highly preferred that the potassium persulfate solution is dissolved in phosphate buffered solution for potassium peroxydisulfate and preparing, it is described
In potassium persulfate solution, phosphatic concentration is 0.1mol/L.
Preferably, step (1) adopts platinum electrode for right using the antibacterial peptide electrochemical luminous sensor as working electrode
Electrode, saturated calomel electrode are that reference electrode constitutes three-electrode system testing electroluminescent intensity.
Preferably, the antibacterial peptide electrochemical luminous sensor is applied to the detection by quantitative of cecropin B.
The present invention adopts L-Cysteine and decorated by nano-gold naked gold electrode, potassium peroxydisulfate to try for a kind of electrochemiluminescence
Agent, applies under certain voltage, during electron transmission to the modified electrode, and L-Cysteine and potassium peroxydisulfate pass through oxidoreduction
Reaction, produces luminous signal, exempts from when the antibacterial peptide antibody for being fixed on electrode surface occurs specificity with antibacterial peptide antigen in solution
After epidemic disease reaction, the immune complex of formation can cause electrode interface electron transmission impaired performance, cause potassium peroxydisulfate electrochemistry to send out
Light intensity weakens, according to determinand (i.e. antibacterial peptide antigen) linear relationship of the log concentration to electrochemiluminescence Strength Changes, can
Realize the immune detection to antibacterial peptide.
The present invention is directly added in detection system with potassium peroxydisulfate as electrochemiluminescence reagent, without the need for being marked at
On antibody or antigen, it is to avoid the antagonist or antigen active that labelling may bring weakens and labeling process, that is, what is prepared is nothing
Mark electrochemistry luminescence sensor.
Sensor of the invention shows excellent accuracy, high sensitivity, high degree of specificity, stability and reproduction
Property, immunoassay detection rapidly, conveniently, can be used for the detection of actual sample.
To sum up, beneficial effect of the present invention includes the following aspects：
(1) antibacterial peptide electrochemical luminous sensor small volume of the present invention, low cost, sensitivity height and good stability, are suitable for
In small size biotic environment, the detection of antibacterial peptide, may be implemented in line analysis；
(2) preparation method of antibacterial peptide electrochemical luminous sensor of the present invention is simple.
Description of the drawings
Electrochemiluminescence design sketchs of the Fig. 1 for naked gold electrode electroplating surface L-Cysteine layer；
Fig. 2 is the electrode surface scanning electron microscope (SEM) photograph that nano gold layer and L-Cysteine layer are modified；
Fig. 3 is electrochemiluminescence Strength Changes and antibacterial of the antibacterial peptide electrochemical luminous sensor when cecropin B is detected
Peptide concentration logarithmic relationship figure.
Specific embodiment
Described below is the preferred embodiment of the present invention, it is noted that for those skilled in the art
For, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications are also considered as
Protection scope of the present invention.
Embodiment 1
A kind of preparation method of antibacterial peptide electrochemical luminous sensor, comprises the following steps：
1. naked gold electrode is provided, naked gold electrode is placed in into the Cys solution that 5mL concentration is 20mmol/L and (is contained 0.1mol/L
HCl in), using cyclic voltammetry in naked gold electrode electroplating surface L-Cysteine layer, voltage is -0.5～1.0V, scanning speed
For 10mV/s, electroplating time is 20min；Then be soaked in water, cleaning electrode, to remove the unreacted Cys molecules in surface, obtain
The electrode of L-Cysteine layer modification；
2. the 40 μ L particle diameters of electrode surface Deca in the modification of above-mentioned L-Cysteine layer are about the nanometer gold dispersion liquid of 10nm,
And 20h is reacted at 4 DEG C, nano gold layer is prepared in L-Cysteine layer surface, obtain nano gold layer and L-Cysteine layer is repaiied
The electrode of decorations；
The preparation method of nanometer gold dispersion liquid is specific as follows：
Take 2mL concentration to be added in the there-necked flask for filling 45mL distilled water for 0.025mol/L chlorauric acid solutions, heat
To seething with excitement, the citric acid three sodium solution that 3mL concentration is 0.030mol/L is then rapidly joined under agitation, keep micro-boiling, and connect
Continuous stirring, when solution gradually becomes colourless, Lycoperdon polymorphum Vitt by light yellow, until when becoming claret, stop heating, be cooled to room temperature,
50mL is settled to, is kept in dark place in 4 DEG C stand-by；
3. the 40 μ L concentration of electrode surface Deca modified in nano gold layer and L-Cysteine layer resists for 5 μ g/mL cecropin Bs
Liquid solution (cecropin B antibody is purchased from abcam companies, and article No. is ab27571), and 12h is reacted at 4 DEG C, in nano gold layer table
Face prepares antibacterial peptide antibody layer, then will soak in phosphate buffered solution that the pH value that electrode is placed in 0.01mol/L is 7.4
5min, then with the buffer solution cleaning electrode 2 times, to remove antibody not to be adsorbed, obtain antibacterial peptide electrochemiluminescence sensing
Device；
4. above-mentioned antibacterial peptide electrochemical luminous sensor is immersed into the bovine serum albumin (BSA) that mass concentration is 1% molten
In liquid, after 30min being reacted under room temperature, antibacterial peptide electrochemical luminous sensor is taken out, then is placed in 0.01mol/L PBS (pH=
7.4) in, 5～8min of immersion, is then cleaned 2 times with 0.01mol/L PBS (pH=7.4) solution, to remove unnecessary BSA point
Son.
The electrode that naked gold electrode and L-Cysteine layer are modified respectively with electrochemiluminescence instrument (the auspicious analytical tool advanced in years in Xi'an
Co., Ltd, model MPI-E type) it is connected, test the electrochemistry of the electrode of naked gold electrode and the modification of L-Cysteine layer
Luminous intensity (ECL intensity), in Fig. 1, a curves are sent out for electrochemistry of the naked gold electrode in 0.1mol/L PBS solutions (pH=7.4)
Light situation；Curve b is naked gold electrode containing 0.1mol/L K2S2O8Phosphate (pH=7.4) solution in electrochemiluminescence
Situation；Curve c is the electrode of L-Cysteine layer modification containing 0.1mol/L K2S2O8PBS (pH=7.4) solution in
Electrochemiluminescence situation.Curve a, b, c from figure is can be seen that without K2S2O8PBS solution in, naked gold electrode surface
Not obvious luminescence phenomenon (shown in curve a)；As addition 0.1mol/L K in solution2S2O8PBS solution when, go out immediately
Existing obvious electrochemiluminescence phenomenon, but light still weaker (shown in curve b)；And further plate in gold electrode surfaces
The electrode of L-Cysteine layer modification is obtained after Cys, the electrode is 7.4 containing 0.1mol/L K in pH2S2O8PBS solution
Middle electrochemiluminescence is remarkably reinforced (shown in curve c).It is hereby understood that K2S2O8With electrochemiluminescence characteristic, in addition, gold electrode
On Cys to K in solution2S2O8Electrochemiluminescence behavior has obviously reinforced effects.This is that have due to Cys in itself
Good electro-chemical activity, and the electron transfer of other materials can be promoted, increase electrode area, strengthen illumination effect, together
When can illustrate that Cys is successfully modified on naked gold electrode surface.
Fig. 2 is the electrode surface scanning electron microscope (SEM) photograph that nano gold layer and L-Cysteine layer are modified；From figure 2 it can be seen that
The electrode surface of L-Cysteine layer modification is uniform-distribution with nanogold particle, illustrates that step (2) is modified in L-Cysteine layer
Electrode surface Deca nanometer gold dispersion liquid after, prepare nano gold layer in L-Cysteine layer surface.
Embodiment 2
A kind of preparation method of antibacterial peptide electrochemical luminous sensor, comprises the following steps：
1. naked gold electrode is provided, naked gold electrode is placed in into the Cys solution that 5mL concentration is 15mmol/L and (is contained 0.1mol/L
HCl in), using cyclic voltammetry in naked gold electrode electroplating surface L-Cysteine layer, voltage is -0.5～1.0V, scanning speed
For 5mV/s, electroplating time is 30min；Then be soaked in water, cleaning electrode, to remove the unreacted Cys molecules in surface, obtain
The electrode of L-Cysteine layer modification；
2. the 40 μ L particle diameters of electrode surface Deca in the modification of above-mentioned L-Cysteine layer are about the nanometer gold dispersion liquid of 10nm,
And 22h is reacted at 4 DEG C, nano gold layer is prepared in L-Cysteine layer surface, obtain nano gold layer and L-Cysteine layer is repaiied
The electrode of decorations；
3. the 60 μ L concentration of electrode surface Deca modified in nano gold layer and L-Cysteine layer resists for 4 μ g/mL cecropin Bs
Liquid solution, and 24h is reacted at 4 DEG C, antibacterial peptide antibody layer is prepared on nano gold layer surface, electrode is placed in into 0.01mol/ then
The pH value of L be 7.4 phosphate buffered solution in soak 8min, then with the buffer solution cleaning electrode 2 times, do not inhaled with removing
Attached antibody, obtains antibacterial peptide electrochemical luminous sensor；
4. above-mentioned antibacterial peptide electrochemical luminous sensor is immersed into the bovine serum albumin (BSA) that mass concentration is 1% molten
In liquid, after 30min being reacted under room temperature, antibacterial peptide electrochemical luminous sensor is taken out, then is placed in 0.01mol/L PBS (pH=
7.4) in, immersion 5min, is then cleaned 3 times with 0.01mol/L PBS (pH=7.4) solution, to remove unnecessary BSA molecules.
Embodiment 3
A kind of preparation method of antibacterial peptide electrochemical luminous sensor, comprises the following steps：
1. naked gold electrode is provided, naked gold electrode is placed in into the Cys solution that 5mL concentration is 25mmol/L and (is contained 0.1mol/L
HCl in), using cyclic voltammetry in naked gold electrode electroplating surface L-Cysteine layer, voltage is -0.5～1.0V, scanning speed
For 15mV/s, electroplating time is 25min；Then be soaked in water, cleaning electrode, to remove the unreacted Cys molecules in surface, obtain
The electrode of L-Cysteine layer modification；
2. the 40 μ L particle diameters of electrode surface Deca in the modification of above-mentioned L-Cysteine layer are about the nanometer gold dispersion liquid of 7nm,
And 24h is reacted at 4 DEG C, nano gold layer is prepared in L-Cysteine layer surface, obtain nano gold layer and L-Cysteine layer is repaiied
The electrode of decorations；
3. the 40 μ L concentration of electrode surface Deca modified in nano gold layer and L-Cysteine layer resists for 8 μ g/mL cecropin Bs
Liquid solution, and 20h is reacted at 4 DEG C, antibacterial peptide antibody layer is prepared on nano gold layer surface, electrode is placed in into 0.01mol/ then
The pH value of L be 7.4 phosphate buffered solution in soak 7min, then with the buffer solution cleaning electrode 3 times, do not inhaled with removing
Attached antibody, obtains antibacterial peptide electrochemical luminous sensor；
4. above-mentioned antibacterial peptide electrochemical luminous sensor is immersed into the bovine serum albumin (BSA) that mass concentration is 1% molten
In liquid, after 30min being reacted under room temperature, antibacterial peptide electrochemical luminous sensor is taken out, then is placed in 0.01mol/L PBS (pH=
7.4) in, immersion 8min, is then cleaned 2 times with 0.01mol/L PBS (pH=7.4) solution, to remove unnecessary BSA molecules.
Application Example
A kind of antibacterial peptide electrochemical luminous sensor is applied to the quantitative detecting method of antibacterial peptide, comprises the following steps：
(1) cecropin B (being purchased from abcam companies) is dissolved in phosphate buffered solution and obtains cecropin B standard solution (pH
=7.4), antibacterial peptide electrochemical luminous sensor obtained in embodiment 1 is placed in the cecropin B standard solution of variable concentrations,
4h is reacted at 37 DEG C, antibacterial peptide electrochemical luminous sensor is taken out and is cleaned, remove unreacted antibacterial peptide, after cleaning
After antibacterial peptide electrochemical luminous sensor is connected with electrochemiluminescence instrument, the persulfuric acid that concentration is 0.1mol/L is then placed on
In the phosphate buffered solution of potassium, using cyclic voltammetry, the electrochemiluminescence intensity of potassium peroxydisulfate recorded, voltage is -2.0～
0.0V, scanning speed 100mV/s；According to electrochemiluminescence intensity and the linear relationship of cecropin B concentration of standard solution logarithm, paint
Standard curve processed；
Fig. 3 be antibacterial peptide electrochemical luminous sensor detect variable concentrations cecropin B when (concentration is respectively x=0.1,
0.2,0.4,0.8,1.2,2,4,6,8,10ng/mL electrochemiluminescence intensity) and cecropin B concentration of standard solution logarithmic relationship
Figure, it can be seen that antibacterial peptide concentration is higher, luminous intensity is lower, the concentration of luminous intensity and antibacterial peptide standard solution
Logarithm is linear, and linear equation is Y=1352.0-520.54lgx, R=0.996, and the sensor is in 0.1～10ng/mL
In the range of have good linear response, detection limit LOD=30pg/mL (S/N=3) to antibacterial peptide；
(2) as the application of current antibacterial peptide is also in initial stage, the also rare listing of pure drug products of commercialization.City
Sell product and mostly be composition and the indefinite antibacterial peptide mixer of content or the feedstuff class product containing antibacterial peptide mixer, it is impossible to
For certain antibacterial peptide detection by quantitative.Therefore the present invention has made the fish meal sample containing cecropin B by oneself, and detects day in the sample
The content of sbombycin B.
Sample Homemade method：0.25g cecropin Bs are accurately weighed, is added in the common fish meals of 50g, is sufficiently stirred for, be obtained
Containing the uniform fish meal sample of cecropin B.
Detection method：1g fish meal samples are accurately weighed, adds 1000mL distilled water, stirring to stand.Remove upper strata to suspend
Thing, takes interstitial fluid in 20mL, is put into centrifuge tube, and centrifugation 10min (10000r/min) takes supernatant 1mL, uses pH=7.4 phosphate
Buffer solution (PBS) is settled in 250mL volumetric flasks, and the sample solution containing cecropin B is obtained.Sample solution 5mL is taken, is used
PBS is settled to 50mL, mixes, and prepare liquid is obtained.Prepare liquid is poured in beaker, antibacterial peptide electrification obtained in insertion embodiment 1
Luminescence sensor is learned, 4h is reacted in 37 DEG C of water-baths, taken out and cleaning sensor.By sensor and electrochemiluminescence after cleaning
Instrument is connected, and is then placed in the PBS solution of the potassium peroxydisulfate that concentration is 0.1mol/L, using cyclic voltammetry, in voltage
Scope is -2.0～0.0V, under the conditions of scanning speed 100mV/s, records the electrochemiluminescence strength signal peak value Y of potassium peroxydisulfate,
Peak value Y is brought in linear equation Y=1352.0-520.54lgx, x values (unit of x is ng/mL) are tried to achieve, i.e., cecropin B exists
Concentration in prepare liquid.Cecropin B percentage composition in fish meal is calculated further according to below equation.
Continuous 5 measure is carried out to homemade 3 samples, 1 is the results are shown in Table.
Cecropin B assay result (n=5) in 1 fish meal of table
During cecropin B in detection sample of the present invention, the relative error of actual content and measured value is less, it can be seen that,
Antibacterial peptide electrochemical luminous sensor of the present invention can accurately detect the content of antibacterial peptide in Feed Sample.
Effect example
1st, the repeatability of antibacterial peptide electrochemical luminous sensor
It is (dense to 3 variable concentrations cecropin B standard sample with antibacterial peptide electrochemical luminous sensor obtained in embodiment 1
Degree is followed successively by 0.5,2.0,5.0ng/mL) detected, each sample concentration parallel assay 5 times, concentration relative standard deviation
(RSDs) it is respectively 4.4%, 4.0%, 3.2%.Again with 1 obtained 5 parallel antibacterial peptide electrochemiluminescence sensing of embodiment
Device is 0.5 respectively to concentration, 2, the cecropin B standard sample of 5ng/mL detect that relative standard deviation is respectively 6.1%,
5.4%, 4.8%.As can be seen here, antibacterial peptide electrochemical luminous sensor batch is interior and differences between batches are less, i.e., repeatability is preferable.
2nd, the stability of antibacterial peptide electrochemical luminous sensor
Antibacterial peptide electrochemical luminous sensor obtained in embodiment 1 is deposited in 4 DEG C of refrigerators, was determined once every 1 day
Its electrochemiluminescence intensity, to observe the stability of sensor.Test result indicate that, after 15 days, electrochemiluminescence intensity (ECL)
Response only declines 6.6%, and change is little, illustrates that sensor has preferable stability.
3rd, the specificity of antibacterial peptide electrochemical luminous sensor
By concentration be the BSA of 100 times of cecropin B concentration, 100 times of soybean protein, 100 times of zein and 20 times
King crab element mix as chaff interference, be then added to jointly in the cecropin B of 6.0ng/mL and form interference sample, made with embodiment 1
The antibacterial peptide electrochemical luminous sensor for obtaining is measured respectively to standard sample (independent cecropin B) and interference sample, be the results are shown in Table
2.From table 2, measure interference of the chaff interference to cecropin B is less.
2 standard sample of table and interference sample Comparative result (n=5)
| Sequence number | Standard sample signal value | Interference sample signal value | Relative error (%) |
| 1 | 949.84 | 907.35 | -4.5 |
| 2 | 948.25 | 896.23 | -5.5 |
| 3 | 924.77 | 884.12 | -4.4 |
| 4 | 953.81 | 901.03 | -5.5 |
| 5 | 918.69 | 876.72 | -4.6 |
Embodiment described above only expresses the several embodiments of the present invention, and its description is more concrete and detailed, but and
Therefore the restriction to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that for one of ordinary skill in the art
For, without departing from the inventive concept of the premise, some deformations and improvement can also be made, these belong to the guarantor of the present invention
Shield scope.Therefore, the protection domain of patent of the present invention should be defined by claims.
Claims (7)
1. a kind of preparation method of antibacterial peptide electrochemical luminous sensor, it is characterised in that comprise the following steps：
(1) naked gold electrode is provided, the naked gold electrode is placed in the L-Cysteine solution that concentration is 15～25mmol/L, is adopted
With cyclic voltammetry in the naked gold electrode electroplating surface L-Cysteine layer, the electrode of L-Cysteine layer modification is obtained；Institute
The voltage range for stating cyclic voltammetry is -0.5～1.0V, and scanning speed is 5～15mV/s, and electroplating time is 20～30min；
(2) the nanometer gold dispersion liquid of the 40 μ L of electrode surface Deca modified in the L-Cysteine layer, the nanometer gold dispersion
The particle diameter of the nanogold particle in liquid is 7～10nm, stands 20～24h, prepare in the L-Cysteine layer surface at 4 DEG C
Nano gold layer, obtains the electrode of nano gold layer and the modification of L-Cysteine layer；
(3) the electrode surface Deca concentration modified in the nano gold layer and L-Cysteine layer is the antibacterial peptide of 4～8 μ g/mL
Antibody-solutions, and 12～24h is reacted at 4 DEG C, antibacterial peptide antibody layer is prepared on the nano gold layer surface, the antibacterial peptide resists
Body layer is combined together by electrostatic adsorption with the nano gold layer, obtains the antibacterial peptide electrochemical luminous sensor.
2. the preparation method of antibacterial peptide electrochemical luminous sensor as claimed in claim 1, it is characterised in that in step (2),
The preparation method of the nanometer gold dispersion liquid is：
Take 1～2mL concentration to be added in 45～49mL distilled water for 0.02～0.03mol/L chlorauric acid solutions, be heated to boiling,
Then the citric acid three sodium solution that 3～6mL concentration is 0.025～0.035mol/L is rapidly joined under agitation, keeps micro-boiling, and
Continuous stirring, when solution gradually becomes colourless, Lycoperdon polymorphum Vitt by light yellow, until when becoming claret, stopping heating, is often cooled to
Temperature, is settled to 50mL, obtains the nanometer gold dispersion liquid.
3. the preparation method of antibacterial peptide electrochemical luminous sensor as claimed in claim 1, it is characterised in that in step (3),
The antibacterial peptide antibody-solutions of 40～60 μ L of electrode surface Deca modified in the nano gold layer and L-Cysteine layer.
4. the preparation method of antibacterial peptide electrochemical luminous sensor as claimed in claim 3, it is characterised in that in step (3),
40～60 μ L concentration of electrode surface Deca modified in the nano gold layer and L-Cysteine layer is 5 μ g/mL antibacterial peptide antibodies
Solution.
5. the preparation method of antibacterial peptide electrochemical luminous sensor as claimed in claim 1, it is characterised in that by step (3)
The antibacterial peptide electrochemical luminous sensor for obtaining is placed in bovine serum albumin solution, and 30～60min is reacted under room temperature, will
The antibacterial peptide electrochemical luminous sensor takes out, then is placed in 5～8min of immersion in the phosphate buffered solution of pH=7.4, so
Cleaned with the phosphate buffered solution 2～3 times afterwards, finally by the antibacterial peptide electrochemical luminous sensor in 4 DEG C of refrigerators
Preserve stand-by.
6. obtained in the preparation method as described in any one of Claims 1 to 5, antibacterial peptide electrochemical luminous sensor is applied to resist
The quantitative detecting method of bacterium peptide, it is characterised in that comprise the following steps：
The antibacterial peptide electrochemical luminous sensor is placed in antibacterial peptide sample, 1.5～4h is reacted at 36～38 DEG C, is taken out
The antibacterial peptide electrochemical luminous sensor is simultaneously cleaned, by antibacterial peptide electrochemical luminous sensor and electrochemiluminescence after cleaning
After instrument is connected, the antibacterial peptide electrochemical luminous sensor is placed in into the potassium persulfate solution that concentration is 0.08～0.12mol/L
In, using cyclic voltammetry, test obtains the electrochemiluminescence intensity of potassium peroxydisulfate, the voltage range of the cyclic voltammetry
For -2.0～0.0V, scanning speed is 80～120mV/s, according to electrochemiluminescence intensity and the standard curve of antibacterial peptide concentration,
Obtain the antibacterial peptide content in antibacterial peptide sample.
7. antibacterial peptide electrochemical luminous sensor as claimed in claim 6 is applied to the quantitative detecting method of antibacterial peptide, and which is special
Levy and be, the antibacterial peptide electrochemical luminous sensor is applied to the detection by quantitative of cecropin B.
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