CN106248742B - Chitosan/gold complex system, preparation method and application - Google Patents
Chitosan/gold complex system, preparation method and application Download PDFInfo
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- CN106248742B CN106248742B CN201610850241.4A CN201610850241A CN106248742B CN 106248742 B CN106248742 B CN 106248742B CN 201610850241 A CN201610850241 A CN 201610850241A CN 106248742 B CN106248742 B CN 106248742B
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Abstract
The invention discloses a kind of chitosan/gold complex systems, preparation method and application, belong to field of material technology.The compound system is to be passed through plasma in the solution containing gold chloride and chitosan to be prepared, and colloidal gold particle is nano-scale in system, and granular size is in irregular shape in 50nm or so.The compound system can as sensitive membrane material modified electrode, after electrode self assembly RAC-anti antibody can specific detection RAC antigen, detection sensitivity is high, and selectivity and stability are good.
Description
Technical field
The present invention relates to a kind of chitosan/gold complex system, also relate to the compound system preparation method and
Using belonging to field of material technology.
Background technique
Colloidal gold is also referred to as aurosol, is gold salt to be reduced into the stabilization formed after gold, uniform nano Au particle suspension, belongs to
In multiphase heterogeneous system, color is from salmon pink to purplish red color change.Colloidal gold particle is based on a golden core (Au), periphery
Coat anion AuCl2-, outermost layer H+, it is dispersed between colloid in solution and maintains colloid in suspended state.Colloidal gold particle
Golden core be not ideal spherical nuclei, particle is lesser substantially spherical in shape, and particle larger (diameter 30nm or more) mostly in ellipse
It is spherical.
Colloidal gold particle has Fluorescence Characteristic, and there are single optical absorption peaks in visible-range, and with
The increase wavelength of partial size is elongated.In addition, being the most significantly the sensitivity to electrolyte in numerous colloid properties of colloidal gold
Property, certain macromolecular substances such as protein not only plays a protective role to colloidal gold, moreover it is possible to improve the stability of colloidal gold.Generally
In the case of, when not destroying the ballasts such as electrolyte, collosol concentration, the stability of colloidal gold is preferable, places also for a long time not
Coacervation can occur.
Currently, colloidal gold is used to detect antigen-antibody mainly as marker or color developing agent, it is immunized as a kind of novel
Labelling technique has potential application prospect in biology, medicine and other fields.Since the technology is interfered and is radiated there is no endogenous enzymes
Property isotope interference etc., and using the colloidal gold of variable grain size as dual or multiple labelling, it is more accurate to position.Glue
The size of gold particle is between 1~100nm in body gold, except the general character (such as biggish specific surface area, quantum ruler with nano material
Very little effect and macro quanta tunnel effect etc.) outside, chemical property is also abnormal active, and it is special generation tool can be adsorbed with nucleopilic reagent
The group of distinguished service energy.Such as the Electrostatic Absorption of non-covalent bond can occur with amino for nanogold, can also generate with thin base stronger
Covalent bond effect, but the optical property of rear nanogold is combined not change, therefore can be used for biological monitoring.In recent years,
The application of colloidal gold is concentrated mainly on dot immunogold filtration assay and colloidal gold fast immune chromatographic method.Quick spot immune
Golden percolation is called the golden immunization of drop, and this method fixes known specific antibody or antigen using microporous barrier as solid phase carrier
Thereon, it and is fitted into the percolating device full of water-absorbent material, sample to be tested is added, make via the capillarity or diafiltration of filter membrane
With making coated antibody or antigen binding on antigen in sample or antibody and film, most mark and reach through colloidal gold conjugate afterwards
To testing goal.And fast immune chromatographic method be also using microporous barrier as solid phase carrier, thereon fixed known specific antibody or
For antigen as detection band, colloid gold label object is dry in glass fibre matter combination release pad, and one end is connected with sample pad, separately
One end is connected with film, and the other end of film is connected with water absorption pad.After sample to be tested is added, sample is moved forward by capillary, and
By the glass fibre containing marker, make marker aquation again, and reacts to each other together forward with colloid gold label object, until
When detection line, detection line intercepts and captures the compound of marker and determinand, to red result occur.
In the prior art, traditional colloidal gold preparation method includes trisodium citrate reduction method, ascorbic acid reduction, boron
Hydrogenate sodium reduction etc..Wherein, the operating procedure of trisodium citrate reduction method are as follows: take 0.01% gold chloride of 50mL and 2mL 1%
Trisodium citrate is sufficiently mixed, and heats 15min, it is cooling after with water mend to original volume to get.The operation of ascorbic acid reduction walks
Suddenly are as follows: take 0.01% gold chloride of 50mL, 0.75mL 0.2M K2CO3It is sufficiently mixed, heats with 0.7% ascorbic acid of 0.5mL
10min, it is cooling after with water mend to original volume to get.The operating procedure of sodium borohydride reduction are as follows: take 0.01% chlorine of 50mL golden
Acid and 0.25mL 0.2M K2CO3It is sufficiently mixed, point 3 to 5 0.5mg/mL hydroborations to mixed solution and dripping Fresh
Sodium, edged oscillation in side is uniformly mixed it, until solution purpling, eventually become it is orange red, after persistent oscillation about 3min to get.So
And the colloidal gold that above-mentioned three kinds of methods are prepared must be stored at 4 DEG C.
The patent of invention of publication No. CN105665740A discloses rubber polymer under a kind of atmospheric air plasma liquid phase
The method of body gold nano grain, comprising: the mixed liquor of gold chloride and reducing agent is added in insulating materials container, to the mixed liquor
In be passed through atmospheric air plasma to get Au colloidal nanoparticles.This method is using air substitution rare gas as sharp
It gets angry body, 2~10min can form the Au colloidal nanoparticles that homogeneity is good, high sensitivity and performance are stable.Publication No.
The patent of invention of CN102430392A also discloses a kind of preparation method of composite nano colloidal gold chitosan carrier, comprising: 1)
The chlorauric acid solution of mass concentration 0.01% is taken to be heated to boiling, 1% citric acid three sodium solution of preheating is added in stirring, until chlorine is golden
Acid solution becomes grey from golden yellow becomes claret again, continues stirring and boils to solution in bright red, obtains colloidal gold
Solution;2) chitosan is dissolved into the chitosan solution of mass concentration 0.01~0.1% with 0.1~1% organic monoacid, filters standby
With;3) colloidal gold solution is uniformly mixed according to mass ratio 1:0.1~1 with chitosan solution, 0.05% carbonic acid is slowly added dropwise
Potassium solution to mixed liquor pH value is 7.0, obtains composite nano colloidal gold chitosan carrier mixed liquor.Wherein, the grain of complex carrier
Diameter is distributed between 20~200nm, and average grain diameter 80nm can be used for immune detection or immunodiagnosis.However, above two method
It is both needed to when preparing colloidal gold using reducing agent, and colloidal gold stability obtained is poor, needs to store at low temperature.
Summary of the invention
The object of the present invention is to provide a kind of chitosan/gold complex systems.
Meanwhile the present invention also provides a kind of chitosan/gold complex system preparation methods.
It ties up to finally, the present invention provides a kind of chitosan/gold complex and prepares electrochemica biological sensor or inspection
Survey the application in Ractopamine.
In order to achieve the goal above, the technical scheme adopted by the invention is that:
Chitosan/gold complex system is that plasma preparation is passed through in the solution containing gold chloride and chitosan
It obtains.
The composition of the solution containing gold chloride and chitosan are as follows: 0.05~0.1g of gold chloride, chitosan 0.1~
0.2g, glacial acetic acid 400~600 μ L, 20~30mL of water.When preparing solution, first chitosan can be dissolved in the aqueous solution of glacial acetic acid,
Add gold chloride.
The plasma is nitrogen gas plasma.It is passed through the technical parameter of plasma are as follows: pulsed RF power source function
100~200W of rate is passed through 5~10min of time.
Chitosan/gold complex system preparation method, comprising the following steps: molten containing gold chloride and chitosan
Plasma is passed through in liquid, ultrasonic mixing obtains chitosan/gold complex system until solution is in aubergine.
The composition of the solution containing gold chloride and chitosan are as follows: 0.05~0.1g of gold chloride, chitosan 0.1~
0.2g, glacial acetic acid 400~600 μ L, 20~30mL of water.
The plasma is nitrogen gas plasma.It is passed through the technical parameter of plasma are as follows: pulsed RF power source function
100~200W of rate is passed through 5~10min of time.Preparation principle are as follows: the present invention prepares colloidal gold using Room-temperature low-pressure plasma,
Wherein chitosan is as stabilizer and reducing agent, and in plasma preparation process, nitrogen gas plasma causes hydrone and is produced from
It is restored by base, and then to gold chloride.
Chitosan/gold complex ties up to the application prepared in electrochemica biological sensor, specifically: by chitosan/
Gold complex system is added dropwise on the electrode, dry, obtains chitosan/colloidal gold modification electrode;Above-mentioned electrode is placed in anti-
In body fluid, in electrode surface self assembly antibody to get electrochemica biological sensor.In above-mentioned application, chitosan per se with
Amino group can provide binding site for subsequent fixed antibody (such as Anti-ractopamine antibody), improve the sensitivity of detection.
When containing Anti-ractopamine antibody in the antibody liquid, gained sensor can be used to detection Ractopamine.
Beneficial effects of the present invention:
Chitosan/gold complex system is to be passed through plasma in the solution containing gold chloride and chitosan in the present invention
Body is prepared, and colloidal gold particle is nano-scale in the system, and granular size is in irregular shape in 50nm or so.This is multiple
Zoarium system can as sensitive membrane material modified electrode, after electrode self assembly RAC-anti antibody can specific detection RAC antigen, examine
High sensitivity is surveyed, selectivity and stability are good.
The present invention prepares chitosan/gold complex system using plasma method, can save chemical reducing agent, at normal temperature
The stable colloidal gold of rapid synthesis is the synthetic method of a kind of " green ".
Detailed description of the invention
Fig. 1 is chitosan/colloidal gold TEM figure in compound system;
Fig. 2 is chitosan/colloidal gold fluorometric investigation spectrogram in compound system;
Fig. 3 is the ultraviolet-visible spectrogram of colloidal gold in compound system;
Fig. 4 is that the CV of electrochemica biological sensor schemes;
Fig. 5 constructs for electrochemica biological sensor and the electrochemical impedance figure of detection process;
Fig. 6 is the EIS figure and matched curve figure that electrochemica biological sensor detects various concentration RAC;
Fig. 7 is the selectivity test result of electrochemica biological sensor;
Fig. 8 is the stability test result of electrochemica biological sensor.
Specific embodiment
Only invention is further described in detail for following embodiments, but does not constitute any limitation of the invention.
Embodiment 1
Chitosan/gold complex system, preparation step are as follows:
1) all glasswares to be used are cleaned with the chloroazotic acid of Fresh, then with water cleaning down, drying for standby;
2) 600 μ L, 99% glacial acetic acid is measured in 25mL volumetric flask with 1mL liquid-transfering gun, 20mL water is added and is diluted to concentration
3% glacial acetic acid solution;
3) 0.2g chitosan is weighed, is added in the glacial acetic acid solution diluted, ultrasound is completely dissolved chitosan, quiet
It sets, until bubble completely disappears;
4) gold chloride is added in deionized water to concentration 100mM, adds 550 μ L chitosan solutions, power 500W,
Ultrasonic mixing 10min under frequency 40kHz, stands to obtain mixed liquor;
5) it is passed through nitrogen gas plasma in mixed liquor, pulsed RF power source power 200W, time 5min, then in power
Ultrasonic mixing 10min under 500W, frequency 40kHz obtains chitosan/gold complex system until solution becomes aubergine.
Chitosan/gold complex ties up to the application in detection Ractopamine, the specific steps are as follows:
1) pretreatment of gold electrode
Using 0.5 μm and 0.3 μm of Al2O3Slurries on chamois leather sanding and polishing gold electrode, until electrode surface is drawn without obvious
Trace, polishing track are the figure of eight, then clean gold electrode with deionized water, by the Al of residual on the electrode2O3Wash clean;It will be electric
Pole is placed in H2O2:H2SO4In the mixed liquor of volume ratio 3:7, the ultrasonication 15min under power 500W, frequency 40kHz, deionization
Water is rinsed well;Electrode is placed in water again: in the mixed liquor of dehydrated alcohol volume ratio 1:1, ibid ultrasound 15min, then be placed in
Ultrasound 15min in ionized water;The gold electrode cleaned up is placed in 0.5M H2SO4In solution, using cyclic voltammetry 0.2~
Activation 2 times in 1.6V voltage range, then rinsed well with deionized water, it dries up spare;
2) chitosan of above-mentioned preparation/gold complex system is diluted to 2 times of volumes, ultrasonic 30min makes to be uniformly mixed;
10 μ L diluted systems are pipetted with 10 μ L liquid-transfering guns to be added dropwise on gold electrode, its electrochemical impedance (EIS) are tested after completely dry, to steady
It is rinsed well and is dried up with deionized water after fixed, obtain chitosan/colloidal gold modification electrode;Above-mentioned electrode is placed in 100ng/
Impregnate 2h in mL Anti-ractopamine antibody (RAC-anti) liquid, guarantee chitosan/colloid metal/composite material have sufficient time with
Anti-ractopamine antibody self assembly, taking-up are rinsed well, test EIS, rinsed and dried up with deionized water after stablizing to get with
In the electrochemica biological sensor of detection Ractopamine;
3) Ractopamine in solution is detected using the sensor.
Embodiment 2
Chitosan/gold complex system, preparation step are as follows:
1) preparation containing gold chloride and chitosan solution
Chitosan is dissolved in dilute glacial acetic acid solution, gold chloride is added after dissolving completely, mixes spare;
Solution composition are as follows: gold chloride 0.05g, chitosan 0.1g, glacial acetic acid 400 μ L, water 20mL;
2) it is passed through nitrogen gas plasma, pulsed RF power source power 100W, time 10min, reaction in the above solution
Become aubergine to solution, obtains chitosan/gold complex system.
The application of chitosan/gold complex system is the same as embodiment 1.
Embodiment 3
Chitosan/gold complex system, preparation step are as follows:
1) preparation containing gold chloride and chitosan solution
Chitosan is dissolved in dilute glacial acetic acid solution, gold chloride is added after dissolving completely, mixes spare;
Solution composition are as follows: gold chloride 0.1g, chitosan 0.2g, glacial acetic acid 600 μ L, water 30mL;
2) it is passed through nitrogen gas plasma in the above solution, pulsed RF power source power 150W, time 5min, reaction is extremely
Solution becomes aubergine, obtains chitosan/gold complex system.
The application of chitosan/gold complex system is the same as embodiment 1.
Embodiment 4
Chitosan/gold complex system, preparation step are as follows:
1) preparation containing gold chloride and chitosan solution
Chitosan is dissolved in dilute glacial acetic acid solution, gold chloride is added after dissolving completely, mixes spare;
Solution composition are as follows: gold chloride 0.075g, chitosan 0.15g, glacial acetic acid 500 μ L, water 25mL;
2) it is passed through nitrogen gas plasma in the above solution, pulsed RF power source power 180W, time 8min, reaction is extremely
Solution becomes aubergine, obtains chitosan/gold complex system.
The application of chitosan/gold complex system is the same as embodiment 1.
Test example
1) pattern of colloidal gold and structured testing in compound system
Test method: chitosan/gold complex system in Example 1 is added dropwise on the copper mesh that carbon film supports, is used for
TEM test, the result is shown in Figure 1, wherein Fig. 1 (a)~1 (c) is followed successively by the figure of the TEM under 10nm, 200nm, 5nm scale, and Fig. 1 (d) is
Electronics selected diffraction, 1 (e) is chitosan/colloidal gold lattice fringe.
As shown in Figure 1, colloidal gold particle is nano-scale, and granular size is in 50nm or so (being in aubergine), irregular shape
Shape, lattice structure are length of looking unfamiliar corresponding to (310) and (400).
2) UV-vis of colloidal gold and fluorometric investigation spectrogram in compound system
Test method: chitosan/gold complex system in Example 1 is added to detection quartz after 10 times of dilution
In cuvette, instrument parameter is set, best launch wavelength is found at excitation wavelength 310nm, as a result sees Fig. 2,3.
As shown in Figure 2, when excitation wavelength is 310nm, colloidal gold has an absorption peak at 400nm.
From the figure 3, it may be seen that there is ultraviolet absorption peak at 543nm in colloidal gold, illustrate the partial size of colloidal gold in 60nm or so, with
TEM test result is almost the same.
3) electrochemical property
Test method: electrochemica biological sensor and a blank electrode in Example 1, using Shanghai Chen Hua CHI600D
Electrochemical workstation, with three traditional electrode work systems, Ag/AgCl is reference electrode, and Pt are auxiliary electrode, electrochemical student
Object sensor is working electrode, under the conditions of open-circuit voltage 0.22V, test scope 0.01Hz~100kHz, amplitude 5mV
Ac impedance spectroscopy is tested, data are acquired and is handled with Zview2, impedance value is obtained, as a result sees Fig. 4.
As shown in Figure 4, compared with blank electrode, the Cyclic voltamogram curve peak of electrochemica biological sensor in embodiment
Value is high, area is big, illustrates that the sensor has preferably electro-chemical activity.
4) electrochemica biological sensor detection Ractopamine analysis
Test method: first in electrode surface drop coating chitosan/colloid metal/composite material, the method for self assembly is used later
Electrode is immersed in 1ng/mL Anti-ractopamine antibody liquid, 4h is reacted, then be soaked in Ractopamine solution, using electrification
The method for learning impedance spectrum tests the variation of electrode surface during differential responses, as a result sees Fig. 5.
As shown in Figure 5, after electrode surface successively assembles chitosan/colloid metal/composite material, RAC-anti and RAC, EIS
Charge transfer resistance Δ Rct is significantly increased in figure, and Cyclic voltamogram colloidal gold material curves area significantly increases in CV figure.
After chitosan/colloidal gold modification after electrode adsorption RAC-anti and RAC, charge transfer resistance Δ Rct increases, and cyclic voltammetric is special
Linearity curve area becomes smaller, and illustrates electrode and RAC-anti generation non-specific adsorption after modification, and RAC-anti and RAC generation is special
Opposite sex absorption.
Test method: electrochemica biological sensor in Example 1 is soaked in the Ractopamine solution of various concentration
In, and its impedance value is tested using Electrode with Electrochemical Impedance Spectroscopy, as a result see Fig. 6, wherein Fig. 6 (a) is that the Lake of test various concentration is more
The Nyquist figure of bar amine, Fig. 6 (b) are the linear relation figure that Rct linear fit corresponding to various concentration is made.
By testing the Ractopamine of various concentration, it is fitted its AC impedance and is obtained about detection Ractopamine
Linear equation: y=200.26LogC+529.5, linearly dependent coefficient R2=0.991, the minimum inspection of electrochemica biological sensor
Survey is limited to 2.27pg/mL.
5) selectivity and stability test of electrochemica biological sensor
Test method: electrochemica biological sensor in Example 1 is soaked in the K of 1 μ g/mL respectively+、Ca2+、Na+、Mg2 +, in urea, UA, CLB, SAC, RAC, impedance Value Data is collected after test, as a result sees Fig. 7.
As shown in Figure 7, electrochemica biological sensor is to the detected level highest of Ractopamine in embodiment, and selectivity is most
It is good.
Test method: 5 identical electrochemica biological sensors are prepared using method in embodiment 1, for detecting Lake
As a result dopamine is shown in Fig. 8.
As shown in Figure 8, different batches electrochemica biological sensors has good stability.
Claims (4)
1. chitosan/gold complex system, it is characterised in that: the compound system is in the solution containing gold chloride and chitosan
In be passed through plasma and be prepared, the composition of the solution containing gold chloride and chitosan are as follows: 0.05 ~ 0.1g of gold chloride,
0.1 ~ 0.2g of chitosan, glacial acetic acid 400 ~ 600 μ L, 20 ~ 30mL of water, the plasma are nitrogen gas plasma, be passed through etc. from
The technical parameter of daughter are as follows: pulsed RF power 100 ~ 200W of source power is passed through 5 ~ 10min of time.
2. chitosan/gold complex system preparation method, it is characterised in that: the following steps are included: containing gold chloride and
Plasma is passed through in the solution of chitosan, ultrasonic mixing obtains chitosan/gold complex system, described to contain gold chloride
With the composition of the solution of chitosan are as follows: 0.05 ~ 0.1g of gold chloride, 0.1 ~ 0.2g of chitosan, 400 ~ 600 μ L of glacial acetic acid, water 20 ~
30mL, the plasma are nitrogen gas plasma, are passed through the technical parameter of plasma are as follows: pulsed RF power source power
100 ~ 200W is passed through 5 ~ 10min of time.
3. electrochemica biological sensor or detection Lake of the compound system as described in claim 1 in preparation detection Ractopamine
Application in dopamine.
4. application according to claim 3, it is characterised in that: chitosan/gold complex system is added dropwise on the electrode,
It is dry, obtain chitosan/colloidal gold modification electrode;Above-mentioned electrode is placed in antibody liquid, in electrode surface self assembly antibody,
Up to electrochemica biological sensor.
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