CN106248742A - Chitosan/gold complex system, preparation method and application - Google Patents
Chitosan/gold complex system, preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of chitosan/gold complex system, preparation method and application, belong to field of material technology.This compound system is to be passed through plasma in the solution containing gold chloride and chitosan to prepare, and in system, colloidal gold particle is nano-scale, and granular size is at about 50nm, in irregularly shaped.This 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, selectivity and good stability.
Description
Technical field
The present invention relates to a kind of chitosan/gold complex system, also relate to this compound system preparation method and
Application, belongs to field of material technology.
Background technology
Gold colloidal is also referred to as aurosol, is gold salt to be reduced into stable, the homogeneous nano Au particle suspension formed after gold, belongs to
In heterogeneous heterogeneous system, color changes from salmon pink to aubergine.Colloidal gold particle is based on a golden core (Au), peripheral
Cladding anion AuCl2-, outermost layer is H+, being dispersed between colloid in solution and maintaining colloid is suspended state.Colloidal gold particle
Golden core be not preferable spherical nuclei, less the most spherical in shape of granule, and granule relatively big (more than diameter 30nm) is many in ellipse
Spherical.
Colloidal gold particle has Fluorescence Characteristic, and it exists single optical absorption peak in visible-range, and along with
The increase wavelength of particle diameter is elongated.It addition, in numerous colloid properties of gold colloidal, be the most significantly the sensitivity to electrolyte
Property, gold colloidal is not only played a protective role by some macromolecular substances such as protein, moreover it is possible to improve the stability of gold colloidal.Typically
In the case of, when not destroying the stabilizing factor such as electrolyte, collosol concentration, the stability of gold colloidal is preferable, and long-time placement is the most not
Coacervation can occur.
At present, gold colloidal is used for detecting antigen-antibody mainly as label or developer, as a kind of novel immunity
Labelling technique has potential application prospect in biological, medicine and other fields.Owing to this technology does not exist endogenous enzymes interference and radiation
Property isotope interference etc., and use the gold colloidal of variable grain size as dual or multiple labelling, its location is more accurate.Glue
In body gold, the size of gold particle is between 1～100nm, except having the general character of nano material (such as bigger specific surface area, quantum chi
Very little effect and macro quanta tunnel effect etc.) outward, its chemical property is also abnormal active, can generate tool spy with nucleopilic reagent absorption
The group of distinguished service energy.Such as nanometer gold can be with the Electrostatic Absorption of amino generation non-covalent bond, it is also possible to produce stronger with thin base
Covalent bond effect, but after combining, the optical property of nanometer gold does not change, and therefore can be used for biological monitoring.In recent years,
The application of gold colloidal is concentrated mainly on dot immunogold filtration assay and gold colloidal fast immune chromatographic method.Quickly spot immune
Gold percolation is named again and drips gold immunization, and known specific antibody or antigen, with microporous membrane as solid phase carrier, are fixed by the method
On it, and loading in the percolating device being full of absorbent material, add sample to be tested, capillarity or diafiltration via filter membrane are made
With, make the antigen in specimen or antibody coated antibody or antigen on film be combined, after reach through colloidal gold conjugate labelling
To testing goal.And fast immune chromatographic method is also with microporous membrane as solid phase carrier, on it fixing known specific antibody or
Antigen is as detection band, and colloid gold label thing combines in glass fibre matter and is dried in release pad, and its one end is connected with sample pad, separately
One end is connected with film, and the other end of film is connected with adsorptive pads.After adding testing sample, sample is moved forward by capillary tube, and
By the glass fibre containing label, make label aquation again, and together with reacting to each other with colloid gold label thing forward, extremely
During detection line, the complex of label and determinand intercepted and captured by detection line, thus red result occurs.
In prior art, traditional gold colloidal preparation method includes trisodium citrate reduction method, ascorbic acid reduction, boron
Sodium hydride reducing process etc..Wherein, the operating procedure of trisodium citrate reduction method is: take 50mL 0.01% gold chloride and 2mL 1%
Trisodium citrate is sufficiently mixed, and heats 15min, mends to original volume with water, to obtain final product after cooling.The operation step of ascorbic acid reduction
Suddenly it is: take 50mL 0.01% gold chloride, 0.75mL 0.2M K2CO3It is sufficiently mixed with 0.5mL 0.7% ascorbic acid, heating
10min, mends to original volume with water after cooling, to obtain final product.The operating procedure of sodium borohydride reduction is: take 50mL 0.01% chlorine gold
Acid and 0.25mL 0.2M K2CO3It is sufficiently mixed, divides 3 to 5 times to mixed solution and dripping freshly prepared 0.5mg/mL hydroboration
Sodium, edged vibration in limit makes its mix homogeneously, until solution purpling, eventually becomes orange red, and continue to vibrate about 3min, to obtain final product.So
And, the gold colloidal 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, including: in insulant container, add the mixed liquor of gold chloride and reducing agent, to this mixed liquor
In be passed through atmospheric air plasma, obtain Au colloidal nanoparticles.The method uses air to substitute rare gas as swashing
Getting angry body, 2～10min can form that homogeneity is good, highly sensitive and the Au colloidal nanoparticles of stable performance.Publication No.
The patent of invention of CN102430392A also discloses that the preparation method of a kind of composite nano colloidal gold chitosan carrier, including: 1)
The chlorauric acid solution taking mass concentration 0.01% is heated to boiling, and stirring adds 1% citric acid three sodium solution of preheating, to chlorine gold
Acid solution is become Lycoperdon polymorphum Vitt from golden yellow and becomes claret again, and continue stirring boiling to solution is bright redness, obtains gold colloidal
Solution；2) the chitosan organic monoacid of 0.1～1% is dissolved into mass concentration 0.01～the chitosan solution of 0.1%, filters standby
With；3) according to mass ratio 1:0.1～1, colloidal gold solution is mixed homogeneously with chitosan solution, be slowly added dropwise the carbonic acid of 0.05%
Potassium solution is 7.0 to mixed liquor pH value, obtains composite nano colloidal gold chitosan carrier mixed liquor.Wherein, the grain of complex carrier
Footpath is distributed between 20～200nm, and mean diameter 80nm can be used for immune detection or immunologic diagnosis.But, above two method
It is both needed to use reducing agent, and the gold colloidal poor stability prepared when preparing gold colloidal, needs to store at low temperatures.
Summary of the invention
It is an object of the invention to provide a kind of chitosan/gold complex system.
Meanwhile, the present invention also provides for the preparation method of a kind of chitosan/gold complex system.
Finally, the present invention provides a kind of chitosan/gold complex to tie up to prepare electrochemica biological sensor or inspection
Survey the application in Ractopamine.
In order to realize object above, the technical solution adopted in the present invention is:
Chitosan/gold complex system, is to be passed through plasma in the solution containing gold chloride and chitosan to prepare
Obtain.
Consisting of of the described solution containing gold chloride and chitosan: gold chloride 0.05～0.1g, chitosan 0.1～
0.2g, glacial acetic acid 400～600 μ L, water 20～30mL.During preparation solution, can first chitosan be dissolved in the aqueous solution of glacial acetic acid,
Add gold chloride.
Described plasma is nitrogen gas plasma.The technical parameter being passed through plasma is: pulsed RF power source merit
Rate 100～200W, is passed through the time 5～10min.
The preparation method of chitosan/gold complex system, comprises the following steps: molten containing gold chloride and chitosan
Liquid is passed through plasma, ultrasonic mixing, is aubergine to solution, obtain chitosan/gold complex system.
Consisting of of the described solution containing gold chloride and chitosan: gold chloride 0.05～0.1g, chitosan 0.1～
0.2g, glacial acetic acid 400～600 μ L, water 20～30mL.
Described plasma is nitrogen gas plasma.The technical parameter being passed through plasma is: pulsed RF power source merit
Rate 100～200W, is passed through the time 5～10min.Preparation principle is: the present invention uses Room-temperature low-pressure plasma to prepare gold colloidal,
Wherein chitosan is as stabilizer and reducing agent, and in plasma preparation process, nitrogen gas plasma causes hydrone to be produced from
By base, and then gold chloride is reduced.
Chitosan/gold complex ties up to the application preparing in electrochemica biological sensor, particularly as follows: by chitosan/
Gold complex system drips on electrode, is dried, and obtains the electrode that chitosan/gold colloidal is modified；Above-mentioned electrode is placed in anti-
In body fluid, at electrode surface self assembly antibody, obtain electrochemica biological sensor.In above-mentioned application, chitosan per se with
Amino group, can be that subsequent fixed antibody (such as Anti-ractopamine antibody) provides binding site, improve the susceptiveness of detection.
Time in described antibody liquid containing Anti-ractopamine antibody, gained sensor i.e. can be used for detecting Ractopamine.
Beneficial effects of the present invention:
In the present invention, chitosan/gold complex system is to be passed through plasma in the solution containing gold chloride and chitosan
Body prepares, and in this system, colloidal gold particle is nano-scale, and granular size is at about 50nm, in irregularly shaped.This is multiple
Fit system can as sensitive membrane material modified electrode, after electrode self assembly RAC-anti antibody can specific detection RAC antigen, inspection
Survey highly sensitive, selectivity and good stability.
The present invention uses plasma method to prepare chitosan/gold complex system, can save chemical reducing agent, at normal temperatures
The gold colloidal that Fast back-projection algorithm is stable, is the synthetic method of a kind of " green ".
Accompanying drawing explanation
Fig. 1 is the TEM figure of chitosan/gold colloidal in compound system；
Fig. 2 is the fluorometric investigation spectrogram of chitosan/gold colloidal in compound system；
Fig. 3 is the ultraviolet-visible spectrogram of gold colloidal in compound system；
Fig. 4 is the CV figure of electrochemica biological sensor；
Fig. 5 is the electrochemical impedance figure that process was constructed and detected to electrochemica biological sensor；
Fig. 6 is EIS figure and the matched curve figure of electrochemica biological sensor detection variable concentrations RAC；
Fig. 7 is the selectivity test result of electrochemica biological sensor；
Fig. 8 is the stability test result of electrochemica biological sensor.
Detailed description of the invention
The present invention is only described in further detail by following embodiment, but does not constitute any limitation of the invention.
Embodiment 1
Chitosan/gold complex system, its preparation process is as follows:
1) clean all glass drying ovens to be used with freshly prepared chloroazotic acid, then use water cleaning down, drying for standby；
2) measure 600 μ L 99% glacial acetic acids in 25mL volumetric flask with 1mL liquid-transfering gun, add 20mL water and be diluted to concentration
The glacial acetic acid solution of 3%；
3) weigh 0.2g chitosan, join in the glacial acetic acid solution diluted, ultrasonic make chitosan be completely dissolved, quiet
Put, be wholly absent to bubble；
4) in deionized water add gold chloride to concentration 100mM, add 550 μ L chitosan solutions, power 500W,
Under frequency 40kHz, ultrasonic mixing 10min, stands to obtain mixed liquor；
5) in mixed liquor, nitrogen gas plasma it is passed through, pulsed RF power source power 200W, time 5min, then at power
Under 500W, frequency 40kHz, ultrasonic mixing 10min, becomes aubergine to solution, obtains chitosan/gold complex system.
Chitosan/gold complex ties up to detect the application in Ractopamine, specifically comprises the following steps that
1) pretreatment of gold electrode
Use 0.5 μm and 0.3 μm Al2O3Serosity sanding and polishing gold electrode on chamois leather, to electrode surface without substantially drawing
Trace, polishing track is the figure of eight, then cleans gold electrode with deionized water, will remain in the Al on electrode2O3Wash clean；By electricity
Pole is placed in H2O2:H2SO4In the mixed liquor of volume ratio 3:7, ultrasonication 15min, deionization under power 500W, frequency 40kHz
Water is rinsed well；Electrode is placed in water again: in the mixed liquor of dehydrated alcohol volume ratio 1:1, the most ultrasonic 15min, then be placed in
Ultrasonic 15min in ionized water；The gold electrode cleaned up is placed in 0.5M H2SO4In solution, use cyclic voltammetry 0.2～
Activation 2 times in 1.6V voltage range, then rinse well with deionized water, dry up standby；
2) chitosan of above-mentioned preparation/gold complex system being diluted to 2 times of volumes, ultrasonic 30min makes mix homogeneously；
Pipette 10 μ L diluted system with 10 μ L liquid-transfering guns to drip on gold electrode, completely dry rear its electrochemical impedance of test (EIS), treat steady
Rinse well with deionized water after Ding and dry up, obtaining the electrode that chitosan/gold colloidal is modified；Above-mentioned electrode is placed in 100ng/
In mL Anti-ractopamine antibody (RAC-anti) liquid soak 2h, it is ensured that chitosan/gold colloidal composite have abundance time with
Anti-ractopamine antibody self assembly, taking-up rinses well, tests EIS, with deionized water rinsing and dry up after stable, the most handy
Electrochemica biological sensor in detection Ractopamine；
3) use this sensor that Ractopamine in solution is detected.
Embodiment 2
Chitosan/gold complex system, its preparation process is as follows:
1) containing gold chloride and the preparation of chitosan solution
Chitosan is dissolved in dilute glacial acetic acid solution, adds gold chloride after dissolving completely, mix standby；
Solution composition is: gold chloride 0.05g, chitosan 0.1g, glacial acetic acid 400 μ L, water 20mL；
2) in above-mentioned solution, nitrogen gas plasma it is passed through, pulsed RF power source power 100W, time 10min, reaction
Become aubergine to solution, obtain chitosan/gold complex system.
The application of chitosan/gold complex system is with embodiment 1.
Embodiment 3
Chitosan/gold complex system, its preparation process is as follows:
1) containing gold chloride and the preparation of chitosan solution
Chitosan is dissolved in dilute glacial acetic acid solution, adds gold chloride after dissolving completely, mix standby；
Solution composition is: gold chloride 0.1g, chitosan 0.2g, glacial acetic acid 600 μ L, water 30mL；
2) being passed through nitrogen gas plasma in above-mentioned 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 with embodiment 1.
Embodiment 4
Chitosan/gold complex system, its preparation process is as follows:
1) containing gold chloride and the preparation of chitosan solution
Chitosan is dissolved in dilute glacial acetic acid solution, adds gold chloride after dissolving completely, mix standby；
Solution composition is: gold chloride 0.075g, chitosan 0.15g, glacial acetic acid 500 μ L, water 25mL；
2) being passed through nitrogen gas plasma in above-mentioned 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 with embodiment 1.
Test example
1) pattern of gold colloidal and structured testing in compound system
Method of testing: chitosan/gold complex system in Example 1, drips on the copper mesh that carbon film supports, is used for
TEM tests, and result is shown in Fig. 1, and the TEM figure under wherein Fig. 1 (a)～1 (c) are followed successively by 10nm, 200nm, 5nm scale, Fig. 1 (d) is
Electronics selected diffraction, 1 (e) is the lattice fringe of chitosan/gold colloidal.
As shown in Figure 1, colloidal gold particle is nano-scale, and granular size is in about 50nm (in aubergine), irregular shape
Shape, its lattice structure is for looking unfamiliar length corresponding to (310) and (400).
2) UV-vis of gold colloidal and fluorometric investigation spectrogram in compound system
Method of testing: chitosan/gold complex system in Example 1, joins detection quartz after diluting 10 times
In cuvette, instrument parameter is set, at excitation wavelength 310nm find most preferably launch wavelength, result see Fig. 2,3.
As shown in Figure 2, when excitation wavelength is 310nm, gold colloidal has an absworption peak at 400nm.
From the figure 3, it may be seen that gold colloidal occurs ultraviolet absorption peak at 543nm, illustrate the particle diameter of gold colloidal at about 60nm, with
TEM test result is basically identical.
3) electrochemical property
Method of testing: electrochemica biological sensor and a blank electrode in Example 1, uses Shanghai occasion China CHI600D
Electrochemical workstation, with three traditional electrode work systems, Ag/AgCl is reference electrode, and Pt silk is auxiliary electrode, electrification student
Thing sensor is working electrode, under the conditions of open-circuit voltage 0.22V, test scope 0.01Hz～100kHz, amplitude 5mV
Test ac impedance spectroscopy, gathers data and with Zview2 process, obtains resistance value, and result is shown in 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 this sensor has preferably electro-chemical activity.
4) electrochemica biological sensor detection Ractopamine analysis
Method of testing: first in electrode surface drop coating chitosan/gold colloidal composite, the method using self assembly afterwards
Electrode is immersed in 1ng/mL Anti-ractopamine antibody liquid, reacts 4h, then be soaked in Ractopamine solution, use electrification
The change of electrode surface during the method test differential responses of impedance spectrum, result is shown in Fig. 5.
As shown in Figure 5, after electrode surface assembles chitosan/gold colloidal composite, RAC-anti and RAC successively, EIS
In figure, charge transfer resistance Δ Rct all significantly increases, and in CV figure, Cyclic voltamogram gold colloidal material curves area significantly increases.
After chitosan/gold colloidal modifies rear electrode absorption RAC-anti and RAC, charge transfer resistance Δ Rct increases, and cyclic voltammetric is special
Linearity curve area diminishes, and illustrates that modifying rear electrode occurs non-specific adsorption with RAC-anti, and RAC-anti Yu RAC occurs spy
Opposite sex absorption.
Method of testing: electrochemica biological sensor in Example 1, is soaked in the Ractopamine solution of variable concentrations
In, and use Electrode with Electrochemical Impedance Spectroscopy to test its resistance value, result is shown in Fig. 6, and wherein Fig. 6 (a) is that the Rec of test variable concentrations is many
The Nyquist figure of bar amine, Fig. 6 (b) is the line relationship figure making the Rct linear fit that variable concentrations is corresponding.
By testing the Ractopamine of variable concentrations, it is worth to about detection Ractopamine through its AC impedance of matching
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 of electrochemica biological sensor and stability test
Method of testing: 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, after test, collect resistance value data, result is shown in Fig. 7.
As shown in Figure 7, in embodiment, electrochemica biological sensor is the highest to the detected level of Ractopamine, and selectivity is
Good.
Method of testing: use method in embodiment 1 to prepare 5 identical electrochemica biological sensors, be used for detecting Rec
Dopamine, result is shown in Fig. 8.
As shown in Figure 8, the having good stability of different batches electrochemica biological sensor.
Claims (10)
1. chitosan/gold complex system, it is characterised in that: this compound system is at the solution containing gold chloride and chitosan
In be passed through plasma and prepare.
Compound system the most according to claim 1, it is characterised in that: described containing gold chloride with the group of the solution of chitosan
Become: gold chloride 0.05～0.1g, chitosan 0.1～0.2g, glacial acetic acid 400～600 μ L, water 20～30mL.
Compound system the most according to claim 1 and 2, it is characterised in that: described plasma is nitrogen gas plasma.
Compound system the most according to claim 3, it is characterised in that: the technical parameter being passed through plasma is: pulse is penetrated
Frequently power source power 100～200W, are passed through the time 5～10min.
5. the preparation method of chitosan/gold complex system, it is characterised in that: comprise the following steps: containing gold chloride and
The solution of chitosan is passed through plasma, ultrasonic mixing, obtains chitosan/gold complex system.
Preparation method the most according to claim 5, it is characterised in that: described containing gold chloride with the group of the solution of chitosan
Become: gold chloride 0.05～0.1g, chitosan 0.1～0.2g, glacial acetic acid 400～600 μ L, water 20～30mL.
7. according to the preparation method described in claim 5 or 6, it is characterised in that: described plasma is nitrogen gas plasma.
Preparation method the most according to claim 7, it is characterised in that: the technical parameter being passed through plasma is: pulse is penetrated
Frequently power source power 100～200W, are passed through the time 5～10min.
9. as according to any one of Claims 1 to 4, compound system is preparing electrochemica biological sensor or detection Rec DOPA
Application in amine.
Application the most according to claim 9, it is characterised in that: chitosan/gold complex system is dripped at electrode
On, it is dried, obtains the electrode that chitosan/gold colloidal is modified；Above-mentioned electrode is placed in antibody liquid, resists in electrode surface self assembly
Body, obtains electrochemica biological sensor.
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| CN101341118A (en) * | 2004-12-22 | 2009-01-07 | Ambrx公司 | Compositions containing, methods involving, and uses of non-natural amino acids and polypeptides |
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2016
- 2016-09-23 CN CN201610850241.4A patent/CN106248742B/en active Active
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| CN101341118A (en) * | 2004-12-22 | 2009-01-07 | Ambrx公司 | Compositions containing, methods involving, and uses of non-natural amino acids and polypeptides |
| CN104458850A (en) * | 2014-11-28 | 2015-03-25 | 青岛海佑海洋生物工程有限公司 | Screen-printed electrode for detecting uric acid, and preparation method of screen-printed electrode |
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| SUELLEN CADORIN FERNANDES ET AL.: "Development of a biomimetic chitosan film-coated gold electrode for determination of dopamine in the presence of ascorbic acid and uric acid", 《ELECTROCHIMICA ACTA》 * |
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