CN108693231A - A kind of electrochemica biological sensor and preparation method thereof of detection carcinomebryonic antigen - Google Patents
A kind of electrochemica biological sensor and preparation method thereof of detection carcinomebryonic antigen Download PDFInfo
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- CN108693231A CN108693231A CN201810513460.2A CN201810513460A CN108693231A CN 108693231 A CN108693231 A CN 108693231A CN 201810513460 A CN201810513460 A CN 201810513460A CN 108693231 A CN108693231 A CN 108693231A
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Abstract
A kind of electrochemica biological sensor of detection carcinomebryonic antigen, the preparation method of the sensor include the following steps:(1) Au@Cu are prepared2O core-shell nanos;(2) carcinomebryonic antigen aptamers modification Au@Cu2O core-shell nanos;(3) glass-carbon electrode of p-aminobenzene sulfonic acid modification is prepared;(4) electrochemica biological sensor is prepared.The present invention is based on Au@Cu2O core-shell nano modified electrodes have good conductive and electric catalyticing characteristic for carcinomebryonic antigen biosensor, substantially increase the sensitivity of the electrochemica biological sensor.
Description
Technical field
The present invention relates to electrochemica biological sensor technical fields, and cancer is detected by electrochemical method more particularly, to one kind
The biosensor of embryonal antigen.
Background technology
One of the main reason for cancer is dead in world wide, because its thorough treatment can not yet be realized.For cancer
The early diagnosis of disease and effectively treatment, detection tumor markers are a kind of very promising directions.As most common
One of biomarker for cancer, the normal contents of carcinomebryonic antigen are 3~5ng/mL in health adult.Carcinomebryonic antigen is measured at present
Method mainly has ELISA, fluoroimmunoassay, radiommunoassay etc..These methods have sensitive reliable excellent
Point, but also suffer from certain drawbacks simultaneously, as ELISA and fluoroimmunoassay cost are higher, and there are kits
The problem of being difficult to preserve, and the reproducibility of radioimmunoassay is poor, there is certain radiation hazradial bundle.Electrochemica biological sensor
Since required instrument and equipment is simple, easy to operate, high sensitivity, selectivity it is good, it is of low cost the advantages that receive extensive pass
Note.Therefore how to build it is a kind of detection carcinomebryonic antigen biosensor become research hot spot.
Invention content
In view of the above-mentioned problems existing in the prior art, the applicant provides a kind of electrochemistry of detection carcinomebryonic antigen
Biosensor and preparation method thereof.The present invention is based on Au@Cu2O core-shell nano modified electrodes are for carcinomebryonic antigen biology
Sensor has good conductive and electric catalyticing characteristic, substantially increases the sensitivity of the electrochemica biological sensor.
Technical scheme is as follows:
A kind of electrochemica biological sensor of detection carcinomebryonic antigen, the preparation method of the sensor include the following steps:
(1) Au@Cu are prepared2O core-shell nanos;
(2) carcinomebryonic antigen aptamers modification Au@Cu2O core-shell nanos;
(3) glass-carbon electrode of p-aminobenzene sulfonic acid modification is prepared;
(4) electrochemica biological sensor is prepared.
The preparation Au@Cu2The method of O core-shell nanos is:Polyvinylpyrrolidone is added into copper nitrate solution,
Add solution of gold nanoparticles and hydrazine hydrate solution after mixing, reaction 2 under the conditions of high-speed stirred~obtain after ten minutes
Au@Cu2O core-shell nanos, constant volume is spare after using water and ethyl alcohol eccentric cleaning respectively.
A concentration of 1~50mM of the copper nitrate solution, volumetric usage are 1~50mL;The polyvinylpyrrolidone is molten
A concentration of 1~8wt% of liquid, volumetric usage are 1~25mL;The particle concentration of the solution of gold nanoparticles is 1 × 10-8mol/
L;The grain size of the gold nanoparticle is 17.8 ± 1.8nm;A concentration of 35wt% of the hydrazine hydrate solution;The high-speed stirred
Rotating speed is 1000~1500r/min;The centrifugal rotational speed is 6500r/min.
The carcinomebryonic antigen aptamers modification Au@Cu2The method of O core-shell nanos is:By Au@made from step (1)
Cu2O core-shell nanoparticle solutions are adapted to liquid solution with carcinomebryonic antigen and are added in tbe buffer liquid simultaneously, are stood after vibrating mixing
12h is centrifuged later, constant volume is spare again after water washing collection precipitation.
The Au@Cu2A concentration of the 1 × 10 of O core-shell nanoparticle solutions-8~10 × 10-8Mol/L, volumetric usage 25
~250 μ L;A concentration of 0.1~5 μm of ol/L of the carcinomebryonic antigen adaptation liquid solution, volumetric usage are 1~20 μ L;The TBE
A concentration of 0.05~0.5mol/L of buffer solution, volumetric usage are 0.1~1mL;The centrifugal rotational speed is 6500r/min.
The method of glass-carbon electrode for preparing p-aminobenzene sulfonic acid modification is:Use alundum (Al2O3) polishing powder to naked first
Glass-carbon electrode is processed by shot blasting, is cleaned by ultrasonic 1 minute with ethyl alcohol and ultra-pure water, and clean rear nitrogen drying is spare;Then dry
P-aminobenzene sulfonic acid is electroplated in net glassy carbon electrode surface;Finally electrode is immersed in phosphorus pentachloride solution and activates 30 minutes, later
Taking-up cleans up spare.
The grain size of the alundum (Al2O3) polishing powder is 1.0,0.5 or 0.3 μm;The system of the plating p-aminobenzene sulfonic acid
For three-electrode system, reference electrode is saturated calomel electrode, is platinum electrode to electrode;The plating p-aminobenzene sulfonic acid
Method is cyclic voltammetry, and scanning range is -0.5~0.5V, and sweep speed 100mV/s, sweep time is 30 minutes;Institute
The molar concentration for stating p-aminobenzene sulfonic acid solution is 5~50mmol/L, and volumetric usage is 1~4mL;The phosphorus pentachloride solution
Molar concentration is 10~100mmol/L, and volumetric usage is 1~4mL.
The method for preparing electrochemica biological sensor is:The glass of p-aminobenzene sulfonic acid modification described in step (3)
With the single stranded DNA solution of carcinomebryonic antigen aptamers complementation in carbon electrodes drop coating, used after reacting 30~180 minutes at room temperature
The Au@Cu that the aptamers made from electrode surface drop coating step (2) are modified again after water is rinsed well2O core-shell nanoparticle solutions,
It is rinsed with water after after same reaction 20~120 minutes spare after extra reactant;It is with the glass-carbon electrode modified finally
Working electrode, the carcinomebryonic antigen solution of various concentration in electrode surface drop coating, according to the Au@Cu of gained2O core-shell nanos
Electrochemical oxidation current value and the logarithm of carcinomebryonic antigen concentration it is in a linear relationship, drawing curve.
A concentration of 2~10 μm of ol/L with the single stranded DNA solution of carcinomebryonic antigen aptamers complementation, volumetric usage 2
~10 μ L;The Au@Cu of the aptamers modification2A concentration of the 5 × 10 of O core-shell nanoparticle solutions-9~1 × 10-8Mol/L, body
Product dosage is 2~10 μ L;The Au@Cu modified with the single stranded DNA solution of carcinomebryonic antigen aptamers complementation, aptamers2O nucleocapsids
The volume ratio of nano-particle solution is 50:1;The system with detection carcinomebryonic antigen is three-electrode system, and reference electrode is
Ag/AgCl electrodes are platinum electrode to electrode;The method of the detection carcinomebryonic antigen is differential pulse voltammetry, scans model
It encloses for -0.2~0.6V;Sweep speed is 4mV/s;The electrolyte solution of the differential pulse voltammetry is phosphate buffer;
The pH of the phosphate buffer is 7.4;The volume of the phosphate buffer is 2mL.
The present invention is beneficial to be had technical effect that:
The present invention can be according to Sensor Design demand by adjusting Au@Cu2The thickness system tune of O core-shell nano shells
Its corresponding electrochemical oxidation peak intensity is controlled, it is easy to operate flexible;
The present invention is acted on by the base pair complementarity between DNA chain by Au@Cu2O core-shell nanos stabilization is quantitatively repaiied
Adorn electrode surface, it is ensured that the stability and accuracy of obtained electrochemical signals;
The present invention passes through Au@Cu2Special parent between O core-shell nanos surface carcinomebryonic antigen aptamers and carcinomebryonic antigen
The quick and precisely detection to carcinomebryonic antigen is realized with effect, is not influenced by other chaff interferents.
Description of the drawings
Fig. 1 is Au@Cu made from the embodiment of the present invention 12The transmission electron microscope picture of O core-shell nanos.
Fig. 2 is electrochemica biological sensor made from the embodiment of the present invention 1 to the standard curve of carcino-embryonic antigen assay.
Specific implementation mode
With reference to the accompanying drawings and examples, the present invention is specifically described.
Embodiment 1:
A kind of electrochemica biological sensor of detection carcinomebryonic antigen, the preparation method of the sensor include the following steps:
(1)Au@Cu2The preparation of O core-shell nanos:4g is added into the copper nitrate solution of a concentration of 40mmol/L of 50mL
It is uniformly mixed after polyvinylpyrrolidone, 2mL a concentration of 1 × 10 is added thereto later-8The grain size of mol/L be 17.8 ±
The hydrazine hydrate solution of the solution of gold nanoparticles of 1.8nm and 68 μ L a concentration of 35% (wt%), it is anti-under the conditions of uniform high-speed stirred
Au@Cu are obtained after answering 3 minutes2O core-shell nanos, with water and ethyl alcohol eccentric cleaning, rear constant volume is 1mL spare twice respectively.Institute
Obtain Au@Cu2The transmission electron microscope picture of O core-shell nanos is as shown in Figure 1, the nano-particle has significantly as seen from Figure 1
Nucleocapsid, grain size are about 138.9 ± 6.5nm, and shell thickness is about 60.55 ± 5.4nm.
(2) carcinomebryonic antigen aptamers modification Au@Cu2The preparation of O core-shell nanos:To 100 a concentration of 0.5mol/L of μ L
Tbe buffer liquid in the Au@Cu that 50 μ L are prepared are added2The cancer of O core-shell nanoparticle solutions and 20 a concentration of 200nmol/L of μ L
Embryonal antigen is adapted to liquid solution, and 12 hours are stood after vibrating mixing, and it is 50 μ to use ultra-pure water constant volume after being washed later by centrifugation again
L is spare.
(3) preparation of p-aminobenzene sulfonic acid modification bare glassy carbon electrode:First by bare glassy carbon electrode respectively with 1.0,0.5 Hes
0.3 μm of alundum (Al2O3) polishing powder polishing treatment, is dried up after being cleaned by ultrasonic respectively 1 minute with ethyl alcohol and ultra-pure water with nitrogen.
Then the bare glassy carbon electrode with smooth mirror surface handled well being used as working electrode, saturated calomel electrode is used as reference electrode,
Platinum electrode is used as, to electrode, cyclic voltammetry curve scanning being carried out in the p-aminobenzene sulfonic acid solution of a concentration of 20mmol/L,
Scanning range is -0.5~0.5V, sweep speed 100mV/s, and scanning has totally been plated after 30 minutes with ultrapure water
The glass-carbon electrode of p-aminobenzene sulfonic acid is finally dipped in the phosphorus pentachloride solution of a concentration of 40mmol/L of 2mL and activates 30 points
Taking-up cleans up spare after clock.
(4) structure of electrochemica biological sensor:The glass carbon electricity of p-aminobenzene sulfonic acid modification first described in step (3)
The single stranded DNA solution of 5 μ L a concentration of 10 μm of ol/L and carcinomebryonic antigen aptamers complementation in pole surface drop coating, after reaction 2 hours
The Au@Cu that aptamers of the 5 μ L as described in step (2) are modified in electrode surface drop coating again after being rinsed with water totally2O core-shell nanos
Particle solution, same reaction are rinsed with water spare after extra reactant after 2 hours.Then in glassy carbon electrode surface drop coating
The carcinomebryonic antigen solution reaction of 5 μ L various concentrations, with the clean electrode of ultrapure water, is with the glass-carbon electrode modified after 2 hours
Working electrode, Ag/AgCl electrodes are reference electrode, and platinum electrode is to scan differential pulse volt-ampere curve to electrode, scans model
It encloses for -0.3~0.5V;Sweep speed is 4mV/s, the phosphate buffer of electrolyte solution 2mL, pH=7.4.According to gained
Au@Cu2The electrochemical oxidation current value and the logarithm of carcinomebryonic antigen concentration of O core-shell nanos are in a linear relationship, draw work
Make curve.Working curve is as shown in Fig. 2, the detection range of this method is 1~500pg/mL as shown in Figure 2.
(5) practical application of electrochemica biological sensor:The accuracy of the electrochemical sensor is by measuring Wuxi the 4th
The concentration of carcinomebryonic antigen is verified in hospital's liver cancer patient blood.First by the blood of 2mL liver cancer patients with the rotating speed of 400g from
The heart obtains 1mL serum after five minutes, and it is spare to be diluted to 2mL with PBS solution.It is obtained by chemical illumination immunity analysis instrument measurement
The concentration of middle carcinomebryonic antigen, is diluted to 60pg/mL, and the dilute serum sample that 5 μ L are prepared is taken to be added drop-wise to Au@Cu2O nucleocapsids
On the electrode of Nanoparticle Modified, Electrochemical Detection is carried out with reference to step (4), 3 groups of parallel determination measures wherein carcinomebryonic antigen
Content is 60.8pg/mL, and error 1.3%, testing result is more accurate.
Embodiment 2
With reference to 1 step of embodiment, a kind of electrochemica biological sensor of detection carcinomebryonic antigen is prepared, clinic is applied to
Experiment:
The blood of 2mL liver cancer patients is obtained into 1mL serum after five minutes with the rotating speed centrifugation of 400g first, it is dilute with PBS solution
It releases spare to 2mL.Then the actual concentrations of wherein carcinomebryonic antigen are obtained by chemical illumination immunity analysis instrument measurement, is diluted
To 200pg/mL, the dilute serum sample that 5 μ L are prepared is taken to be added drop-wise to Au@Cu2On the electrode of O core-shell nanos modification, ginseng
It examines step (4) and carries out Electrochemical Detection, 3 groups of parallel determination, the content for measuring wherein carcinomebryonic antigen is 196.3pg/mL, and error is
1.9%, testing result is more accurate.
Embodiment 3
With reference to 1 step of embodiment, a kind of electrochemica biological sensor of detection carcinomebryonic antigen is prepared, clinic is applied to
Experiment:
The blood of 2mL liver cancer patients is obtained into 1mL serum after five minutes with the rotating speed centrifugation of 400g first, it is dilute with PBS solution
It releases spare to 2mL.Then the actual concentrations of wherein carcinomebryonic antigen are obtained by chemical illumination immunity analysis instrument measurement, is diluted
To 360pg/mL, the dilute serum sample that 5 μ L are prepared is taken to be added drop-wise to Au@Cu2On the electrode of O core-shell nanos modification, ginseng
It examines step (4) and carries out Electrochemical Detection, 3 groups of parallel determination, the content for measuring wherein carcinomebryonic antigen is 346.7pg/mL, and error is
3.7%, testing result is more accurate.
Claims (9)
1. a kind of electrochemica biological sensor of detection carcinomebryonic antigen, which is characterized in that the preparation method of the sensor includes
Following steps:
(1) Au@Cu are prepared2O core-shell nanos;
(2) carcinomebryonic antigen aptamers modification Au@Cu2O core-shell nanos;
(3) glass-carbon electrode of p-aminobenzene sulfonic acid modification is prepared;
(4) electrochemica biological sensor is prepared.
2. electrochemica biological sensor according to claim 1, which is characterized in that the preparation Au@Cu2O core-shell nano grains
Son method be:Polyvinylpyrrolidone is added into copper nitrate solution, add after mixing solution of gold nanoparticles and
Hydrazine hydrate solution, reaction 2 under the conditions of high-speed stirred~obtain Au@Cu after ten minutes2O core-shell nanos use water and second respectively
Constant volume is spare after alcohol eccentric cleaning.
3. electrochemica biological sensor according to claim 2, which is characterized in that a concentration of the 1 of the copper nitrate solution
~50mM, volumetric usage are 1~50mL;A concentration of 1~8wt% of the polyvinylpyrrolidonesolution solution, volumetric usage be 1~
25mL;The particle concentration of the solution of gold nanoparticles is 1 × 10-8mol/L;The grain size of the gold nanoparticle be 17.8 ±
1.8nm;A concentration of 35wt% of the hydrazine hydrate solution;The high-speed stirred rotating speed is 1000~1500r/min;The centrifugation
Rotating speed is 6500r/min.
4. electrochemica biological sensor according to claim 1, which is characterized in that the carcinomebryonic antigen aptamers modification
Au@Cu2The method of O core-shell nanos is:By Au@Cu made from step (1)2O core-shell nanoparticle solutions are suitable with carcinomebryonic antigen
Ligand solution is added in tbe buffer liquid simultaneously, and 12h is stood after vibrating mixing, and constant volume is standby again after centrifugation later, water washing collection precipitation
With.
5. electrochemica biological sensor according to claim 4, which is characterized in that the Au@Cu2O core-shell nanos are molten
A concentration of the 1 × 10 of liquid-8~10 × 10-8Mol/L, volumetric usage are 25~250 μ L;The carcinomebryonic antigen adaptation liquid solution
A concentration of 0.1~5 μm of ol/L, volumetric usage are 1~20 μ L;A concentration of 0.05~0.5mol/L of the tbe buffer liquid, volume
Dosage is 0.1~1mL;The centrifugal rotational speed is 6500r/min.
6. electrochemica biological sensor according to claim 1, which is characterized in that described to prepare p-aminobenzene sulfonic acid modification
The method of glass-carbon electrode be:Bare glassy carbon electrode is processed by shot blasting with alundum (Al2O3) polishing powder first, with ethyl alcohol and is surpassed
Pure water is cleaned by ultrasonic 1 minute, and clean rear nitrogen drying is spare;Then p-aminophenyl sulphur is electroplated in clean glassy carbon electrode surface
Acid;Finally electrode is immersed in phosphorus pentachloride solution and is activated 30 minutes, taking-up later cleans up spare.
7. requiring the electrochemica biological sensor according to right 6, which is characterized in that the grain of the alundum (Al2O3) polishing powder
Diameter is 1.0,0.5 or 0.3 μm;The system of the plating p-aminobenzene sulfonic acid is three-electrode system, and reference electrode is that saturation is sweet
Mercury electrode is platinum electrode to electrode;It is described plating p-aminobenzene sulfonic acid method be cyclic voltammetry, scanning range be-
0.5~0.5V, sweep speed 100mV/s, sweep time are 30 minutes;The molar concentration of the p-aminobenzene sulfonic acid solution is
5~50mmol/L, volumetric usage are 1~4mL;The molar concentration of the phosphorus pentachloride solution is 10~100mmol/L, and volume is used
Amount is 1~4mL.
8. requiring the electrochemica biological sensor according to right 1, which is characterized in that described to prepare electrochemica biological sensor
Method be:Described in step (3) p-aminobenzene sulfonic acid modification glassy carbon electrode surface drop coating on carcinomebryonic antigen aptamers
Complementary single stranded DNA solution, again in electrode surface drop coating step after being rinsed with water after reacting 30~180 minutes at room temperature totally
(2) the Au@Cu that aptamers made from are modified2O core-shell nanoparticle solutions, equally react 20~120 minutes after after be rinsed with water
Fall spare after extra reactant;It is different dense in electrode surface drop coating finally using the glass-carbon electrode modified as working electrode
The carcinomebryonic antigen solution of degree, according to the Au@Cu of gained2The electrochemical oxidation current value and carcinomebryonic antigen of O core-shell nanos are dense
The logarithm of degree is in a linear relationship, drawing curve.
9. electrochemica biological sensor according to claim 8, which is characterized in that described complementary with carcinomebryonic antigen aptamers
Single stranded DNA solution a concentration of 2~10 μm of ol/L, volumetric usage be 2~10 μ L;The Au@Cu of the aptamers modification2O cores
A concentration of the 5 × 10 of core/shell nanoparticles solution-9~1 × 10-8Mol/L, volumetric usage are 2~10 μ L;It is described suitable with carcinomebryonic antigen
The single stranded DNA solution of ligand complementation, the Au@Cu of aptamers modification2The volume ratio of O core-shell nanoparticle solutions is 50:1;It is described
System with detection carcinomebryonic antigen is three-electrode system, and reference electrode is Ag/AgCl electrodes, is platinum electrode to electrode;Institute
The method for stating detection carcinomebryonic antigen is differential pulse voltammetry, and scanning range is -0.2~0.6V;Sweep speed is 4mV/s;
The electrolyte solution of the differential pulse voltammetry is phosphate buffer;The pH of the phosphate buffer is 7.4;The phosphorus
The volume of phthalate buffer is 2mL.
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