CN110487869A - Electrochemical luminescence biosensor and preparation method thereof based on nitridation carbon quantum dot - Google Patents
Electrochemical luminescence biosensor and preparation method thereof based on nitridation carbon quantum dot Download PDFInfo
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Abstract
The invention discloses a kind of electrochemical luminescence biosensors and preparation method thereof based on nitridation carbon quantum dot, the electrochemica biological sensor is used for cancer diagnosis, it include glass-carbon electrode, the glassy carbon electrode surface has one layer of g-CNQDs film, g-CNQDs film is equipped with gold nano grain-lung cancer lesion hairpin dna, hairpin dna end is connected on gold nano grain, in the detection process hairpin dna and lung cancer ct DNA complementary pairing.The present invention has the advantage that (1) the present invention be directed to the approach and technology of the noninvasive pathological diagnosis of cancer compared with prior art, and compared with aspiration biopsy, which will not generate secondary harm to patient；(2) this sensor is constituted simple, compared with current mainstream detection method, has the advantages such as easy to detect, quick and at low cost；(3) due to can designed, designed DNA probe, kinds cancer can be detected simultaneously, have very big potential applicability in clinical practice.
Description
Technical field
The invention discloses a kind of electrochemical luminescence biosensors and preparation method thereof based on nitridation carbon quantum dot, should
Electrochemica biological sensor is used for cancer diagnosis, belongs to new function material and biosensor technique field.
Background technique
How to realize the early detection of cancer and staging diagnosis is still an arduous challenge.Cancer hair generally can companion
With specific gene mutation, this is we provide the new approaches of cancer diagnosis.
Electrochemical luminescence (ECL) refers generally under three-electrode system, causes electrochemistry anti-by applying voltage on the electrode
It answers, the substance generated at electrode carries out high energy electron transfer reaction and forms excitation state, and luminescence phenomenon when ground state is returned in transition.Electricity
Chemiluminometry has many advantages, such as that the range of linearity is wide, instrument is simple, high sensitivity, controllability are good, selectivity is high.Quantum dot
There is unique physicochemical property as a kind of novel electrochemiluminescent, be electrochemiluminescence analysis field research hotspot it
One.G-CNQDs has chemical inertness, good stability and biocompatibility, and hypotoxicity is easily prepared and environmental-friendly etc. excellent
Point.In the past few years, the exploitation of the electrochemical luminescence bioprobe based on g-CNQDs obtains important achievement.
Due to no detection program appropriate and treatment method, 8,200,000 people are had more than every year and die of cancer, wherein lung cancer is
One of most common malignant tumour in the world, it has also become first of China's urban population Death Cause for Malignant Tumors.Study people
Member is exploring always through screening for cancer, and Index for diagnosis and monitoring are to detect and cure the method for cancer.However, to being at present
Only, there are no known diagnostic methods during cancer detection will not patient harm health.Therefore, develop it is noninvasive and
Accurate early diagnosis of cancer method has important practical significance.
Summary of the invention
It is a kind of based on the electrochemical luminescence biosensor for nitrogenizing carbon quantum dot and its production the purpose of the present invention is constructing
Method has important practical significance for noninvasive and accurate early diagnosis of cancer method.
In order to achieve the above objectives, the solution of use of the invention is: the electrochemical luminescence based on nitridation carbon quantum dot
Biosensor includes glass-carbon electrode, and the glassy carbon electrode surface has one layer of g-CNQDs (nitridation carbon quantum dot) film, g-
CNQDs film is equipped with gold nano grain-lung cancer lesion hairpin dna, and hairpin dna end is connected on gold nano grain, In
Hairpin dna and lung cancer ct DNA complementary pairing in detection process.
According to the above scheme, the hairpin dna is according to designed by the DNA sequence dna of lung carcinoma cell mutation.
According to the above scheme, the average grain diameter of the nitridation carbon quantum dot is 5nm.
The production method of the electrochemical luminescence biosensor based on nitridation carbon quantum dot, includes following step
It is rapid:
(1) it by glass-carbon electrode polishing powder polishing treatment, then cleans；
(2) g-CNQDs, gold nano grain-lung cancer lesion hairpin dna solution are modified to glassy carbon electrode surface respectively,
It is dried at room temperature and 37 DEG C respectively；
(3) above-mentioned electrode is rinsed through PBS solution to get the electricity based on nitridation carbon quantum dot for detecting lung cancer ct DNA
Chemiluminescence biosensor.
According to the above scheme, the preparation method of the g-CNQDs is: weighing the urea and citric acid three that molar ratio is 6:1
Sodium is pulverized, and is washed product after cooling in 160~190 DEG C of reaction 1h, is then dialysed to get g-CNQDs.
According to the above scheme, the preparation method of the gold nano grain-lung cancer lesion hairpin dna solution includes following
Step:
1) HAuCl for being 1mmol/L by 100mL concentration4·4H2O aqueous solution is heated to fluidized state, in the effect of stirring
Lower addition 20mL trisodium citrate continues boiling mixture, and mixture color becomes claret by colourless, stops heating, refrigerates standby
With；
2) hairpin dna for taking 10 μ L 0.1mmol/L lung cancer lesions, adds 0.15 μ L 10mmol/L tri- (2- carboxyethyl)
Phosphine solution, solvent are the PBS solution of 0.01mol/L NaCl containing 0.1mol/L, react 1h, then, 100 μ L steps 1) are added
Gained AuNPs solution reacts 0.5h in said mixture.
The detection method of the electrochemical luminescence biosensor based on nitridation carbon quantum dot, includes following step
It is rapid:
1) it is added dropwise 1 × 10 respectively on glass-carbon electrode-12~1 × 10-15The lung cancer lesion ct DNA of mol/L is trained at 37 DEG C
Educate 2h；
2) it is tested using the three-electrode system of electrochemical workstation, using traditional three-electrode system, with Ag/AgCl
For reference electrode, it is to electrode, using above-mentioned steps 1 with platinum plate electrode) treated glass-carbon electrode is working electrode, by electrochemistry
Work station and superweak luminescence survey meter link together, and using cyclic voltammetry, scanning range is -2.0~0V, sweep speed
For 0.1V/S；
It 3) is 0.1mol/L K in the concentration that contains of 10mL, pH 7.42S2O8Phosphate buffer solution in, pass through electrochemistry
Luminescent system detects the electrochemical luminescence signals intensity generated to the lung cancer target dna of various concentration, draws working curve.
The present invention will nitrogenize carbon quantum dot as electrochemical luminescence material, it and co-reactant K2S2O8In phosphate-buffered
Stablized in solution (PBS) and strong cathode ECL emits.Hairpin dna end is connect and modified with gold nano grain
On g-CNQDs, resonance energy transfer system can be constructed between the electrochemical luminescence and AuNPs of g-CNQDs, when target dna and hair
Block DNA and match clock synchronization, hairpin dna ring is opened, nitrogenize carbon quantum dot between gold nano grain at a distance from increase, Resonance energy transfer
System is obstructed, and electrochemical luminescence signals is caused to restore.
Due to the application of the above technical scheme, the present invention has the advantage that compared with prior art
(1) the present invention be directed to the approach and technology of the noninvasive pathological diagnosis of cancer, and compared with aspiration biopsy, the sensor is not
Secondary harm can be generated to patient；
(2) this sensor is constituted simple, compared with current mainstream detection method, has easy to detect, quick and cost
Low advantage；
(3) due to can designed, designed DNA probe, kinds cancer can be detected simultaneously, have very big clinical application
Prospect.
Detailed description of the invention
Fig. 1 is the ECL response curve target dna detection after the target dna cultivation of biosensor and various concentration
Working curve.
Specific embodiment
Below with reference to embodiment, the present invention will be further described in detail.
The preparation method of g-CNQDs of the invention is: weighing the urea and trisodium citrate that molar ratio is 6:1, is ground into
Product is washed, is then dialysed to get g-CNQDs after cooling in 160~190 DEG C of reaction 1h by powder.
The preparation method of gold nano grain-lung cancer lesion hairpin dna solution is: 1) being 1mmol/L by 100mL concentration
HAuCl4·4H2O aqueous solution is heated to fluidized state, and 20mL trisodium citrate is added under the action of stirring and continues to boil mixing
Object, mixture color become claret by colourless, stop heating, refrigerate spare；2) 10 μ L 0.1mmol/L lung cancer lesions are taken
Hairpin dna, adds 0.15 μ L 10mmol/L tri- (2- carboxyethyl) phosphine solution, and solvent is that 0.01mol/L contains 0.1mol/
The PBS solution of LNaCl reacts 1h, then, AuNPs solution obtained by 100 μ L steps 1) is added in said mixture, reaction
0.5h。
Embodiment 1
A kind of preparation method of the electrochemical luminescence biosensor based on nitridation carbon quantum dot
1) glass-carbon electrode of diameter 3mm is successively taken turns doing into polishing treatment with 0.3 μm, 0.05 μm of aluminum oxide polishing powder, so
It is successively cleaned up afterwards with ethyl alcohol, nitric acid, ultrapure water；
2) 5~10 μ L g-CNQDs (nitridation carbon quantum dot), AuNPs-hairpin (gold nano grain-lung cancer are added dropwise respectively
The hair fastener of lesion) DNA solution to electrode surface, dries at room temperature and 37 DEG C respectively, the average grain of the nitridation carbon quantum dot
Diameter is 5nm；
3) above-mentioned electrode is rinsed with PBS solution, is then added dropwise 1 × 10-12The target dna of mol/L, in 37 DEG C of cultivation 2h,
Being detected its electrochemical luminescence test intensity is 5800.
Its detection method, includes following steps:
A) 1 × 10 is added dropwise on glass-carbon electrode-12Lung cancer lesion ct DNA, in 37 DEG C of cultivation 2h；
B) it is tested using the three-electrode system of electrochemical workstation, using traditional three-electrode system, with Ag/AgCl
For reference electrode, it is to electrode, using above-mentioned steps 1 with platinum plate electrode) treated glass-carbon electrode is working electrode, by electrochemistry
Work station and superweak luminescence survey meter link together, and using cyclic voltammetry, scanning range is -2.0~0V, sweep speed
For 0.1V/S；
It c) is 0.1mol/L K in the concentration that contains of 10mL, pH 7.42S2O8Phosphate buffer solution in, pass through electrochemistry
Luminescent system detects the electrochemical luminescence signals intensity generated to the lung cancer target dna of various concentration, draws working curve.
Embodiment 2
1) glass-carbon electrode of diameter 3mm is successively taken turns doing into polishing treatment with 0.3 μm, 0.05 μm of aluminum oxide polishing powder, so
It is successively cleaned up afterwards with ethyl alcohol, nitric acid, ultrapure water；
2) 5~10 μ L g-CNQDs, AuNPs-hairpin DNA solutions are added dropwise respectively to electrode surface, respectively in room temperature
It is dried at 37 DEG C；
3) above-mentioned electrode is rinsed with PBS solution, is then added dropwise 1 × 10-15The target dna of mol/L, in 37 DEG C of cultivation 2h,
Its electrochemical luminescence test intensity is 4300.
The target dna of various concentration is detected, obtained electrochemical luminescence response curve is as shown in Figure 1A, Ke Yifa
Existing, ECL intensity is gradually increased with the increase of target DNA concentration.Calibration curve such as Figure 1B shows ECL intensity and DNA concentration
Logarithm (log c) within the scope of 1fM~1pM have good linear relationship R2Exist for the detection limit of 0.9922, ECL sensor
Down to 0.33fM.
The sensor of embodiment 3 is compared with same type of sensor
| ECL system | Linear range | LOD | Ref. |
| Ru(bpy)3 2+/TPA | 0.1pM-1.0nM | 0.091pM | 1 |
| CdS:Mn NCs/S2O8 2- | 50aM-5fM | 50aM | 2 |
| CdTe@SiO2/S2O8 2- | 0.1nM-2μM | 0.03nM | 3 |
| g-CNQDs/S2O8 2- | 1fM to 1pM | 0.33fM | This work |
The comparison of 1. difference ECL biosensor of table detection DNA
As shown in table 1, which is greatly improved in sensitivity compared with same type of sensor, with good
Good application prospect.
This sensor is used for the type cancer plasma detection, has obtained good response effect in optimal conditions
Fruit.Meanwhile compared with current mainstream detection method, sensor has many advantages, such as easy to detect, quick and at low cost, shows this
Sensor has very big clinical potentials.
Claims (7)
1. it include glass-carbon electrode based on the electrochemical luminescence biosensor of nitridation carbon quantum dot, the glassy carbon electrode surface
There is one layer of g-CNQDs (nitridation carbon quantum dot) film, g-CNQDs film is equipped with gold nano grain-lung cancer lesion hair fastener
DNA, hairpin dna end are connected on gold nano grain, in the detection process hairpin dna and lung cancer ct DNA complementary pairing.
2. the electrochemical luminescence biosensor according to claim 1 based on nitridation carbon quantum dot, it is characterised in that institute
The hairpin dna stated is according to designed by the DNA sequence dna of lung carcinoma cell mutation.
3. the electrochemical luminescence biosensor according to claim 1 based on nitridation carbon quantum dot, it is characterised in that institute
The average grain diameter for stating nitridation carbon quantum dot is 5nm.
4. the production method of the electrochemical luminescence biosensor described in claim 1 based on nitridation carbon quantum dot, includes
Following steps:
(1) it by glass-carbon electrode polishing powder polishing treatment, then cleans；
(2) g-CNQDs, gold nano grain-lung cancer lesion hairpin dna solution are modified to glassy carbon electrode surface, difference respectively
It is dried at room temperature and 37 DEG C；
(3) above-mentioned electrode is rinsed through PBS solution to get the electrochemistry based on nitridation carbon quantum dot for detecting lung cancer ct DNA
Luminescence biosensor.
5. the production method of the electrochemical luminescence biosensor according to claim 4 based on nitridation carbon quantum dot,
Being characterized in that the preparation method of the g-CNQDs is: the urea and trisodium citrate that molar ratio is 6:1 are weighed, is pulverized,
Product is washed, is then dialysed to get g-CNQDs after cooling in 160~190 DEG C of reaction 1h.
6. the production method of the electrochemical luminescence biosensor according to claim 4 based on nitridation carbon quantum dot,
The preparation method for being characterized in that gold nano grain-lung cancer lesion hairpin dna solution includes following steps:
1) HAuCl for being 1mmol/L by 100mL concentration4·4H2O aqueous solution is heated to fluidized state, adds under the action of stirring
Enter 20mL trisodium citrate and continue boiling mixture, mixture color becomes claret by colourless, stops heating, refrigerates spare；
2) it is molten to add 0.15 μ L 10mmol/L tri- (2- carboxyethyl) phosphine for the hairpin dna for taking 10 μ L 0.1mmol/L lung cancer lesions
Liquid, solvent are the PBS solution of 0.01mol/L NaCl containing 0.1mol/L, react 1h, then, are added obtained by 100 μ L steps 1)
AuNPs solution reacts 0.5h in said mixture.
7. the detection method of the electrochemical luminescence biosensor described in claim 1 based on nitridation carbon quantum dot, includes
Following steps:
1) it is added dropwise 1 × 10 respectively on glass-carbon electrode-12~1 × 10-15The lung cancer lesion ct DNA of mol/L, in 37 DEG C of cultivation 2h；
2) it is tested using the three-electrode system of electrochemical workstation, is ginseng with Ag/AgCl using traditional three-electrode system
Than electrode, it is to electrode, using above-mentioned steps 1 with platinum plate electrode) treated glass-carbon electrode is working electrode, by electrochemical operation
It stands and superweak luminescence survey meter links together, using cyclic voltammetry, scanning range is -2.0~0V, and sweep speed is
0.1V/S；
It 3) is 0.1mol/L K in the concentration that contains of 10mL, pH 7.42S2O8Phosphate buffer solution in, pass through electrochemical luminescence
System detects the electrochemical luminescence signals intensity generated to the lung cancer target dna of various concentration, draws working curve.
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Cited By (4)
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| CN112577948A (en) * | 2020-12-03 | 2021-03-30 | 武汉理工大学 | Copper ion rapid detection method and device |
| CN112710709A (en) * | 2020-12-22 | 2021-04-27 | 武汉理工大学 | Cadmium sulfide quantum dot glassy carbon electrode for target DNA detection, preparation method thereof, electrochemical luminescence sensor system and application |
| CN115184428A (en) * | 2022-06-27 | 2022-10-14 | 东南大学 | Electrochemical luminescence sensor for detecting mutant BRAF gene and preparation method and application thereof |
| CN115980163A (en) * | 2023-03-17 | 2023-04-18 | 武汉理工大学 | Portable tumor DNA electrochemiluminescence detection device and method |
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