CN102818826B - Electrochemical cell-based biosensor based on nanometer Ag@BSA biomimetic interface and preparation method thereof - Google Patents
Electrochemical cell-based biosensor based on nanometer Ag@BSA biomimetic interface and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an electrochemical cell-based biosensor based on a nanometer Ag@BSA biomimetic interface and a preparation method thereof. According to the invention, nanometer Ag@BSA microballoons are immobilized onto the surface of a gold electrode through the Au-S chemical bond and then covalently bond with folic acid molecules. Compared with traditional electrochemical biosensors, the electrochemical cell-based biosensor based on the nanometer Ag@BSA biomimetic interface in the invention has the following advantages: the electrochemical cell-based biosensor is used for detecting tumor cells, has good biocompatibility, electrochemical response, a wide detection range and high detection sensitivity and is capable of distinguishing cancer cells from normal cells at a certain concentration. Furthermore, the preparation method provided by the invention has the advantages of speed, simpleness, convenience, stability and low cost and poses on pollution to the environment.
Description
Technical field
The present invention relates to a kind of preparation of the novel galvanochemistry cell sensor based on the bionical interface of nanometer Ag@BSA.
Background technology
Cancer is one of major disease threatening human health.The death that cancer causes is mainly because the growth of cancer cell and split speed are higher than normal cell, and is transferred to its hetero-organization.Still lack in the world at present effective treatment means and capture cancer.To cancer, effective early diagnosis has very important meaning to the treatment in later stage in realization.Yet modern diagnosis technology great majority all exist the deficiencies such as instrument and equipment costliness, complicated, the consuming time length of operating process.Therefore, develop a kind of low cost, quick, easy biology sensor carries out accurately, detects delicately cancer cell, and to protect mankind health is had to suitable major and immediate significance.Along with the fast development of nanoscale science and technology, select suitable nano composite material, be the key point of the high performance biology sensor of preparation.
It is a green route of synthesis that has potentiality that the bionic of development nano material synthesizes, and meets the domestic demand of Preen nono science and Environmental Health development.The nano material that possesses good biocompatibility is that can it be widely used in biomedical basic premise, and bionic is synthetic to be expected to give security to realizing this prerequisite.Bovine serum albumin(BSA) (BSA) is a kind of globular preteins, in biosome serum, exists in a large number, is widely used in biological nano technology, as PCR (PCR), drug delivery, molecular image, molecular self-assembling, metal nanoparticle auxiliary synthetic etc.Nano silver grain has broad application prospects in fields such as photoelectricity, catalysis, Molecular Detection, biomedicines.Folacin receptor (FR) is expressed (as the cancer cell of ovary, colon, mammary gland, prostate, nasopharynx, brain etc.) at most of tumor cell surface height, yet in normal structure, do not express or very little distribute, based on having very strong affinity between folic acid (FA) and folacin receptor, folic acid can be used as the targeted molecular of capturing tumour cell.The present invention utilizes the BSA of single component to substitute poisonous surfactant and complicated plant solution, in aqueous phase system, the synthetic microballoon that obtains the Nano silver grain assembling of the coated BSA in surface, and in gold electrode conductive substrates, construct Ag@BSA nano bionic interface, for the fixing and propagation of cell provides good bio-compatible environment.Utilize the premium properties such as biological safety that Ag@BSA microballoon is good, electric conductivity, and by introduce targeted molecular on bionical interface, to improve detection sensitivity and the target recognition function of biology sensor.
Summary of the invention
The object of the invention is to a kind of galvanochemistry cell sensor based on the bionical interface of nanometer Ag@BSA, with low-cost, realize cancer cell carried out accurately, detected delicately fast, easily.
Technical scheme of the present invention is as follows:
A galvanochemistry cell sensor based on the bionical interface of nanometer Ag@BSA, is characterized in that, fixedly nanometer Ag@BSA microballoon of Au-S chemical bond is passed through on the surface of gold electrode, and nanometer Ag@BSA microballoon is by covalent bonding folate molecule.
The preparation of above-mentioned galvanochemistry cell sensor comprises the following steps:
(1) preparation of nanometer Ag@BSA microballoon: by BSA solution and AgNO
3solution is according to BSA:AgNO
3for the mass ratio of 1:1-2 mixes, the standing preservation of nitrogen protection 2-3h, adds excessive hydrazine hydrate under stirring, ageing 10-24h, and centrifuging, by gained solids wash, freeze drying at 0-4 ℃, obtains nanometer Ag@BSA microballoon;
(2) preparation of Ag@BSA modified electrode: step (1) gained nanometer Ag@BSA microballoon is scattered in to the aqueous dispersions that forms 1-2mg/mL in distilled water, according to 0.1-0.15mL/cm
2drop to the gold electrode surfaces through polishing and cleaning, be placed in freeze drying at 0-4 ℃, obtain Ag@BSA modified electrode;
(3) preparation of the galvanochemistry cell sensor based on the bionical interface of nanometer Ag@BSA: the folic acid solution of processing through activated carboxylic is dropped on step (2) gained Ag@BSA modified electrode, be placed in freeze drying at 0-4 ℃, spend the night, with PBS solution, rinse, obtain the galvanochemistry cell sensor based on the bionical interface of nanometer Ag@BSA tumour cell to target identification.
The step of cleaning described in step (2) is: by gold electrode ultrasonic 3-5min in the mixed solution of distilled water and ethanol, with nitrogen, dry up; Described distilled water is redistilled water, and the volume ratio of distilled water and ethanol is preferably 1:1.
The method that activated carboxylic described in step (3) is processed is: folic acid (FA) is dissolved in DMSO according to the solid-to-liquid ratio of 1-2g/L, then in above-mentioned folic acid solution, add EDC and NHS, shaking table reaction 3-4h, the mass ratio of FA:EDC:NHS is 8:4-5:4-5, preferably 8:5:5.
In step (3) according to 50-100 μ L/cm
2on Ag@BSA modified electrode, drip folic acid solution.
The silver-colored microballoon that the present invention synthesizes BSA template is as biomimetic material, by Au-S chemical bond, be fixed to gold electrode surfaces, construct the bionical interface that biocompatibility is good, by covalent bonding effect, folate molecule is loaded to Ag@BSA microsphere surface again, form galvanochemistry cell sensor tumour cell to target recognition function.By AC impedance electrochemical technology, tested and shown, this galvanochemistry cell sensor has high detection sensitivity and targeting to tumour cell, for the screening of tumour early detection and cell provides new method.
Compare with traditional electrochemica biological sensor, the application tool of the galvanochemistry cell sensor based on the bionical interface of nanometer Ag BSA of the present invention has the following advantages: for detection of tumour cell, there is good biocompatibility, electrochemical response, wide sensing range, higher detection sensitivity, and can distinguish cancer cell and the normal cell under finite concentration; And preparation method of the present invention have advantages of quick, easy, stable, cost is low, and environmentally safe.
Accompanying drawing explanation
Fig. 1 is the SEM figure of the nanometer Ag@BSA microballoon prepared of the embodiment of the present invention.
Fig. 2 is Zeta potential phenogram, and wherein a is the current potential (+4.73mV) of Ag@BSA, and b is the current potential (9.23mV) of FA/Ag@BSA.
Fig. 3 is the ac impedance spectroscopy that the galvanochemistry cell sensor based on Ag BSA quantitatively detects normal cell HFL-I.(a) naked gold electrode, the gold electrode that (b) FAAg@BSA modifies, the gold electrode surfaces propagation 2h:(c that the HFL-I cell of variable concentrations is modified at FA/Ag@BSA) 5.2 * 10
3, (d) 5.2 * 10
4, (e) 5.2 * 10
5, (f) 5.2 * 10
6, (g) 5.2 * 10
7cells mL
-1.Interior illustration: the calibration curve that the galvanochemistry cell sensor based on Ag BSA quantitatively detects normal cell HFL-I.
Fig. 4 is the ac impedance spectroscopy that the galvanochemistry cell sensor based on Ag@BSA quantitatively detects cancer cell KB.(a) naked gold electrode, the gold electrode that (b) FA/Ag@BSA modifies, the gold electrode surfaces propagation 2h:(c that the KB cell of variable concentrations is modified at FA/Ag@BSA) 6.0 * 10
1, (d) 6.0 * 10
2, (e) 6.0 * 10
3, (f) 6.0 * 10
4, (g) 6.0 * 10
5, (h) 6.0 * 10
6, (i) 6.0 * 10
7, (j) 1.2 * 10
8cellsmL
-1.Interior illustration: the calibration curve that the galvanochemistry cell sensor based on Ag BSA quantitatively detects cancer cell KB.
Embodiment
Experiment reagent: silver nitrate (AgNO
3, AR), absolute ethyl alcohol (CH
3cH
2oH, AR), hydrazine hydrate (N
2h
4h
2o) buy from Solution on Chemical Reagents in Shanghai product company limited.Bovine serum albumin(BSA) (BSA, purity >99.8%, molecular weight 68000) is bought from Xiamen Xing Longda chemical reagent company limited.Folic acid (FA), ethyl-(3-dimethyl propyl) carbodiimide (EDC), N-hydroxyl succinyl 256 imines (NHS), dimethyl sulfoxide (DMSO) are bought from Sigma-Aldrich company.Test institute water is deionized water (18.2M Ω).
Embodiment:
(1) preparation of Ag@BSA microballoon:
To (3mg mL in the BSA aqueous solution of 10mL
-1), add 5mL liquor argenti nitratis ophthalmicus (0.05mol/L), mix, and vigorous stirring 10min at room temperature; Gained solution is vacuumized, with the standing preservation 2～3h of nitrogen.Under intense agitation, add rapidly the hydrazine hydrate of 0.2mL, reaction 30min.After complete reaction, ageing is spent the night.Product is carried out to centrifuging, obtain black solid.With distilled water and ethanol, wash 3-5 time respectively, lyophilized overnight, obtains Ag@BSA microballoon.
Its SEM figure as shown in Figure 1, gained Ag@BSA bionic composite material pattern homogeneous as seen from the figure, particle diameter is mainly at 300-500nm.
This microballoon has water wettability, chemical stability and bio-compatibility, is suitable for fixing of cell and being cross-linked of biomolecule.
(2) preparation of Ag@BSA modified electrode:
Use 1,0.3,0.05 μ m Al first respectively
2o
3burnishing powder is by gold electrode (diameter 2.0mm) polishing; Then by gold electrode ultrasonic 3min in redistilled water and ethanol (volume ratio 1:1) mixed solution, to remove the Al that remains in gold electrode surfaces
2o
3particle; Finally by the electrode high-purity N of handling well
2dry up.
The preparation process of Ag@BSA modified electrode is as follows:
Get Ag@BSA microballoon aqueous dispersions (the 1.5mg mL of 3 μ L
-1), drop to the gold electrode surfaces of handling well, be positioned over 4 ℃ of refrigerator and cooled freeze-drying dry.
(3) preparation of FA/Ag@BSA modified electrode:
Preparation principle: carboxyl and amino amidation coupling reaction.
First the FA of 4mg is dissolved in the DMSO of 2mL, then the NHS of the EDC of 2.5mg and 2.5mg is added in above-mentioned folic acid solution, shaking table reaction 3h, to activate the carboxyl of FA; Get 2 μ L the FA solution of activated carboxyl drop on Ag@BSA modified electrode, by the carboxyl on FA surface and the amino on Ag@BSA surface, carry out amidation coupling reaction; Finally above-mentioned modified electrode is positioned over to 4 ℃ of refrigerator and cooled freeze-drying dry, spends the night, with PBS solution, rinse, to remove the not FA of stable bond.
The Zeta potential figure of Fig. 2 has proved that folic acid is successfully attached to the surface of Ag@BSA microballoon by amido link.
Tumour cell in exponential phase (as KB cell) is diluted to the cell suspension of variable concentrations, with the sensor, carry out cell sequestration, in constant incubator, cultivate 2h, adopt AC impedance electrochemical technology quantitatively to detect the KB cell of variable concentrations, experiment is measured normal cell (as HFL-I cell) with this sensor under same experimental conditions in contrast.
The cultivation of KB cell and HFL-I cell:
KB oral cavity epidermoid carcinoma cell line and HFL-I HELF strain are purchased from Chinese Academy of Sciences's Shanghai cell bank.
KB cell is containing the MEM(Gibco of 10% calf serum) cultivate in nutrient culture media.HFL-I cell is being cultivated containing in α-MEM (Thermo) nutrient culture media of 10% calf serum.In above-mentioned nutrient culture media all containing 100 μ g mL
-1mycillin.Cell culture environment is 5%CO
237 ℃ of constant incubators.Exponential phase cell is selected in experiment.
The preparation of KB cell and HFL-I cell suspension:
The KB cell of taking the logarithm growth period and HFL-I cell, carry out cell dissociation with 0.25% pancreatin solution.Above-mentioned celliferous nutrient culture media, at 1200rpm centrifuging 6min, and is cleaned 2 times with the PBS solution of aseptic pH7.4.Gained sediment is scattered in the PBS(10mM of the aseptic pH7.4 of 0.5mL) in, cell suspension obtained.Through cell count, obtaining KB cell concentration is 1.2 * 10
8cells mL
-1, HFL-I cell concentration is 5.2 * 10
7cells mL
-1.With above-mentioned PBS solution stepwise dilution, can obtain the cell suspension of variable concentrations.
The quantitative detection of FA/Ag@BSA modified electrode to HFL-I cell and KB cell
First by FA/Ag@BSA modified electrode after ultraviolet-sterilization, the cell suspension of variable concentrations is dropped to respectively to FA/Ag@BSA modified electrode surface, 37 ℃ of constant temperature are cultivated 2h.Again modified electrode is rinsed gently with PBS solution, to remove cell and the dead cell of not absorption, for electrochemical quantitative, detect.
By AC impedance electrochemical technology, measure demonstration, this cell sensor is 5.2 * 10 to the sensing range of HFL-I cell
4～5.2 * 10
6cells mL
-1, and be 6.0 * 10 to the sensing range of KB cell
1～1.2 * 10
8cells mL
-1, under detection, be limited to 20cells mL
-1.As shown in Figure 3, Figure 4, show that this sensor is applicable to the quantitative detection of cancer cell, has wide sensing range and low detectability.And reach 10 at cell concentration
3cells mL
-1time, this sensor can be used for distinguishing cancer cell and normal cell.
The above is preferred embodiment of the present invention, but the present invention should not be confined to the disclosed content of this embodiment.So every, do not depart from the equivalence completing under spirit disclosed in this invention or revise, all falling into the scope of protection of the invention.
Claims (5)
1. the preparation method of the galvanochemistry cell sensor based on the bionical interface of nanometer Ag BSA, fixedly nanometer Ag@BSA microballoon of Au-S chemical bond is passed through on the surface of the gold electrode of described sensor, nanometer Ag@BSA microballoon is by covalent bonding folate molecule, it is characterized in that, comprise the following steps:
(1) preparation of nanometer Ag@BSA microballoon: by BSA solution and AgNO
3solution is according to BSA:AgNO
3for the mass ratio of 1:1-2 mixes, the standing preservation of nitrogen protection 2-3h, adds excessive hydrazine hydrate under stirring, ageing 10-24h, and centrifuging, by gained solids wash, freeze drying at 0-4 ℃, obtains nanometer Ag@BSA microballoon;
(2) preparation of Ag@BSA modified electrode: step (1) gained nanometer Ag@BSA microballoon is scattered in to the aqueous dispersions that forms 1-2mg/mL in distilled water, according to 0.1-0.15mL/cm
2drop to the gold electrode surfaces through polishing and cleaning, be placed in freeze drying at 0-4 ℃, obtain Ag@BSA modified electrode;
(3) preparation of the galvanochemistry cell sensor based on the bionical interface of nanometer Ag@BSA: the folic acid solution of processing through activated carboxylic is dropped on step (2) gained Ag@BSA modified electrode, be placed in freeze drying at 0-4 ℃, spend the night, with PBS solution, rinse, obtain the galvanochemistry cell sensor based on the bionical interface of nanometer Ag@BSA tumour cell to target identification.
2. the preparation method of the galvanochemistry cell sensor based on the bionical interface of nanometer Ag@BSA claimed in claim 1, it is characterized in that, the step of cleaning described in step (2) is: by gold electrode ultrasonic 3-5min in the mixed solution of distilled water and ethanol, with nitrogen, dry up; Described distilled water is redistilled water, and the volume ratio of distilled water and ethanol is 1:1.
3. the preparation method of the galvanochemistry cell sensor based on the bionical interface of nanometer Ag@BSA claimed in claim 1, it is characterized in that, the method that activated carboxylic described in step (3) is processed is: folic acid (FA) is dissolved in DMSO according to the solid-to-liquid ratio of 1-2g/L, then in above-mentioned folic acid solution, add EDC and NHS, shaking table reaction 3-4h, the mass ratio of FA:EDC:NHS is 8:4-5:4-5.
4. the preparation method of the galvanochemistry cell sensor based on the bionical interface of nanometer Ag@BSA claimed in claim 3, is characterized in that, the mass ratio of FA:EDC:NHS is 8:5:5.
5. the preparation method of the galvanochemistry cell sensor based on the bionical interface of nanometer Ag@BSA claimed in claim 1, is characterized in that, in step (3) according to 50-100 μ L/cm
2on Ag@BSA modified electrode, drip folic acid solution.
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