CN102818826B - Electrochemical cell-based biosensor based on nanometer Ag@BSA biomimetic interface and preparation method thereof - Google Patents

Abstract

The invention discloses an electrochemical cell sensor based on a nano-AgBSA bionic interface and a preparation method thereof. Nano-AgBSA microspheres are fixed on the surface of a gold electrode through Au-S chemical bonds, and the nano-AgBSA microspheres are covalently bonded to folic acid molecules. Compared with the traditional electrochemical biosensor, the application of the electrochemical cell sensor based on the nano-AgBSA biomimetic interface of the present invention has the following advantages: it is used to detect tumor cells, has good biocompatibility, electrochemical response, wide detection range range, high detection sensitivity, and can distinguish cancer cells and normal cells at a certain concentration; and the preparation method of the invention has the advantages of fast, simple, stable, low cost, and has no pollution to the environment.

Description

Electrochemical cell sensor based on nano-AgBSA biomimetic interface and its preparation method

technical field

The invention relates to the preparation of a novel electrochemical cell sensor based on nano AgBSA bionic interface.

Background technique

Cancer is one of the major diseases threatening human health. Death from cancer is mainly due to the fact that cancer cells grow and divide faster than normal cells and can metastasize to other tissues. At present, there is still a lack of effective treatments to overcome cancer in the world. It is of great significance to realize effective early diagnosis of cancer for later treatment. However, most modern diagnostic techniques have the disadvantages of expensive instruments and equipment, complicated operation procedures, and long time-consuming. Therefore, the development of a low-cost, fast, and simple biosensor for accurate and sensitive detection of cancer cells is of great practical significance for the protection of human health. With the rapid development of nanoscience and technology, choosing suitable nanocomposite materials is the key to prepare high-performance biosensors.

The development of biomimetic synthesis of nanomaterials is a promising green synthesis approach, which meets the inherent needs of green nanoscience and the healthy development of the environment. Nanomaterials with good biocompatibility are the basic premise of whether they can be widely used in biomedicine, and biomimetic synthesis is expected to provide a guarantee for the realization of this premise. Bovine serum albumin (BSA) is a globular protein that exists in large amounts in the serum of living organisms and is widely used in bionanotechnology, such as polymerase chain reaction (PCR), drug delivery, molecular imaging, molecular self-assembly, Assisted synthesis of metal nanoparticles, etc. Silver nanoparticles have broad application prospects in photoelectricity, catalysis, molecular detection, biomedicine and other fields. Folate receptor (FR) is highly expressed on the surface of most tumor cells (such as cancer cells of ovary, colon, breast, prostate, nasopharynx, brain, etc.), but it is not expressed or distributed in a very small amount in normal tissues, based on folic acid ( FA) has a strong affinity with folate receptors, and folic acid can be used as a targeting molecule to capture tumor cells. In the present invention, single-component BSA is used to replace toxic surfactants and complex plant solutions. In the aqueous phase system, microspheres assembled with silver nanoparticles coated with BSA on the surface are synthesized, and AgBSA is constructed on the conductive substrate of gold electrodes. The nano-bionic interface provides a good biocompatible environment for cell fixation and proliferation. Utilize the excellent properties of AgBSA microspheres such as good biological safety and electrical conductivity, and introduce targeting molecules on the bionic interface to improve the detection sensitivity and target recognition function of biosensors.

Contents of the invention

The object of the present invention is an electrochemical cell sensor based on nano-AgBSA biomimetic interface, which can realize accurate and sensitive detection of cancer cells at low cost, quickly and easily.

Technical scheme of the present invention is as follows:

An electrochemical cell sensor based on a nano-AgBSA bionic interface is characterized in that the surface of a gold electrode is fixed with nano-AgBSA microspheres through Au-S chemical bonds, and the nano-AgBSA microspheres are covalently bonded to folic acid molecules.

The preparation of the above-mentioned electrochemical cell sensor comprises the following steps:

(1) Preparation of nano-AgBSA microspheres: Mix BSA solution and AgNO ₃ solution evenly according to the mass ratio of BSA:AgNO ₃ of 1:1-2, store it under nitrogen protection for 2-3 hours, add excess hydrazine hydrate under stirring , aged for 10-24 hours, centrifuged, washed the obtained solid, and freeze-dried at 0-4°C to obtain nano-AgBSA microspheres;

(2) Preparation of AgBSA modified electrode: Disperse the nano-AgBSA microspheres obtained in step (1) in distilled water to form a 1-2mg/mL aqueous dispersion, and drop them on the polished and cleaned gold at 0.1-0.15mL/ ^cm2 The surface of the electrode was freeze-dried at 0-4°C to obtain an AgBSA-modified electrode;

(3) Preparation of electrochemical cell sensor based on nano-AgBSA biomimetic interface: Drop the carboxy-activated folic acid solution onto the AgBSA-modified electrode obtained in step (2), freeze-dry at 0-4°C overnight, wash with PBS The solution was washed to obtain an electrochemical cell sensor based on nano-AgBSA biomimetic interface with targeted recognition for tumor cells.

The cleaning step in step (2) is: ultrasonically 3-5min the gold electrode in the mixed solution of distilled water and ethanol, and dry it with nitrogen; the distilled water is double distilled water, and the volume ratio of distilled water and ethanol is preferably 1 :1.

The carboxyl group activation treatment method in step (3) is: dissolve folic acid (FA) in DMSO at a solid-to-liquid ratio of 1-2g/L, then add EDC and NHS to the above-mentioned folic acid solution, and shake the table for 3- 4h, the mass ratio of FA:EDC:NHS is 8:4-5:4-5, preferably 8:5:5.

In step (3), add folic acid solution dropwise to the AgBSA modified electrode at 50-100 μL/cm ² .

In the present invention, silver microspheres synthesized by BSA template method are used as biomimetic materials, fixed on the surface of gold electrodes through Au-S chemical bonds, and a biomimetic interface with good biocompatibility is constructed, and folic acid molecules are loaded onto AgBSA microspheres through covalent bonding. The surface of the sphere forms an electrochemical cell sensor with a target recognition function for tumor cells. The electrochemical cell sensor has high detection sensitivity and targeting ability for tumor cells, and provides a new method for early detection of tumors and cell screening.

Compared with the traditional electrochemical biosensor, the application of the electrochemical cell sensor based on the nano-AgBSA biomimetic interface of the present invention has the following advantages: it is used to detect tumor cells, has good biocompatibility, electrochemical response, wide detection range range, high detection sensitivity, and can distinguish cancer cells and normal cells at a certain concentration; and the preparation method of the invention has the advantages of fast, simple, stable, low cost, and has no pollution to the environment.

Description of drawings

Fig. 1 is the SEM picture of the nano-AgBSA microsphere prepared by the embodiment of the present invention.

Figure 2 is a characterization diagram of Zeta potential, where a is the potential of AgBSA (+4.73mV), and b is the potential of FA/AgBSA (-9.23mV).

Fig. 3 is the AC impedance spectrum of the quantitative detection of HFL-I in normal cells by the electrochemical cell sensor based on AgBSA. (a) Bare gold electrode, (b) FAAgBSA modified gold electrode, HFL-I cells with different concentrations proliferated on the surface of FA/AgBSA modified gold electrode for 2h: (c) 5.2×10 ³ , (d) 5.2×10 ⁴ , (e) 5.2×10 ⁵ , (f) 5.2×10 ⁶ , (g) 5.2×10 ⁷ cells mL ⁻¹ . Inset: Calibration curve of AgBSA-based electrochemical cell sensor for quantitative detection of HFL-I in normal cells.

Fig. 4 is the AC impedance spectrum of the quantitative detection of cancer cell KB by the electrochemical cell sensor based on AgBSA. (a) bare gold electrode, (b) FA/AgBSA modified gold electrode, KB cells with different concentrations proliferate on the surface of FA/AgBSA modified gold electrode for 2h: (c) 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 ⁸ cellsmL ^{- 1} . Inset: Calibration curve of AgBSA-based electrochemical cell sensor for quantitative detection of cancer cell KB.

Detailed ways

Experimental reagents: Silver nitrate (AgNO ₃ , AR), absolute ethanol (CH ₃ CH ₂ OH, AR), and hydrazine hydrate (N ₂ H ₄ ·H ₂ O) were purchased from Shanghai Chemical Reagent Co., Ltd. Bovine serum albumin (BSA, purity >99.8%, molecular weight 68,000) was purchased from Xiamen Xinglongda Chemical Reagent Co., Ltd. Folic acid (FA), ethyl-(3-dimethylpropyl)carbodiimide (EDC), N-hydroxysuccinyl 256 imide (NHS), dimethylsulfoxide (DMSO) were purchased from Sigma-Aldrich . The water used in the test is deionized water (18.2MΩ).

Example:

(1) Preparation of AgBSA microspheres:

Add 5mL of silver nitrate solution (0.05mol/L) to 10mL of BSA aqueous solution (3mg mL ^-1 ), mix well, and vigorously stir at room temperature for 10min; vacuumize the resulting solution, and store it under nitrogen for 2-3h . Under the condition of vigorous stirring, 0.2mL of hydrazine hydrate was quickly added and reacted for 30min. After the reaction was complete, it was aged overnight. The product was centrifuged to obtain a black solid. Wash with distilled water and ethanol for 3-5 times respectively, and freeze-dry overnight to obtain AgBSA microspheres.

The SEM image is shown in Figure 1, from which it can be seen that the obtained AgBSA biomimetic composite material has a uniform appearance, and the particle size is mainly in the range of 300-500nm.

The microsphere has hydrophilicity, chemical stability and biocompatibility, and is suitable for fixing cells and cross-linking biomolecules.

(2) Preparation of AgBSA modified electrode:

Firstly, the gold electrode (2.0 mm in diameter) was polished with 1, 0.3, 0.05 μm Al ₂ O ₃ polishing powder; then the gold electrode was sonicated in a mixed solution of twice distilled water and ethanol (volume ratio 1:1) for 3 minutes to remove Al ₂ O ₃ particles remaining on the surface of the gold electrode; finally, the treated electrode was blown dry with high-purity N ₂ .

The preparation process of AgBSA modified electrode is as follows:

Take 3 μL of the AgBSA microsphere aqueous dispersion (1.5 mg mL ^-1 ), drop it on the surface of the treated gold electrode, and place it in a refrigerator at 4°C to freeze-dry.

(3) Preparation of FA/AgBSA modified electrode:

Preparation principle: Amidation coupling reaction between carboxyl and amino groups.

Firstly, 4 mg of FA was dissolved in 2 mL of DMSO, then 2.5 mg of EDC and 2.5 mg of NHS were added to the above folic acid solution, and the shaking table was reacted for 3 hours to activate the carboxyl group of FA; On the AgBSA modified electrode, the carboxyl group on the surface of FA and the amino group on the surface of AgBSA were subjected to amidation coupling reaction; finally, the above modified electrode was placed in a refrigerator at 4°C to freeze-dry overnight, and washed with PBS solution to remove unstabilized Fa.

The Zeta potential diagram in Figure 2 demonstrates that folic acid was successfully bound to the surface of AgBSA microspheres through amide bonds.

Dilute tumor cells in the logarithmic growth phase (such as KB cells) into cell suspensions of different concentrations, use the above sensors to capture cells, and cultivate them in a constant temperature incubator for 2 hours. For quantitative detection, as a control experiment, the sensor is used to measure normal cells (such as HFL-I cells) under the same experimental conditions.

Culture of KB cells and HFL-I cells:

KB oral epidermoid carcinoma cell line and HFL-I human embryonic lung fibroblast cell line were purchased from Shanghai Cell Bank of Chinese Academy of Sciences.

KB cells were cultured in MEM (Gibco) medium containing 10% calf serum. HFL-I cells were cultured in α-MEM (Thermo) medium containing 10% calf serum. All the above mediums contained 100 μg mL ^-1 of penicillin and streptomycin. The cell culture environment was a 37°C incubator with 5% CO ₂ . Cells in logarithmic growth phase were used in the experiment.

Preparation of KB cell and HFL-I cell suspension:

KB cells and HFL-I cells in the logarithmic growth phase were taken and digested with 0.25% trypsin solution. The above-mentioned cell-containing medium was centrifuged at 1200 rpm for 6 min, and washed twice with sterile pH 7.4 PBS solution. The resulting precipitate was dispersed in 0.5 mL of sterile PBS (10 mM) at pH 7.4 to obtain a cell suspension. By cell counting, the concentration of KB cells was 1.2×10 ⁸ cells mL ^-1 , and the concentration of HFL-I cells was 5.2×10 ⁷ cells mL ^-1 . Different concentration of cell suspension can be obtained by serially diluting with the above PBS solution.

Quantitative detection of HFL-I cells and KB cells by FA/AgBSA modified electrode

First, after the FA/AgBSA modified electrode was sterilized by ultraviolet light, cell suspensions of different concentrations were dropped onto the surface of the FA/AgBSA modified electrode, and incubated at a constant temperature of 37°C for 2 hours. Then the modified electrode was gently rinsed with PBS solution to remove unadsorbed cells and dead cells for electrochemical quantitative detection.

The detection range of the cell sensor for HFL-I cells was 5.2×10 ⁴ ～5.2×10 ⁶ cells mL ^-1 by electrochemical impedance spectroscopy, and the detection range for KB cells was 6.0×10 ¹ ～1.2× 10 ⁸ cells mL ^-1 , the detection limit is 20 cells mL ^-1 . As shown in Figure 3 and Figure 4, it shows that the sensor is suitable for the quantitative detection of cancer cells, with a wide detection range and low detection limit. And when the cell concentration reaches 10 ³ cells mL ^-1 , the sensor can be used to distinguish cancer cells from normal cells.

The above description is only a preferred embodiment of the present invention, but the present invention should not be limited to the content disclosed in this embodiment. Therefore, all equivalents or modifications that do not deviate from the spirit disclosed in the present invention fall within the protection scope of the present 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.