CN111855624B - Electrochemical biosensor for cell epithelial-mesenchymal transition detection and preparation method and application thereof - Google Patents
Electrochemical biosensor for cell epithelial-mesenchymal transition detection and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides an electrochemical biosensor for detecting cell epithelial-mesenchymal transition, a preparation method and application thereof, belonging to the technical field of molecular biology and biosensor preparation. The electrochemical biosensor prepared by the invention can specially detect the change of the marker protein (E-cadherin) of the EMT, and can effectively analyze different stages of the EMT. The signal of the EMT is greatly amplified due to the transmission of molecular information to the electronic device. Meanwhile, due to the synergistic effect of QD and the carbon nano tube-gold nanoparticles, when the differential pulse voltammetry is used for detection, the response of an electrochemical signal is quick and sensitive, and the electrochemical biosensing technology can be effectively promoted to be applied to the research of complex biological behaviors, so that the method has good practical application value.
Description
Technical Field
The invention belongs to the technical field of molecular biology and biosensor preparation, and particularly relates to an electrochemical biosensor for detecting epithelial-mesenchymal transition of cells, and a preparation method and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Epithelial-mesenchymal transition (EMT) plays an important role in a variety of biological processes, such as embryonic development, tissue growth and wound healing, among others. There is increasing evidence that EMT plays a key role in tumor progression, promoting infiltration of benign tumor cells into surrounding tissues and metastasis to distant sites. During the development of EMT, epithelial cells lose polarity and intercellular junctions, assume a slender morphology, and acquire cell motility. A number of molecules, such as Transforming Growth Factor (TGF) and Epidermal Growth Factor (EGF), have been identified that are capable of inducing the development of EMT. In addition, several molecular events are involved in the EMT process, including: activation of transcription factors, expression of specific proteins, and rearrangement of the cytoskeleton.
EMT is currently detected mainly by traditional methods such as Western blotting and immunofluorescence microscopy. However, the inventor finds that the method has the defects of long detection time, complicated steps, high cost and the like.
Disclosure of Invention
In view of the above-mentioned deficiencies of the prior art, the present invention provides an electrochemical biosensor for detecting epithelial-mesenchymal transition of cells, which is capable of specifically detecting a change in a marker protein (E-cadherin) of EMT, thereby effectively analyzing different stages of EMT, and a method for preparing the same and applications thereof. In the invention, as molecular information is transmitted to electronic equipment, signals of EMT are greatly amplified, and meanwhile, due to the synergistic effect of CdSe/ZnS Quantum Dots (QDs) and carbon nano tubes-gold nano particles, when the differential pulse voltammetry is used for detection, the response of electrochemical signals is rapid and sensitive, and based on the unique performance of the QDs, the fluorescence detection can be realized while the electrochemical signals are detected. Therefore, the technical scheme of the invention has good value in practical application.
The invention realizes the technical purpose based on the following technical scheme:
in a first aspect of the present invention, there is provided an electrochemical biosensor for the detection of epithelial-to-mesenchymal transition of cells, said electrochemical biosensor comprising at least a carboxylated carbon nanotube-gold nanoparticle (CNT-AuNP) modified electrode; and an E-cad-antibody-QD probe.
In a second aspect of the present invention, there is provided a method for preparing the above electrochemical biosensor for detecting epithelial-to-mesenchymal transition of cells, the method comprising:
preparing a carboxylated carbon nanotube-gold nanoparticle modified electrode; and (c) and (d),
preparation of E-cad-antibody-QD Probe.
The preparation method of the carboxylated carbon nanotube-gold nanoparticle modified electrode comprises the following steps:
mixing the suspension of carboxylated carbon nanotubes with HAuCl 4 The solution is mixed to obtain a modifier, the electrode is placed in the modifier, and the carboxylated carbon nanotube-gold nanoparticle modified electrode is obtained by adopting a chronoamperometry.
The preparation method of the E-cad-antibody-QD probe comprises the following steps:
adding the E-cad-antibody and the coupling agent 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) into the carboxyl quantum dot solution, stirring at room temperature for 1-2 hours for conjugation reaction, and purifying to obtain the product.
In a third aspect of the present invention, there is provided the use of the above electrochemical biosensor in the detection of epithelial-to-mesenchymal transition of cells.
In a fourth aspect of the present invention, there is provided a method for detecting epithelial-to-mesenchymal transition of cells, the method comprising detecting a change in E-cadherin based on electrochemical and fluorescent signals using the electrochemical biosensor to analyze the epithelial-to-mesenchymal transition of cells;
specifically, the carboxylated carbon nanotube-gold nanoparticle modified electrode is a working electrode, ag/AgCl (saturated KCl) is used as a reference electrode, a platinum wire is used as a counter electrode, and the counter electrode is connected to an electrochemical workstation, hg 2+ -taking an acetic acid buffer solution as a supporting electrolyte, incubating a sample to be detected with the E-cad-antibody-QD probe, and performing electrochemical detection by using a Differential Pulse Voltammetry (DPV); meanwhile, based on the immunofluorescence technique, the fluorescence detection is carried out.
The invention has the advantages and positive effects that:
the invention designs a novel electrochemical biosensor which can detect EMT by using E-cadherin as a biomarker. QD and CNT-AuNP nanocomposites are used to enhance the sensitivity of EMT biosensors. The EMT electrochemical biosensor prepared by the present invention has several advantages over conventional methods. First, the E-cad-antibody probe contained in the EMT biosensor can specifically recognize E-cadherin in living cells. Thus, EMT biosensors can distinguish between EMT phases after TGF β treatment at different time points.
In addition, due to the unique properties of QDs, fluorescence detection can be achieved while detecting electrochemical signals. Finally, the electrochemical EMT biosensor is an ideal choice for detecting EMT in-situ cells, and has short detection time and high sensitivity. Electrochemical detection does not require cell lysis, immobilization, and secondary antibody incubation, thereby saving time and cost.
The electrochemical biosensor prepared according to the present invention can detect EMT in a shorter time and has higher sensitivity in living cells than the conventional standard method. Meanwhile, the electrochemical biosensor can be expanded to various cell processes, such as cell migration, cell apoptosis, necrosis, multi-drug resistance and other related fields, and can effectively promote the application of the electrochemical biosensor technology to the research of complex biological behaviors, so that the electrochemical biosensor has good practical application value.
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The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are included to illustrate an exemplary embodiment of the invention and not to limit the invention.
FIG. 1 is a flow chart of electrochemical detection based on an EMT biosensor, in which an EMT model is established by using A549 cells in example 1 of the present invention; wherein FIG. 1 (a) is a drawing of A549 cells before and after TGF-beta treatment by Western blot analysis using E-cadherin, N-cadherin, ZO-1, vimentin, and a-tubulin antibodies; fig. 1 (b) is a bright field image of a549 cells before and after EMT; FIG. 1 (c) is a graph of the Transwell invasion assay (scale bar, 200 μm) in A549 cells before and after TGF β induction; FIG. 1 (d) is a graph showing the statistical analysis of the number of cells in visual fields before and after TGF-beta treatment; FIG. 1 (E) is a graph of the levels of E-cadherin, N-cadherin, ZO-1, vimentin and a-tubulin detected by immunofluorescence microscopy (scale bar, 10 μm).
FIG. 2 is a graphical representation of the morphology, structure and electrochemical performance of the nanocomposite material of example 1 of the present invention; among them, FIG. 2 (a-c) AuNP(a) Transmission electron microscope images of CNT (b) and CNT-AuNP (scale bar, 50 nm); FIG. 2 (d) is an X-ray diffraction pattern of CNT-AuNP; FIG. 2 (e) is a plot of the scan rate at 50mV/s at 10mM K 3 [Fe(CN) 6 ]Cyclic voltammograms of bare GCE, CNT/GCE and CNT-AuNPs/GCE in (1); FIG. 2 (f) is a graph of kinetic analysis of CNT-AuNPs/GCE with scan rates ranging from 10-100mV/s, indicating that the reaction modifying the electrode is a diffusion-controlled surface reaction; FIG. 2 (g) shows the oxidation peak current (Ipa) and the reduced peak current (Ipc) versus the square root of the scan rate (v @) 1/2 ) Linear fit of (a).
FIG. 3 is a graph showing the analysis of specificity and sensitivity of the probe prepared in example 1 of the present invention; wherein, FIG. 3 (a) is a Western blot of E-cadherin from cells incubated at different concentrations with standard antibody and E-cad-antibody-QD probe; FIG. 3 (b) is a graph of fluorescence intensity from cells at different concentrations; FIG. 3 (c) is a probe with HNO through E-cad-antibody-QD 3 Immunofluorescence microscopy images of E-cad-antibody-QD probes and pure QD (scale bar, 25 μm) stained cells of the solution; FIG. 3 (d) response current plots of electrochemical biosensors prepared using differential pulse voltammetry measurements, with and without cell sensor measurements; FIG. 3 (E) is a graph of the incubation time from 0 min to 180 min for the optimized E-cad-QD probe.
FIG. 4 is an electrochemical measurement of protein levels and different cell concentrations using EMT biosensors as in example 1 of the present invention; wherein, FIG. 4 (a) is a graph of E-cadherin detected at different concentrations by differential pulse voltammetry, ranging from 1ng/mL to 900ng/mL; fig. 4 (b) is a calibration graph showing the linear relationship of the biosensor in detecting E-cadherin, with error bars representing mean ± standard deviation (n = 5); FIG. 4 (c) is a diagram of differential pulse voltammetry measurements of cells at different concentrations, ranging from 7.0X 10 2 -4.1×10 5 Individual cells/mL.
FIG. 5 is a diagram showing a stage of EMT detection using an EMT biosensor in example 1 of the present invention; wherein FIG. 5 (a) is a Western blot of E-cadherin and N-cadherin in cells induced by TGF for 0 to 72 hours; FIG. 5 (b) is a graph of E-cadherin expression after TGF-beta treatment at different time points assessed by immunofluorescence microscopy (scale bar, 50 μm); FIG. 5 (c) is an electrochemical detection of different degrees of EMT by differential pulse voltammetry using a biosensor; FIG. 5 (d) is a graph showing the relationship between current and induction time for a calibration curve. Error bars represent mean ± standard deviation (n = 3).
FIG. 6 is a diagram showing that the EMT biosensor in example 1 of the present invention is applied to various cell lines; wherein, FIG. 6 (a) is an immunofluorescence photograph of RBE, MCF7, SKOV3 and PANC1 before and after TGF β treatment; FIG. 6 (b-d) is a Western blot showing E-cadherin in RBE, MCF7, SKOV3 and PANC1 cells before and after TGF β treatment; the prepared biosensor was used to perform EMT electrochemical detection on RBE of fig. 6 (b), MCF7 of fig. 6 (c), SKOV3 of fig. 6 (d), and PANC1 of fig. 6 (e).
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
As described in the background, EMT is detected mainly by conventional methods such as Western blotting and immunofluorescence microscopy. However, the method has the defects of long detection time, complicated steps, high cost and the like.
The down-regulation of the cadherin family member, E-cadherin (E-cad), is known to be one of the key events for the initiation of EMT. Expression of E-cadherin is inversely correlated with the pathological classification and staging of lung, liver, ovarian and stomach-related cancers. Meanwhile, cdSe/ZnS Quantum Dots (QDs) are good tools for preparing EMT electrochemical biosensors, and can realize synchronous detection of fluorescence and electrochemical signals in cells because of the unique characteristics of high electron density and size adjustability and narrow fluorescence emission spectrum.
In view of the above, in one exemplary embodiment of the present invention, there is provided an electrochemical biosensor for epithelial-mesenchymal transition detection of cells, the electrochemical biosensor including at least a carboxylated carbon nanotube-gold nanoparticle (CNT-AuNP) modified electrode; and an E-cad-antibody-QD probe.
In another embodiment of the present invention, the preparation method of the carboxylated carbon nanotube-gold nanoparticle modified electrode comprises:
mixing the suspension of carboxylated carbon nanotubes with HAuCl 4 The solution is mixed to obtain a modifier, the electrode is placed in the modifier, and the carboxylated carbon nanotube-gold nanoparticle modified electrode is obtained by adopting a chronoamperometry.
In another embodiment of the present invention, the modified agent is carboxylated carbon nanotubes and HAuCl 4 The mass molar ratio of (b) is 2 to 4 mg.
In another embodiment of the present invention, the preparation method of the carboxylated carbon nanotube suspension comprises: ultrasonically dispersing CNT in 0.4% polydiallyldimethylammonium chloride for 0.5-2 hours (preferably 1 hour), centrifuging and washing with double distilled water for at least 3 times.
In still another embodiment of the present invention, the HAuCl-containing material is 4 The solution of (A) is composed of L-cysteine and H 2 SO 4 And HAuCl 4 And (4) forming.
In still another embodiment of the present invention, the HAuCl-containing material is 4 The composition of the solution is as follows: 0.1mM L-cysteine, 0.1M H 2 SO 4 ,5mM HAuCl 4 。
In still another embodiment of the present invention, the method for preparing the E-cad-antibody-QD probe comprises: and adding the E-cad-antibody and the coupling agent 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride into the carboxyl quantum dot solution for carrying out a conjugation reaction, and purifying to obtain the product.
In yet another embodiment of the invention, the purification is carried out by passing the conjugate through a 0.2 μm filter to remove large aggregates and washing at least 5 times with borate buffer.
In another embodiment of the present invention, there is provided a method for preparing the electrochemical biosensor for detecting epithelial-to-mesenchymal transition of cells, the method comprising:
preparing a carboxylated carbon nanotube-gold nanoparticle modified electrode; and, preparation of E-cad-antibody-QD probe.
In another embodiment of the present invention, the preparation method of the carboxylated carbon nanotube-gold nanoparticle modified electrode comprises:
mixing the suspension of carboxylated carbon nanotubes with HAuCl 4 The solution is mixed to obtain a modifier, the electrode is placed in the modifier, and the carboxylated carbon nanotube-gold nanoparticle modified electrode is obtained by adopting a chronoamperometry.
In another embodiment of the present invention, the chronoamperometry processing conditions are: the fixed deposition voltage is 300-500 mv (preferably 400 mv); the treatment time is 100 to 130s (preferably 120 s); by optimizing the treatment conditions, the carbon nano tube adsorbed with the nano gold particles is favorably deposited and attached on the electrode.
In yet another embodiment of the present invention, the electrode is a pretreated glassy carbon electrode.
In another embodiment of the present invention, the pretreatment method comprises: polishing the surface of the glassy carbon electrode GCE using alumina powders having diameters of 0.3 μm and 0.05 μm in this order to remove the oxide layer; meanwhile, in order to remove other physical adsorbed substances, double distilled water and ethanol are used for ultrasonically cleaning the surface of the electrode; the glassy carbon electrode GCE is then immediately dried under nitrogen.
In another embodiment of the present invention, the modifier comprises carboxylated carbon nanotubes and HAuCl 4 The mass molar ratio of (a) is 2 to 4mg (preferably 3 mg.
In another embodiment of the present invention, the preparation method of the carboxylated carbon nanotube suspension comprises: ultrasonically dispersing CNT in 0.4% polydiallyldimethylammonium chloride for 0.5-2 hours (preferably 1 hour), centrifuging and washing with double distilled water for at least 3 times.
In still another embodiment of the present invention, the HAuCl-containing material is 4 The solution of (a) is composed of L-cysteine, H 2 SO 4 And HAuCl 4 And (4) forming.
In still another embodiment of the present invention, the HAuCl-containing material is 4 The composition of the solution is as follows: 0.1mM L-cysteine, 0.1M H 2 SO 4 ,5mM HAuCl 4 。
In another embodiment of the present invention, the method for preparing the E-cad-antibody-QD probe comprises:
adding the E-cad-antibody and the coupling agent 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) into the carboxyl quantum dot solution, stirring at room temperature for 1-2 hours for conjugation reaction, and purifying to obtain the product.
In yet another embodiment of the invention, the purification is performed by passing the conjugate through a 0.2 μm filter to remove large aggregates and washing at least 5 times with borate buffer.
In another embodiment of the present invention, the electrochemical biosensor is used for detecting epithelial-mesenchymal transition of cells.
In yet another embodiment of the present invention, there is provided a method for detecting epithelial-to-mesenchymal transition of a cell, the method comprising detecting a change in E-cadherin based on electrochemical and fluorescent signals using the electrochemical biosensor to analyze the epithelial-to-mesenchymal transition of the cell;
the modified electrode of the carboxylated carbon nano tube-gold nano particles is used as a working electrode, ag/AgCl (saturated KCl) is used as a reference electrode, a platinum wire is used as a counter electrode, the counter electrode is connected to an electrochemical workstation, and Hg is added 2+ An acetic acid buffer solution is used as a supporting electrolyte, and after a sample to be detected and the E-cad-antibody-QD probe are incubated (preferably for 2 hours), the electrochemical detection is carried out by adopting Differential Pulse Voltammetry (DPV); meanwhile, fluorescence detection is carried out based on an immunofluorescence technique.
The inventionIn yet another embodiment, the Hg 2+ The pH of the acetate buffer solution is 5.2.
The present invention will be further described with reference to the following examples, but the present invention is not limited thereto.
Example 1
1. Chemicals and reagents
Carboxylated carbon nanotubes (CNTs, 30-50nm in diameter, 10-20 μm in average length) were purchased from Xfnano Materials Tech (Nanjing, china). Qdot TM 655ITK TM carboxy quantum dots (Q21321 MP) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Boric acid, potassium chloride (KCl) and phosphate were purchased from Sinopharm Group Chemical Reagent Co. An anti-E-cad antibody, an anti-N-cad antibody, an anti-ZO-1 antibody, an anti-vimentin antibody are provided by antibody cam. E-cadherin protein and chloroauric acid (HAuCl) 4 ) Purchased from Sigma-Aldrich. All detection systems were prepared using double distilled water.
2. Cell culture and construction of EMT model
A549, PANC1 and MCF7 cells were cultured in DMEM medium supplemented with 10% FBS. RBE and SKOV3 cells were cultured in RPMI1640 medium supplemented with 10% FBS. All cells were purchased from the cell bank of the Chinese academy of sciences. The cells were grown in a humid atmosphere at 5% CO2 and 37 ℃. Cells were seeded in 6-well plates for Western blot analysis and 24-well plates for immunofluorescence staining and electrochemical detection. EMT was induced by addition of TGF to the medium at a final concentration of 15ng/mL and incubated for 48 hours.
3. Immunofluorescence microscope
Cells grown on slides incubated with or without TGF (15 ng/ml) were washed twice with PBS and then fixed with 4% Paraformaldehyde (PFA) for 20 minutes. After permeabilization with 0.2% Triton X-100/PBS and blocking with 4% BSA at room temperature for 1 hour, cells were incubated and stained with antibody overnight. After washing with PBS, the slides were incubated with secondary antibodies for 2 hours at room temperature. Nuclei were visualized with 4, 6-diamidino-2-phenylindole (DAPI). Viable cell staining was performed without a secondary antibody and incubated with E-cad-antibody-QD for 2 hours prior to the fixation step.
4. Western blot analysis
The whole cell extracts were resolved separately on SDS-PAGE gels and transferred to polyvinylidene difluoride membranes. The membrane was blocked with 5% skim milk powder for 2 hours and then incubated with primary antibody in 5% skim milk powder overnight at 4 ℃. After removing excess primary antibody by washing the membrane in TBST (3 × 5 minutes each) containing 0.1% tween-100, secondary antibody-coupled horseradish peroxidase was incubated with the membrane for 1 hour at room temperature. The membranes were then washed in TBST (3 × 5 min each) and developed with enhanced chemiluminescent detection reagent (Pierce Biotechnology). Western blot analysis using E-cad-antibody-QD was performed without secondary antibody and excited by blue light.
5. Transwell migration and invasion assay
Cells starved for 24 hours (5X 10) 4 ～1×10 5 Individual cells/well) were placed in a transwell with 500 μ L of medium containing 10% fbs in the lower layer of a 24-well plate. After 24 hours incubation at 37 ℃, migrating cells were fixed with methanol and stained with 0.1% crystal violet. Invaded cells were photographed under a microscope and counted from five random fields of view.
6. Preparation of E-cad-antibody-QD Probe
According to the QD product specification, an E-cad-antibody-QD nanocomposite was prepared and modified. Briefly, 10. Mu.L of stock solution of carboxy quantum dots was diluted to 2mL using 10mM borate buffer (pH 7.4). To the above solution, a total of 30. Mu.L of E-cad-antibody was added, followed immediately by 1.2. Mu.L of a 5mg/mL EDC stock solution. The solution was gently stirred at room temperature for 1-2 hours for conjugation. The conjugate solution was filtered through a 0.2 μmPES syringe filter to remove large aggregates and washed at least 5 times with 50mM borate buffer (ph 8.3). The solution was concentrated to 20. Mu.L and stored at 4 ℃.
7. Electrode modification
The surface of the glassy carbon electrode GCE was polished with alumina powders having diameters of 0.3 μm and 0.05 μm to remove the oxide layer. In order to remove other physisorbed substances, the electrode surface was ultrasonically cleaned using double distilled water and ethanol. The GCE was immediately dried under nitrogen. Ultrasonic dispersion of CNTs at 04% polydiallyldimethylammonium chloride for 1 hour, then centrifuged and washed at least 3 times with double distilled water. By placing 10. Mu.L of CNT suspension (3 mg/mL) in 20mL of solution (0.1 mM L-cysteine, 0.1 MH) 2 SO4,5mMHAuCl 4 ) In (1), the CNT-gold nanocomposite modified GCE was prepared by placing GCE in the mixed solution and then measuring at 400mV for 120 seconds by chronoamperometry.
8. Electrochemical measurements
Electrochemical measurements were performed with a CHI400C potentiostat electrochemical workstation (CH Instruments, chenhua, china). A CNT-AuNP modified Glassy Carbon Electrode (GCE) was used as the working electrode, ag/AgCl (saturated KCl) as the reference electrode, and a platinum wire (1 mm diameter) as the counter electrode. Cyclic voltammetric measurements were performed between-0.1V and 0.6V at 50mV/s in potassium ferricyanide solution (10 mM).
Cells were incubated with the E-cad-antibody-QD probe for 2 hours and then washed three times with PBS. With 0.1MHNO 3 The QDs are dissolved. After a pre-concentration step of-1.1V for 300 seconds, at a frequency of 15Hz Hg 2+ Differential Pulse Voltammetry (DPV) measurements of QDs were performed from-1.0V to 0V in a solution of-acetic acid buffer (pH 5.2) with an amplitude of 25mV and a potential step of 4mV. All electrochemical experiments were performed at room temperature.
And (4) analyzing results:
1. characterization of EMT model construction
In view of the intensive studies of EMT in lung cancer, a well-established a549 human lung cancer cell was used in this example. Various markers of EMT before and after TGF treatment were analyzed to characterize the successful construction of a cellular EMT model system for electrochemical detection. Immunoblot analysis showed that E-cadherin levels were down-regulated and N-cadherin expression was up-regulated 48 hours after TGF β treatment, demonstrating classical cadherin turnover during the development of EMT. In addition, down-regulation of ZO-1 expression and up-regulation of vimentin levels also followed changes in classical EMT markers (fig. 1 a). Morphological images of cells before and after TGF treatment were also compared in the bright field (figure 1 b). After TGF β treatment, the morphology of a549 changed from cuboidal epithelial to long spindle mesenchymal, which can promote cell migration and is a behavioral characteristic of cells undergoing EMT. In the transwell invasion assay, the number of cells treated with TGF + and stained with crystal violet was significantly higher than in the TGF-group (fig. 1 c). The invasion rate between the two groups was also analyzed by counting the number of invading TGF β + cells by inverted microscopy, which increased from 63.7% to 76.7% compared to TGF β -cells (fig. 1 d). Immunofluorescent staining results were also consistent with immunoblot analysis showing that the expression of EMT markers varied as expected, including E-cadherin, N-cadherin, ZO-1 and vimentin (fig. 1E). These results indicate successful establishment of the EMT model system.
2. Characterization of nanocomposites and electrocatalytic activity of modified electrodes
To improve the electrode performance, the surface of the electrode was modified using a carboxylated multiwalled carbon nanotube-gold nanoparticle (MWCNT-AuNP) nanocomposite. The MWCNT-AuNP morphology and structure were first characterized using transmission electron microscopy, and pure AuNPs proved to be homogeneous spherical structures with an average diameter of about 5nm (fig. 2 a). As shown in fig. 2b, the typical core-shell structure of MWCNTs without aggregation is clearly shown with the help of poly (diallyldimethylammonium chloride) (PDDA), a strong cationic polyelectrolyte. Aunps having negative charges can be attached to the surface of CNTs due to electrostatic adsorption (fig. 2 c). In addition, EDX experiments were performed to analyze the elemental composition of the nanomaterials and showed significant peaks of Au and C corresponding to AuNPs and CNTs, respectively, where the peaks associated with Cu and O came from the substrate (fig. 2 d).
Electrochemical properties of bare GCE, CNT/GCE and CNT-AuNPs/GCE were studied using cyclic voltammetry and the results showed that each modified electrode had a distinct redox peak formed by ferricyanide ions (FIG. 2 e). The microscopic electroactive area of CNT-AuNPs/GCE was calculated according to the Randles-Sevcik equation, which is 2.6 and 1.7 times higher than that of bare GCE and CNT/GCE, respectively. These results indicate that the modified nanomaterial can enhance the conductivity and sensitivity of the electrode.
The kinetics of the modified electrode was studied by analyzing the effect of the scan rate on the redox current. The electrochemical performance of CNT-AuNPs/GCE was examined when the scanning rate was 10-100mV/s in 10mM potassium ferricyanide solution, and the maximum current value of the redox reaction was found to follow the sweepThe increase in the scan rate increases linearly. Furthermore, the distance between the redox peaks becomes more and more distant (fig. 2 f). Based on these results, the square root (v) of the scan rate is compared 1/2 ) The associated oxidation peak (Ipa) and reduction (Ipc) peak currents were fitted linearly (fig. 2 g). Determine final linear equation as Ipa =13.79v 1/2 (mV/s)-4.19(R 2 ＝0.99675)、Ipc＝-18.09v 1/2 (mV/s)-11.08(R 2 = 0.99991). The results of these calculations indicate that the electrochemical signal is the result of diffusion-controlled surface reactions.
3. Characterization of E-cad-antibody-QD probes and optimization of the experimental conditions
To explore the effectiveness of such probes, it was first considered whether E-cad-antibody-QD is able to specifically recognize E-cadherin, a prerequisite for EMT detection. E-cadherin lysed from different concentrations of cells was compared using E-cad-antibody-QD and a standard E-cad antibody (FIG. 3 a). The membrane stained by E-cad-antibody-QD was brighter than the membrane stained with standard antibody, and the fluorescence intensity increased with increasing cell concentration (fig. 3 b). Immunofluorescence also demonstrated that the prepared probes can specifically bind to E-cadherin and are bound by HNO at QD 3 The fluorescence signal disappeared after dissolution. Furthermore, cells incubated with pure QDs lacked a fluorescent signal (fig. 3 c). Electrochemical measurements were also performed with and without a549 cells, indicating that specific electrochemical signals for the probe at-0.65V are related to E-cadherin on the cell surface (fig. 3 d). The effect of incubation time on the E-cad-antibody-QD probe was studied, which can directly affect the performance of the electrochemical biosensor. Figure 3e shows the results of DPV experiments using electrochemical EMT systems prepared at different incubation times. The results show that the oxidation current increases from 0 to 120 minutes and then decreases with time, so 120 minutes is the optimal time for probe incubation.
4. Electrochemical detection at protein level and at different concentrations in living cells using EMT sensing system
The analytical performance of the proposed EMT sensing system was first evaluated at the protein level. Differential pulse voltammetry measurements showed that the peak current signal of the QDs was intense with increasing E-cadherin concentrationAnd (fig. 4 a). The increase in current was attributed to the greater adsorption of E-cadherin antigen on the CNT-AuNP modified electrode to the E-cad-antibody-QD probe. The proposed biosensor shows significant linearity, with E-cadherin concentrations from 1ng/mL to 900ng/mL, linear regression equations calculated as Y =0.01X +1.83, pearson coefficient 0.99 (RSD changes from 2.3% to 3.9%, n = 5) (FIG. 4 b). To demonstrate that detection of the oxidation peak is an effective means to assess E-cadherin expression in cells, different concentrations of a549 cells were detected using a biosensor. When the number of A549 cells is from 7.0X 10 2 Gradually increase to 4.1 × 10 5 After that, the current DPV peak was significantly increased (fig. 4 c), which corresponds to the results for Western blot (fig. 3 a). These results indicate that the proposed sensing system can be used to detect E-cadherin and to analyze EMT processes.
5. Detecting changes in current during EMT by an EMT electrochemical biosensor
To study the electrochemical performance of EMT biosensors, different stages of EMT models were obtained by TGF- β treatment and detected using western blot, immunofluorescence microscopy and electrochemical methods. Western blot showed that E-cadherin and N-cadherin levels were altered as expected, demonstrating the EMT process (fig. 5 a). There was no significant change in alpha-tubulin levels. FIG. 5b shows the gradual disappearance of the fluorescence intensity of E-cadherin with increasing induction time. Differential pulse voltammetry measurements showed that the peak current signal became small at the later stages of EMT (fig. 5 c). Calibration curves of current and induction time were fitted after five independent replicates giving a linear current response in the late phase of EMT with a correlation coefficient of 0.98 (RSD <3.0%, n = 5) (fig. 5 d). The current signal shows a sharp decrease in early EMT from 0 to 24 hours, which corresponds to Western blot results (fig. 5 a). These data demonstrate that EMT electrochemical sensors can distinguish between different stages of EMT.
6. EMT detection in different cell lines
To verify the universality of the application of the prepared EMT electrochemical sensor, the expression levels of E-cadherin in RBE bile duct cancer, MCF7 breast cancer, SKOV3 ovarian cancer, and PANC1 pancreatic cancer cell lines before and after TGF treatment were examined using western blot, immunofluorescence microscopy, and the EMT sensor prepared in this study. Immunofluorescence microscopy (fig. 6 a) demonstrated that all four cell lines were induced into EMT. Electrochemical signals in RBE (fig. 6 b), MCF7 (fig. 6 c), SKOV3 (fig. 6 d) and PANC1 (fig. 6 e) cells were significantly attenuated after TGF treatment, consistent with western blot results. This phenomenon demonstrates that EMT electrochemical sensors can be used in multiple cell lines for EMT detection.
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited thereto, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or equivalents thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Although the present invention has been described with reference to the specific embodiments, it should be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.
Claims (19)
1. An electrochemical biosensor for detecting epithelial-mesenchymal transition of cells, wherein the electrochemical biosensor at least comprises a carboxylated carbon nanotube-gold nanoparticle modified electrode; and an E-cad-antibody-QD probe; wherein QD is a carboxyl quantum dot.
2. The electrochemical biosensor as claimed in claim 1, wherein the carboxylated carbon nanotube-gold nanoparticle modified electrode is prepared by the following method:
mixing the suspension of carboxylated carbon nanotubes with HAuCl 4 The solution is mixed to obtain a modifier, the electrode is placed in the modifier, and the carboxylated carbon nanotube-gold nanoparticle modified electrode is obtained by adopting a chronoamperometry.
3. The electrochemical biosensor of claim 1, wherein the E-cad-antibody-QD probe is prepared by a method comprising: and adding the E-cad-antibody and the coupling agent 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride into the carboxyl quantum dot solution for carrying out a conjugation reaction, and purifying to obtain the product.
4. A method for preparing an electrochemical biosensor for detecting epithelial-to-mesenchymal transition of a cell according to any one of claims 1 to 3, wherein the method comprises:
preparing a carboxylated carbon nanotube-gold nanoparticle modified electrode; and (c) and (d),
preparation of E-cad-antibody-QD Probe.
5. The method of claim 4, wherein the carboxylated carbon nanotube-gold nanoparticle modified electrode is prepared by a method comprising:
mixing the suspension of carboxylated carbon nanotubes with HAuCl 4 Mixing the solution to obtain a modifier, putting the electrode into the modifier, and obtaining the carboxylated carbon nanotube-gold nanoparticle modified electrode by adopting a chronoamperometry;
the electrode is a pretreated glassy carbon electrode;
the pretreatment method comprises the following steps: polishing the surface of the glassy carbon electrode GCE using alumina powder to remove the oxide layer; meanwhile, in order to remove other physical adsorbed substances, double distilled water and ethanol are used for ultrasonically cleaning the surface of the electrode; the glassy carbon electrode GCE was then immediately dried under nitrogen.
6. The method of claim 5, wherein the modifier is carboxylated carbon nanotubes and HAuCl 4 The mass molar ratio of (b) is 2 to 4mg, and 8 to 12mmol.
7. The method of claim 6, wherein the modifier is carboxylated carbon nanotubes and HAuCl 4 OfThe molar ratio was 3mg.
8. The method of claim 5, wherein the carboxylated carbon nanotube suspension is prepared by: ultrasonically dispersing CNT in 0.4% polydiallyldimethylammonium chloride for 0.5-2 hours, centrifuging and washing with double distilled water for at least 3 times.
9. The method of claim 8, wherein the suspension of carboxylated carbon nanotubes is prepared by ultrasonically dispersing CNTs in 0.4% polydiallyldimethylammonium chloride for 1 hour.
10. The method of claim 5, wherein the HAuCl-containing compound is present in the sample 4 The solution of (a) is composed of L-cysteine, H 2 SO 4 And HAuCl 4 And (4) forming.
11. The method of claim 10, wherein the HAuCl-containing compound is present in the sample 4 The composition of the solution of (A) is as follows: 0.1mM L-cysteine, 0.1M H 2 SO 4 ，5 mM HAuCl 4 。
12. The method of claim 5, wherein the chronoamperometric treatment conditions are: the fixed deposition voltage is 300 to 500mv; the treatment time was 100 to 130s.
13. The method of claim 12, wherein the chronoamperometric treatment conditions are: the fixed deposition voltage is 400mv; the treatment time was 120s.
14. The method of claim 4, wherein the method of preparing the E-cad-antibody-QD probe comprises:
and adding the E-cad-antibody and the coupling agent 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride into the carboxyl quantum dot solution, stirring at room temperature for 1 to 2 hours to perform a conjugation reaction, and purifying to obtain the target product.
15. The method of claim 14, wherein the conjugate is purified by passing the conjugate through a 0.2 μm filter to remove large aggregates and washing at least 5 times with borate buffer.
16. Use of the electrochemical biosensor according to any one of claims 1 to 3 or the electrochemical biosensor prepared by the method according to any one of claims 4 to 15 for detecting epithelial-to-mesenchymal transition of cells.
17. A method for detecting epithelial-to-mesenchymal transition of cells, comprising analyzing epithelial-to-mesenchymal transition of cells by detecting changes in E-cadherin based on electrochemical and fluorescent signals using the electrochemical biosensor according to any one of claims 1 to 3 or the electrochemical biosensor prepared by the preparation method according to any one of claims 4 to 15.
18. The method of claim 17, wherein the carboxylated carbon nanotube-gold nanoparticle modified electrode is a working electrode, ag/AgCl is used as a reference electrode, platinum wire is used as a counter electrode, and is connected to an electrochemical workstation, hg 2+ -taking an acetic acid buffer solution as a supporting electrolyte, incubating a sample to be detected and the E-cad-antibody-QD probe, and performing electrochemical detection by adopting a differential pulse voltammetry method; meanwhile, based on the immunofluorescence technique, the fluorescence detection is carried out.
19. The method of claim 18, wherein the reference electrode is an Ag/AgCl electrode containing saturated KCl; the Hg is 2+ -the pH of the acetate buffer solution is 5.2; the time for incubating the sample to be tested with the E-cad-antibody-QD probe was 2 hours.
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| CN108196060A (en) * | 2017-12-07 | 2018-06-22 | 山东大学齐鲁医院(青岛) | Applications of the soluble E-cadherin as cancer Epithelial and stromal conversion marker |
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