CN110057799B - Kit for detecting cell C/EBP alpha by using amino modified gold nano-carrier, detection method and application thereof - Google Patents

Abstract

The invention discloses a kit, a detection method and an application of an amino-modified gold nanocarrier for detecting cell C/EBPα. The kit contains amino-modified gold nanocarriers, membrane-penetrating proteins and double-stranded DNA reagents; the DNA reagents contain C/EBPα binding sites and fluorescent labels. Positively charged aminated AuNPs can adsorb negatively charged double-stranded DNA containing C/EBPα-specific binding sites, and the fluorescent molecules carried by them will be quenched by the unique optical effect of AuNPs. In the presence of C/EBPα, C/EBPα can specifically bind to double-stranded DNA, thereby hindering the adsorption of AuNPs to dsDNA, resulting in the recovery of fluorescence intensity. This method enables the detection of C/EBPα as low as 29 pM without any signal amplification or other synthetic complexes.

Description

Kit for detecting cell C/EBP alpha by using amino modified gold nano-carrier, detection method and application thereof

Technical Field

The invention relates to the field of genetic engineering, in particular to a kit for detecting cell C/EBP alpha by using an amino modified gold nano-carrier, a detection method and application thereof.

Background

CCAAT/Enhancer Binding Proteins (C/EBPs) are a family of basic leucine zipper transcription factors, including 6 elements named from C/EBP α to C/EBP ζ, that are ubiquitously expressed in different species.

C/EBPs are a family of transcription factors that have the effects of inhibiting proliferation, promoting differentiation, and regulating DNA damage response. Mainly takes part in important life activities such as cell proliferation and differentiation, tumorigenesis and apoptosis, cell cycle regulation and the like through the regulation of target cell gene transcription.

Traditional C/ebpa protein detection techniques are mainly ELISA and immunoblotting, and recognition and signal generation of these methods rely on antibodies and enzymes (e.g., horseradish peroxidase), respectively, resulting in expensive experiments, requiring multiple incubation and washing steps, tedious and time-consuming.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide a kit for detecting C/EBP alpha by using an amino modified gold nano carrier, and also aims to provide application of the kit in-situ detection of adipose precursor cells and mature adipocytes; the invention also aims to provide the application of the kit in the in-situ detection of the C/EBP alpha protein expression cells; still another object of the present invention is to provide a method for detecting C/ebpa of ex vivo cells based on amino-modified gold nanocarriers with short time and high sensitivity.

The kit for detecting C/EBP alpha by using the amino modified gold nano-carrier comprises the amino modified gold nano-carrier, membrane penetrating protein and a double-stranded DNA reagent; the necessary PBS buffer system may also be included. The double-stranded DNA contains a C/EBP alpha binding site, and the 3' end of the C/EBP alpha chain in the double-stranded DNA is marked by a fluorescent dye.

As one embodiment, the nucleotide sequence of the double-stranded DNA is:

C/EBPα1：5’-TTTTTTTGCGCAAGTTTTT-M-3’；

C/EBPα2：5’-AAAAACTTGCGCAAAAAAA-3’，

the nucleotide sequence of the above DNA can be adapted by those skilled in the art according to actual needs, but should contain at least a C/EBP α binding site and have a fluorescent dye label at its 3' end.

In a preferred embodiment, the fluorescent dye is Cy3, and the detection is performed by setting the excitation wavelength and the emission wavelength to 548nm and 562nm using a fluorescence spectrometer. The fluorescent dye used in the present invention includes but is not limited to Cy3, and other fluorescent dyes such as FITC, CY5, Pacific, PE, APC, etc. can be used, using the excitation and emission wavelengths recommended by the dye manufacturer.

Recent advances in nanotechnology have made nanoparticles a key component of various bioassays. Gold nano-carriers are often used to amplify the analytical signal due to their higher current and number of active sites. The gold nano-carrier provided by the invention comprises, but is not limited to, any one or more combinations of gold nano-particles (AuNPs), gold nano-rods (AuNRs) and gold nano-particles (AuNSs). Gold nanoparticles are preferably used as support for in situ detection.

Referring to fig. 1, after the surface of the gold nanocarrier modified by the amino group has a positive charge, the double-stranded DNA containing the C/ebpa binding site is linked and the 3' end is fluorescently labeled, when the C/ebpa in the nucleus is absent, the double-stranded DNA will be adsorbed to the surface of the gold nanocarrier due to the attraction of the positive charge. On the contrary, the presence of C/EBP alpha will block the interaction between double-stranded DNA and gold nanocarrier. The present invention is to verify the presence and amount of C/EBP alpha by quenching and recovering the fluorescence of the label and its intensity. Compared with the prior art such as ELISA, immunoblotting and the like, the method utilizes cheap short-chain DNA, does not need expensive antibodies and enzymes, has low cost, simple detection steps (only needs one-step incubation with a sample), and does not need repeated incubation and washing.

The membrane-penetrating protein is used for mediating the crossing of nuclear pores, and the membrane-penetrating peptide is selected from any one or more of but not limited to transcription transactivator (TAT), polyarginine (Stearyl-R8), drosophila homeotic transcription factor (Antp) and Transportan 10 (TP 10). TAT is preferably used as a membrane-penetrating protein.

The kit can be used for in-situ detection of in-vitro fat precursor cells and mature fat cells. The method is also suitable for other cells expressing the C/EBP alpha protein, such as isolated leukemia cells, liver cells, colon cancer cells, keratinocytes, adipocytes and the like, but the method is particularly suitable for in-situ detection of fat precursor cells and mature adipocytes, and the fluorescence intensity of the method is remarkably improved.

The method for detecting the isolated cell C/EBP alpha by using the amino modified gold nano-carrier comprises the following steps:

(1) adding PEG-NH₂Dissolving the membrane-penetrating protein and the membrane-penetrating protein in a PBS solution, adding the solution into a gold nano-carrier solution, and incubating to obtain an amino-modified gold nano-carrier;

(2) incubating the gold nano-carrier modified by the amino group with double-stranded DNA containing a C/EBP alpha binding site for 5 minutes, marking the 3' end containing a C/EBP alpha chain in the double-stranded DNA by using a fluorescent dye, adding the double-stranded DNA into cell lysate, and continuously incubating for 1 hour;

(3) fluorescence measurements were performed using a fluorescence spectrometer and curves were drawn.

And establishing a standard curve of concentration and fluorescence intensity based on the fluorescence intensity curve drawn by the C/EBP alpha with different concentrations, so as to be beneficial to further detecting the concentration of the C/EBP alpha.

Preferably, the gold nano-carrier and PEG-NH in the step (1)₂The mass ratio of (A) to (B) is 0.1-2: 10. More preferably, gold nanocarriers are coupled to PEG-NH₂The mass ratio of (A) to (B) is 1: 10.

Preferably, TAT is selected as the membrane penetrating protein, and the final concentration of TAT is 0.02 mmol.

Has the advantages that: the invention utilizes the characteristic that C/EBP alpha is specifically combined with double-stranded DNA to block the adsorption of AuNPs on the double-stranded DNA, thereby leading to the recovery of fluorescence intensity. The method achieves detection of C/EBP alpha as low as 29pM without any signal amplification or other synthetic complex involvement.

Drawings

FIG. 1 is a schematic diagram of the C/EBP alpha detection method of the present invention;

fig. 2A is the result of fluorescence spectrum reaction (0.05 ng/mL-1500 ng/mL) for detecting different concentrations of C/ebpa in example 2 of the present invention, fig. 2B is a standard curve corresponding to fig. 2A, fig. 2C is a linear concentration range, σ standard deviation (n = 3);

FIG. 3 shows the fluorescence intensity of C/EBP alpha detected in the extracts of the adipocyte precursor cells and the mature adipocytes of example 3;

FIG. 4A is a fluorescence plot of mature adipocytes incubated with dsDNA-AuNPs for various time periods; FIG. 4B is a graph of fluorescence of preadipocytes and mature adipocytes after incubation with dsDNA-AuNPs.

Detailed Description

The present invention is further illustrated by the following specific examples.

Example 1

In this example, the test subjects were isolated cells expressing C/EBP alpha protein (hepatocytes, lung cells, bone marrow cells, keratinocytes, etc.), and C/EBP alpha protein of these cells was tested.

₂1. PEG-NH modified gold nano-carrier

Adding PEG-NH₂And a membrane-penetrating protein covalently coupled to the gold nanocarrier. Specifically, PEG-NH₂And membrane-penetrating protein in PBS (10 mM PBS, pH 7.4) to give a homogeneous solution: adding PEG-NH₂Adding into gold nano-carrier. The concentration of membrane-penetrating proteins is adjusted to ensure the effect of penetrating the cell membrane. The solutions were mixed and incubated at room temperature and the amino-modified gold nanocarriers were stored at 4 ℃ for further use.

2. Detection of C/EBP alpha by using amino-modified gold nano-carrier

And (3) incubating the gold nano-carrier modified by the amino group with double-stranded DNA containing the C/EBP alpha binding site, wherein the 3' end of the double-stranded DNA containing the C/EBP alpha chain is marked by a fluorescent dye.

3. Fluorescence measurements were performed with a fluorescence spectrometer.

And respectively adjusting the excitation wavelength and the emission wavelength of the fluorescence spectrometer to the wavelengths corresponding to the fluorescent dyes. Fluorescence detection was performed at room temperature. And (3) drawing a fluorescence intensity curve based on different C/EBP alpha concentrations, and establishing a standard curve of linear concentrations of the fluorescence intensity curve.

Example 2

This example detects C/EBP alpha protein in isolated adipocytes.

The kit contains pre-prepared PEG-NH₂Modified AuNPs, membrane penetrating proteins, double stranded DNA reagents and the necessary PBS buffer system. Wherein, PEG-NH₂The modified AuNPs were prepared as follows:

adding PEG-NH₂And TAT is covalently coupled to AuNPs. Specifically, PEG-NH₂And TAT dissolved in PBS (10 mM PBS, pH 7.4) to give a homogeneous solution: 50 μ L of PEG-NH₂（1mg/mL）Added to 100. mu. LAuNPs at a concentration of 0.05 mg/mL. The final concentration of TAT was 0.02 mmol. The solutions were mixed and incubated at room temperature for 1 hour and the amino-modified AuNPs were stored at 4 ℃ for further use.

And (3) incubating the gold nano-carrier modified by the amino group with double-stranded DNA containing the C/EBP alpha binding site for 5 minutes, marking the 3' end of a C/EBP alpha chain in the double-stranded DNA by using a fluorescent dye, and then continuously incubating with cell lysate for 1 hour.

Fluorescence detection is carried out by selecting a Hitachi F-2500 fluorescence spectrometer, and the excitation wavelength and the emission wavelength of Cy3 are respectively adjusted to be 548nm and 562 nm. The excitation and emission gap was 5nm and the PMT voltage was 800V. Fluorescence detection was performed at room temperature. And (3) drawing a fluorescence intensity curve based on different C/EBP alpha concentrations, and establishing a standard curve of linear concentrations of the fluorescence intensity curve.

As shown in FIG. 2, the fluorescence spectra of the double-stranded DNA after C/EBP α treatment at different concentrations were obtained. When the concentration of C/EBP alpha is increased from 0.05 to 1500 ng/mL, the fluorescence intensity signal is increased. The high concentration of C/EBP alpha can obviously improve the intensity of a fluorescence intensity signal. In the range of 0.05-600 ng/mL, the fluorescence intensity is linearly related to the C/EBP alpha concentration, and the detection limit is expected to be 29 pg/mL. The method is equivalent to the detection limit of 20pg/mL which can be achieved by the currently accepted EILISA technology, but the experimental operation steps and time are greatly reduced.

Example 3

The C/EBP alpha detection of the fat precursor cells and the mature fat cells has important guiding significance for identifying the causes of the obesity and developing targeted treatment technologies.

₂1. Preparation of PEG-NH modified AuNPs

Adding PEG-NH₂And TAT dissolved in PBS (10 mM PBS, pH 7.4) to give a homogeneous solution: 50 μ L of PEG-NH₂(1 mg/mL) was added to 100. mu. LAuNPs at a concentration of 0.05 mg/mL. The final concentration of TAT was 0.02 mmol. The solutions were mixed and incubated at room temperature for 1 hour and the amino-modified AuNPs were stored at 4 ℃ for further use.

2. Detection of adipose precursors using amino-modified AuNPsC/EBP alpha in cells and mature adipocytes

a. Equal amounts of ex vivo adipocyte precursor cells and mature adipocytes were added to RIPA lysis buffer, mixed and incubated on ice for 30 min. The mixture was then centrifuged at 12000 rpm for 30 minutes to obtain a cell extract. And (3) incubating the gold nano-carrier modified by the amino group with double-stranded DNA containing the C/EBP alpha binding site for 5 minutes, marking the 3' end of a C/EBP alpha chain in the double-stranded DNA by using a fluorescent dye, and then continuously incubating with cell lysate for 1 hour. Fluorescence measurements were performed using a Hitachi F-2500 fluorescence spectrometer, method example 2.

b. C/EBP alpha in the fat precursor cells and mature fat cells is detected in situ, the cells are cultured to an exponential phase, and an analysis experiment is carried out. After incubation with dsDNA-AuNPs for a certain period of time, the cells were observed for their fluorescent properties under a fluorescent microscope.

As shown in FIG. 3, the fluorescence intensity of the mature cell group was significantly higher than that of the undifferentiated cell group, indicating that C/EBP α was highly expressed mainly in the differentiated tissues.

As shown in FIG. 4, the mature adipocytes did not see signal within the initial 10 min, showed weak fluorescence after 60 min, and reached maximum intensity at 120 min (FIG. 4A). Microscopic observations also indicated that C/EBP α expression was higher in mature adipocytes than in preadipocytes (FIG. 4B).

Claims (8)

1. A kit for detecting C/EBP alpha by using an amino modified gold nano-carrier is characterized in that: the kit comprises an amino-modified gold nano-carrier, membrane-penetrating protein and a double-stranded DNA reagent;

the double-stranded DNA contains a C/EBP alpha binding site, and the 3' end of the C/EBP alpha chain in the double-stranded DNA is marked by a fluorescent dye; the fluorescent dye is Cy3, and the excitation wavelength and the emission wavelength are set to 548nm and 562nm by using a fluorescence spectrometer for detection; the nucleotide sequence of the double-stranded DNA is as follows:

C/EBPα1：5’-TTTTTTTGCGCAAGTTTTT-M-3’；

C/EBPα2：5’-AAAAACTTGCGCAAAAAAA-3’，

wherein M represents a fluorescent dye label.

2. The kit of claim 1, wherein: the gold nano-carrier is AuNPs.

3. The kit of claim 2, wherein: the membrane penetrating protein is any one or combination of multiple of transcription trans-activator polyarginine, Drosophila homeotic transcription factor and Transportan 10.

4. Use of the kit of claim 1 for in situ detection of adipogenic precursor cells and mature adipocytes.

5. Use of the kit of claim 1 for the in situ detection of C/EBP α protein-expressing cells.

6. A method for detecting in vitro cell C/EBP alpha by using an amino modified gold nano-carrier is characterized by comprising the following steps:

(1) adding PEG-NH₂Dissolving the membrane-penetrating protein and the membrane-penetrating protein in a PBS solution, adding the solution into a gold nano-carrier solution, and incubating to obtain an amino-modified gold nano-carrier;

(2) incubating the amino-modified gold nanoparticles with double-stranded DNA containing a C/EBP alpha binding site, wherein the 3' end of a C/EBP alpha chain in the double-stranded DNA is marked by a fluorescent dye;

(3) fluorescence measurements were performed using a fluorescence spectrometer and curves were drawn.

7. The method of claim 6, wherein: the gold nano-carrier and PEG-NH₂The mass ratio of (A) to (B) is 0.1-2: 10.

8. The method of claim 7, wherein: the final concentration of the membrane penetrating protein was 0.02 mmol.

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