Method for detecting MUC1 mucoprotein by combining aptamer and terahertz metamaterial sensor

Abstract

The invention relates to the technical field of terahertz biological detection, and particularly discloses a method for detecting MUC1 mucoprotein by combining an aptamer and a terahertz metamaterial sensor, which comprises the following steps: testing the no-load spectrum of the terahertz metamaterial sensor; preparing gold nanoparticles; preparing gold nanoparticles marked by the aptamer; preparing a double-aptamer sandwich structure; testing the spectrum when the terahertz metamaterial sensor covers the test sample; and establishing a relational expression according to the frequency shift amount of the spectrum and calculating the MUC1 concentration. The terahertz metamaterial sensor disclosed by the invention has high sensitivity, and the sensor modified by the aptamer has high-specificity MUC1 mucoprotein recognition capability. Therefore, the method for detecting the MUC1 mucoprotein by combining the aptamer and the terahertz metamaterial sensor has an important application value in the field of high-sensitivity and high-specificity MUC1 mucoprotein detection.

Description

Technical Field

[0001] The present invention relates to the technical field of terahertz biological detection, and in particular to a method for detecting MUC1 mucin by combining an aptamer and a terahertz metamaterial sensor. Background Art

[0002] Aptamers: Aptamers are unique molecular diagnostic elements that are single-stranded DNA (ssDNA), RNA, or modified nucleic acids that are isolated through an in vitro selection process called SELEX (Systematic Evolution and Exponential Enrichment of Ligands). Aptamers have high specificity and binding affinity similar to antibodies because they fold into unique three-dimensional structures by producing various secondary structures such as loops, hairpins, and probes. In addition, they have the advantages of rapid in vitro selection, cell-free chemical synthesis, smaller size and low complexity, resistance to denaturation and high stability, and ease of chemical modification. Therefore, aptamers can become molecular probes for specific detection, as well as cancer treatment. The use of aptamers can specifically identify cancer-specific markers by targeting proteins overexpressed in tumors. And many aptamers with high affinity for MUC1 have been isolated.

[0003] Terahertz waves (THz) refer to electromagnetic waves with a frequency of 0.1THz-10THz. Their photon energy is very low and they do not produce ionizing radiation. They are particularly suitable for label-free and non-destructive biological detection and have important application value in the biomedical field.

[0004] MUC1 (also known as PEM, H23Ag, EMA, CA15-3, and MCA) is a mucin that is normally present on the apical surface of most normal secretory epithelial cells. MUC1 is overexpressed in most adenocarcinomas, so it not only appears on the entire cell surface, but is also shed into the blood in the form of MUC1 fragments. Therefore, MUC1 can be used as a biomarker indicator for early cancer detection. There are colorimetric, fluorescent, electrochemical, and electrochemical methods for detecting MUC1. These methods provide favorable conditions for obtaining qualitative and quantitative information on bioactive molecules, but lack highly sensitive sensors and highly specific molecular recognition capabilities.

[0005] Therefore, a method for detecting MUC1 quickly, conveniently, with high sensitivity and specificity will have very important application value in the field of biomedical testing. Summary of the invention

[0006] The object of the present invention is to provide a method for detecting MU C1 mucin by combining an aptamer and a terahertz metamaterial sensor, aiming to solve the problem in the prior art that MU C1 cannot be detected with high sensitivity and high specificity.

[0007] To achieve the above object, the present invention adopts a method for detecting MUC1 mucin by combining an aptamer and a terahertz metamaterial sensor, comprising the following steps:

[0008] Testing the unloaded spectrum of terahertz metamaterial sensors;

[0009] Preparation of gold nanoparticles;

[0010] preparing aptamer-labeled gold nanoparticles;

[0011] Preparation of dual aptamer sandwich structures;

[0012] Spectrum of the terahertz metamaterial sensor when covering the test sample;

[0013] A relationship was established based on the frequency shift of the spectrum and the MUC1 concentration was calculated.

[0014] The gold nanoparticles serve as a bridge connecting the terahertz metamaterial sensor and the aptamer, providing highly sensitive quantitative analysis through the terahertz metamaterial sensor and providing highly specific molecular recognition capabilities through the aptamer; the frequency shift of the spectrum of the covered test sample caused by the capture of the MUC1 molecule by the aptamer in the dual aptamer structure is used to determine whether the MUC1 molecule is contained; the frequency shift of the absorption peak is obtained by comparing the empty spectrum and the covered sample spectrum, and a relationship is established to calculate the MUC1 concentration. The method of combining the aptamer and the terahertz metamaterial sensor to detect MUC1 mucin is a method that can perform highly specific and sensitive quantitative analysis on MUC1 and can also achieve rapid detection of the content of the adenocarcinoma marker MUC1.

[0015] The method of testing the no-load spectrum of the terahertz metamaterial sensor comprises the following steps:

[0016] Clean the terahertz metamaterial sensor and dry it with nitrogen;

[0017] The terahertz time domain spectrum of the terahertz metamaterial sensor was tested, and the no-load frequency domain spectrum was obtained by fast Fourier transform.

[0018] Wherein, the preparation of gold nanoparticles comprises the following steps:

[0019] Heat the chloroauric acid solution to boiling under magnetic stirring, and add the sodium citrate solution to the boiling chloroauric acid solution;

[0020] When the colorless solution gradually turns blue and finally wine red, it is cooled to room temperature to obtain gold nanoparticles.

[0021] Wherein, the preparation of aptamer-labeled gold nanoparticles comprises the following steps:

[0022] The prepared gold nanoparticles were fully mixed with two different aptamer molecule solutions with the same specific recognition sites at a volume ratio of 1:400;

[0023] The mixed solution was incubated at -20°C for 2 hours and thawed to obtain two aptamer-coupled gold nanoparticles.

[0024] The two aptamers in the aptamer-labeled gold nanoparticles are both modified with thiol -SH at the ends, and the sequences of the two aptamers are 5'-CCCCCCGATCCTTTGGATA-3' and 5'-GCAGTTGATCC TTTGGATACCCTGG-3', respectively.

[0025] The preparation of the dual aptamer sandwich structure comprises the following steps:

[0026] Using APTES to activate the terahertz metamaterial sensor as claimed in claim 2 at room temperature for 20 minutes;

[0027] The prepared first aptamer-labeled gold nanoparticles, the target solution to be tested, and the prepared second aptamer-labeled gold nanoparticles were sequentially added to the surface of the terahertz metamaterial sensor and incubated at room temperature for 30 minutes;

[0028] Use BSA solution to block the active sites; use PBS to wash the free gold nanoparticles, aptamers, and detection objects, and dry them to obtain the dual aptamer sandwich structure.

[0029] During the preparation of the dual aptamer sandwich structure, the volume ratio of APTES to anhydrous ethanol was 2%, the target solution to be tested was a solution containing different concentrations of MUC1, the mass concentration of BSA was 1wt%, and the molar concentration of PBS solution was 0.01 M. The active sites blocked by the BSA solution were gold nanoparticles without aptamers.

[0030] The testing of the spectrum of the terahertz metamaterial sensor when covering the test sample comprises the following steps:

[0031] Prepare more than 10 concentrations of MUC1 solution;

[0032] In the process of preparing the dual-aptamer sandwich structure, the configured MUC1 solution was dropped onto the surface of the terahertz metamaterial sensor respectively;

[0033] The prepared dual aptamer sandwich structure is dried naturally or placed in a constant temperature oven to form a molecular film;

[0034] The terahertz metamaterial sensor covered with the molecular film was placed in the test system for terahertz time-domain spectroscopy testing, and then the terahertz frequency-domain spectrum of the "terahertz metamaterial sensor plus MUC1 molecular film" was obtained through fast Fourier transform;

[0035] The method of establishing a relationship based on the spectral frequency shift and calculating the MUC1 concentration comprises the following steps:

[0036] The terahertz frequency domain spectrum of each concentration is subtracted from the empty frequency domain spectrum to obtain the absorption peak frequency shift corresponding to the MUC1 solution of different concentrations;

[0037] Establish the relationship between MUC1 concentration and frequency shift; calculate the concentration of MUC1.

[0038] The present invention discloses a method for detecting MUC1 mucin by combining an aptamer and a terahertz metamaterial sensor. A dual-mode detection platform is constructed to detect MUC1 with high specificity and high sensitivity. First, aptamer-modified gold nanoparticles are directly connected to the surface of a terahertz metamaterial sensor by physical adsorption. Subsequently, the aptamer acts as a specific recognition element to specifically recognize MUC1. The capture of MUC1 molecules by the aptamer in the dual aptamer structure causes a greater spectral frequency shift in the test sample, thereby determining whether the sample contains MUC1 molecules. By comparing the frequency shift of the absorption peak obtained by the empty spectrum and the spectrum, a relationship is established to calculate the MUC1 concentration. This method has the advantages of low cost, high sensitivity and good specificity, and solves the problems of low sensitivity and poor high specificity in existing MUC1 detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0040] Figure 1 This is a schematic diagram of the detection principle of a method for detecting MUC1 mucin by combining an aptamer and a terahertz metamaterial sensor according to the present invention;

[0041] Figure 2 is a schematic diagram of absorption spectra of different MUC1 concentrations according to a specific embodiment of the present invention;

[0042] Figure 3 It is a schematic diagram of a process of the method for detecting MUC1 mucin by combining an aptamer and a terahertz metamaterial sensor according to the present invention;.

[0043] Figure 4 It is a flow chart of calculating MUC1 concentration according to a specific embodiment of the present invention. DETAILED DESCRIPTION

[0044] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

[0045] See also Figure 3 The present invention proposes a method for detecting MUC1 mucin by combining an aptamer and a terahertz metamaterial sensor, comprising the following steps:

[0046] S11: Testing the unloaded spectrum of the terahertz metamaterial sensor;

[0047] S12: Preparation of gold nanoparticles;

[0048] S13: preparation of aptamer-labeled gold nanoparticles;

[0049] S14: Preparation of dual aptamer sandwich structure;

[0050] S15: Spectrum of the test terahertz metamaterial sensor covering the test sample;

[0051] S16: Establish a relationship based on the frequency shift of the spectrum and calculate the MUC1 concentration.

[0052] The method of testing the no-load spectrum of the terahertz metamaterial sensor comprises the following steps:

[0053] S101: clean the terahertz metamaterial sensor and dry it with nitrogen;

[0054] S102: Testing the terahertz time domain spectrum of the terahertz metamaterial sensor, and obtaining the frequency domain spectrum through fast Fourier transform.

[0055] Wherein, the preparation of gold nanoparticles comprises the following steps:

[0056] S21: heating the chloroauric acid solution to boiling under magnetic stirring, and adding the sodium citrate solution to the boiling chloroauric acid solution;

[0057] S22: When the colorless solution gradually turns blue and finally wine red, it is cooled to room temperature to obtain gold nanoparticles.

[0058] Wherein, the preparation of aptamer-labeled gold nanoparticles comprises the following steps:

[0059] S31: fully mixing the prepared gold nanoparticles with two different aptamer molecule solutions having the same specific recognition site at a volume ratio of 1:400;

[0060] S32: The mixed solution was incubated at -20°C for 2 hours, and two aptamer-coupled gold nanoparticles were obtained after thawing.

[0061] S33: Both aptamers in the aptamer-labeled gold nanoparticles are modified with thiol -SH at the ends, and the sequences of the two aptamers are 5'-CCCCCCGATCCTTTGGATA-3' and 5'-GCAGTTG ATCCTTTGGATACCCTGG-3', respectively.

[0062] The preparation of the dual aptamer sandwich structure comprises the following steps:

[0063] S41: activating the terahertz metamaterial sensor as claimed in claim 2 using APTES at room temperature for 20 minutes;

[0064] S42: dropping the prepared first aptamer-labeled gold nanoparticles, the target solution to be tested, and the prepared second aptamer-labeled gold nanoparticles onto the surface of the terahertz metamaterial sensor in sequence and incubating at room temperature for 30 minutes;

[0065] S43: Use BSA solution to block the active site; use PBS to wash the free aptamer and dry it to obtain the double aptamer sandwich structure.

[0066] S44: The volume ratio of APTES to anhydrous ethanol is 2%, the target solution to be tested is a solution containing different concentrations of MUC1, the mass concentration of BSA is 1wt%, and the molar concentration of PBS solution is 0.01M.

[0067] S45: Use BSA solution to block the active sites of gold nanoparticles without aptamers.

[0068] The testing of the spectrum of the terahertz metamaterial sensor when covering the test sample comprises the following steps:

[0069] S51: prepare 10 or more concentrations of MUC1 solution;

[0070] S52: In the process of preparing the dual aptamer sandwich structure, the configured MUC1 solution is respectively added dropwise to the surface of the terahertz metamaterial sensor;

[0071] S53: drying the prepared dual aptamer sandwich structure naturally or placing it in a constant temperature oven to dry to form a molecular film;

[0072] S54: Put the silicon wafer covered with the molecular film into the test system for terahertz time domain spectroscopy test, and then obtain the terahertz frequency domain spectrum of the "terahertz metamaterial sensor plus MUC1 molecular film" through fast Fourier transform;

[0073] The method of establishing a relationship based on the frequency shift of the spectrum and calculating the MUC1 concentration comprises the following steps:

[0074] S61: subtract the empty-load frequency domain spectrum from the terahertz frequency domain spectrum of each concentration to obtain the absorption peak frequency shift corresponding to the MUC1 solution of different concentrations;

[0075] S62: Establish a relationship between MUC1 concentration and frequency shift; calculate the MUC1 concentration each time.

[0076] The method for detecting MUC1 mucin by combining aptamers and terahertz metamaterial sensors constructs a dual-mode detection platform to detect MUC1 with high specificity and high sensitivity, directly connects aptamer-modified gold nanoparticles to the surface of the terahertz metamaterial sensor through physical adsorption, and then, as a specific recognition element, the aptamer specifically recognizes MUC1, and then uses the frequency shift of the spectrum of the covered test sample caused by the capture of MUC1 molecules by the aptamer in the dual aptamer structure to determine whether the sample contains MUC1 molecules; the frequency shift of each absorption peak is obtained by comparing the empty spectrum with the spectrum, and a relationship is established to calculate the MUC1 concentration. This method uses two aptamer-modified gold nanoparticles to amplify the frequency shift of the absorption peak, and can detect MUC1 with high sensitivity and high specificity.

[0077] The present invention also proposes a specific embodiment:

[0078] Firstly, a terahertz absorber unit structure based on the all-dielectric metasurface is established, the transmission parameter S21 is calculated using the finite integration technique, and the absorption peak position is determined according to the transmission parameter S21.

[0079] Furthermore, the present embodiment provides a method for detecting MUC1 mucin by combining an aptamer and a terahertz metamaterial sensor, and the specific steps are as follows:

[0080] (1) The terahertz metamaterial sensor was repeatedly cleaned with anhydrous ethanol for three times, and then dried with nitrogen. The sensor was placed in a CCT-1800 multifunctional terahertz time-domain spectrometer to test the terahertz time-domain spectrum of the terahertz metamaterial sensor, and the no-load frequency domain spectrum was obtained by fast Fourier transform.

[0081] (2) Preparation of gold nanoparticles: 100 ml of 0.01 wt% chloroauric acid solution was heated to boiling under magnetic stirring, and then 2 ml of 1 wt% sodium citrate solution was added to the boiling chloroauric acid solution. The reaction was allowed to boil for 20 min. During this process, the colorless solution gradually turned blue and finally wine red. The solution was cooled to room temperature to obtain gold nanoparticles with a particle size of 50 nm.

[0082] (3) Preparation of aptamer-labeled gold nanoparticles: 8 ml of the gold nanoparticles prepared above were mixed with 20 ul of two different aptamer molecule solutions with the same specific recognition site, the sequences of the two aptamers were 5'-CCCCCCGATCCTTTGGATA-3' and 5'-GCAGTTGATCC TTTGGATACCCTGG-3', respectively, and the mixture was incubated at -20°C for 2 h and then thawed to obtain two aptamer-labeled gold nanoparticles;

[0083] (4) Preparation of dual aptamer sandwich structure: 20ul of 2% APTES by volume was added to the surface of the washed and dried terahertz metamaterial sensor, and activated at room temperature for 20 minutes. Then, 20ul of the first aptamer-labeled gold nanoparticles prepared above were continued to be added to the surface of the terahertz metamaterial sensor, and incubated at room temperature for 30 minutes. Subsequently, 20ul of a 1wt% BSA solution was added to block the active sites of the gold nanoparticles. After incubation at room temperature for 30 minutes, 20ul of MUC 1 samples of different concentrations were continued to be added, and incubated at 37°C for 30 minutes. Finally, 20ul of the second aptamer-labeled gold nanoparticles were added, and incubated at 37°C for 30 minutes. Then, PBS with a pH of 7.2 and a molar concentration of 0.01M was used to slowly wash the free gold nanoparticles, aptamers and MUC1. After drying at room temperature, a dual aptamer sandwich structure was obtained. Figure 1 shown.

[0084] (5) Testing the spectrum of the terahertz metamaterial sensor when covering the test sample: preparing more than 10 concentrations of MUC1 solution;

[0085] In the process of preparing the dual-aptamer sandwich structure, the configured MUC1 solution was dropped onto the surface of the terahertz metamaterial sensor respectively;

[0086] The prepared dual aptamer sandwich structure is dried naturally or placed in a constant temperature oven to form a molecular film;

[0087] The silicon wafer covered with the molecular film was placed in the test system for terahertz time-domain spectroscopy testing, and then the terahertz frequency domain spectrum of the "terahertz metamaterial sensor plus MUC1 molecular film" was obtained by fast Fourier transform. Figure 2 As shown, Figure 2 The three curves shown from right to left are the terahertz frequency domain spectra corresponding to MUC1 concentrations of 1 ng / ml, 10 ng / ml, and 100 ng / ml. As the concentration increases, the number of captured MUC1 molecules increases, and the frequency shift also increases;

[0088] (6) Establish a relationship based on the frequency shift of the spectrum and calculate the MUC1 concentration: subtract the above-mentioned covered test sample absorption spectrum from the empty load absorption spectrum to obtain the frequency shift of the absorption peak corresponding to the sample absorption spectrum, establish a relationship between the frequency shift and the MUC1 concentration according to different MUC1 concentrations, and use the relationship to calculate the corresponding MUC1 concentration. The calculation process is as follows: Figure 3 shown.

[0089] What is disclosed above is only a preferred embodiment of the present invention, and it certainly cannot be used to limit the scope of rights of the present invention. Ordinary technicians in this field can understand that all or part of the processes of the above embodiment and equivalent changes made according to the claims of the present invention still fall within the scope of the invention.

Claims

1. A method for detecting MUC1 mucin by combining an aptamer and a terahertz metamaterial sensor, characterized in that: The following steps are involved: Testing the unloaded spectrum of terahertz metamaterial sensors; Preparation of gold nanoparticles; preparing aptamer-labeled gold nanoparticles; Preparation of dual aptamer sandwich structures; Spectrum of the terahertz metamaterial sensor when covering the test sample; A relationship was established based on the frequency shift of the spectrum and the MUC1 concentration was calculated.

2. The method for detecting MUC1 mucin by combining an aptamer and a terahertz metamaterial sensor as claimed in claim 1, characterized in that: The method for testing the unloaded spectrum of the terahertz metamaterial sensor comprises the following steps: Clean the terahertz metamaterial sensor and dry it with nitrogen; The terahertz time domain spectrum of the terahertz metamaterial sensor was tested, and the no-load frequency domain spectrum was obtained by fast Fourier transform.

3. The method for detecting MUC1 mucin by combining an aptamer and a terahertz metamaterial sensor as claimed in claim 1, characterized in that: The preparation of gold nanoparticles includes the following steps: Heat the chloroauric acid solution to boiling under magnetic stirring, and add the sodium citrate solution to the boiling chloroauric acid solution; When the colorless solution gradually turns blue and finally wine red, it is cooled to room temperature to obtain gold nanoparticles.

4. The method for detecting MUC1 mucin by combining an aptamer and a terahertz metamaterial sensor as claimed in claim 1, characterized in that: The preparation of aptamer-labeled gold nanoparticles includes the following steps: The prepared gold nanoparticles were fully mixed with two different aptamer solutions with the same specific recognition sites at a volume ratio of 1:400; The mixed solution was incubated at -20°C for 2 hours and thawed to prepare two aptamer-labeled gold nanoparticles.

5. The method for detecting MUC1 mucin by combining an aptamer and a terahertz metamaterial sensor as claimed in claim 4, characterized in that: The two aptamers in the aptamer-labeled gold nanoparticles are both modified with thiol -SH at the ends, and the sequences of the two aptamers are 5'-CCCCCCGATCCTTT GGATA-3' and 5'-GCAGTTGATCCTTTGGATACCCTGG-3' respectively.

6. The method for detecting MUC1 mucin by combining an aptamer and a terahertz metamaterial sensor as claimed in claim 1, characterized in that: The preparation of the dual aptamer sandwich structure includes the following steps: Activate the terahertz metamaterial sensor according to claim 2 using APTES at room temperature for 20 minutes; The prepared gold nanoparticles labeled with the first aptamer, the target solution to be tested, and the gold nanoparticles labeled with the second aptamer were sequentially added to the terahertz metamaterial sensor and incubated at room temperature for 30 minutes; The active sites were blocked with BSA solution, and the free gold nanoparticles, aptamers, and test objects were washed with PBS, and the dual aptamer sandwich structure was obtained after natural drying.

7. The method for detecting MUC1 mucin by combining an aptamer and a terahertz metamaterial sensor as claimed in claim 6, characterized in that: In the preparation process of the dual aptamer sandwich structure, the volume ratio of APTES to anhydrous ethanol is 2%, the target solution to be tested is a MUC1 solution with different concentrations, the BSA mass concentration is 1wt%, and the molar concentration of the PBS solution is 0.01M.

8. The method for detecting MUC1 mucin by combining an aptamer and a terahertz metamaterial sensor as claimed in claim 6, characterized in that: In the preparation process of the dual-aptamer sandwich structure, the active sites blocked by the BS A solution are gold nanoparticles that are not connected to the aptamer.

9. The method for detecting MUC1 mucin by combining an aptamer and a terahertz metamaterial sensor as claimed in claim 1, characterized in that: Testing the spectrum of a terahertz metamaterial sensor over a test sample involves the following steps: Prepare more than 10 concentrations of MUC1 solution; In the process of preparing the dual-aptamer sandwich structure, the configured MUC1 solution was dripped onto the surface of the terahertz metamaterial sensor respectively; The prepared dual aptamer sandwich structure is dried naturally or placed in a constant temperature oven to form a molecular film; The terahertz metamaterial sensor covered with the molecular film was placed in the test system for terahertz time-domain spectroscopy testing, and then the terahertz frequency domain spectrum of the "terahertz metamaterial sensor plus MUC1 molecular film" was obtained through fast Fourier transform.

10. The method for detecting MUC1 mucin by combining an aptamer and a terahertz metamaterial sensor as claimed in claim 1, characterized in that: According to the frequency shift of the spectrum covering different concentrations of MUC1, a relationship is established to calculate the MUC1 concentration, including the following steps: The terahertz frequency domain spectrum of each concentration was subtracted from the empty frequency domain spectrum to obtain the frequency shift of the corresponding absorption peak of MUC1 solution with different concentrations; A relationship between the frequency shift and the concentration of MUC1 was established according to different MUC1 concentrations, and the concentration of MUC1 was calculated using the relationship.

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