A method of identifying a base

Abstract

The application provides a method for identifying bases, drops a DNA solution on a molybdenum disulfide film to prepare a DNA testing device, uses a fluorescence spectrometer to detect fluorescence spectrum data of the molybdenum disulfide under the influence of bases, carries out characteristic matching degree comparison analysis based on the fluorescence spectrum curve of the molybdenum disulfide under the influence of unknown bases and the fluorescence spectrum curve of the molybdenum disulfide under the influence of known bases, and determines which kind of known base the unknown base is according to the matching degree comparison analysis result; the application can effectively identify bases by using the DNA and MoS2 adsorption and charge transfer mechanism, and has a lower requirement for the concentration of the used DNA solution to be tested; meanwhile, the MoS2 has the advantages of low cost and simple preparation, and compared with the prior art, the application has the advantages of low-concentration trace DNA base effective identification, simple device structure, simple operation and low cost.

Description

Technical Field

[0001] This invention relates to the field of gene detection technology, and in particular to a method for identifying bases. Background Technology

[0002] Genes are the code of life, and gene sequencing is a crucial technological means for humans to understand and alter life. Currently, gene sequencing methods include end-termination sequencing (first generation), cyclic SBS sequencing (second generation), and nanopore sequencing (third generation). First-generation sequencing offers long reads and high accuracy, but low throughput; second-generation sequencing offers short reads, high throughput, and high accuracy, but offers no advantage in genome assembly or structural variation analysis; third-generation sequencing offers long reads, high throughput, but low accuracy, which cannot be compensated for by sequencing depth. Gene sequencing requires base identification. Using the above three generations of methods for base identification requires high DNA sample concentrations, reaching above 100 ng / μl. Therefore, developing low-concentration DNA sample base identification methods is of great significance for studying novel DNA fragments. The article "Optical Fingerprints and electron transports properties of DNA bases absorbed on monolayer MoS2" theoretically simulates that DNA modulates the photoelectric properties of MoS2, but this has not been experimentally verified. The article "Tunable excitonic emission of monolayer WS2 for their optical detection of DNA nucleobases" reported the effect of DNA on the fluorescence spectrum of WS2, but could not quantify the analysis.

[0003] It should be noted that the information disclosed in the background section above is only for understanding the background of this application, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0004] The main objective of this invention is to overcome the deficiencies of the aforementioned background technology and provide a method for identifying bases.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A method for identifying bases includes the following steps:

[0007] S1. A DNA test device is prepared by dropping a DNA solution containing known different bases onto a molybdenum disulfide film. The fluorescence spectrum data of molybdenum disulfide under the influence of known bases is detected by a fluorescence spectrometer.

[0008] S2. Perform curve fitting analysis on the fluorescence spectrum data of molybdenum disulfide under the influence of known bases to obtain the fluorescence spectrum curve of molybdenum disulfide under the influence of known bases.

[0009] S3. Drop the DNA solution containing unknown bases onto a molybdenum disulfide film to prepare a target DNA testing device. Use a fluorescence spectrometer to detect the fluorescence spectrum of the molybdenum disulfide in the target DNA testing device.

[0010] S4. Perform curve fitting analysis on the fluorescence spectrum data of molybdenum disulfide obtained in step S3 to obtain the fluorescence spectrum curve of molybdenum disulfide under the influence of the unknown base.

[0011] S5. Based on the fluorescence spectrum curve of molybdenum disulfide under the influence of the unknown base, a feature matching degree comparison analysis is performed with the fluorescence spectrum curve of molybdenum disulfide under the influence of the known base. Based on the matching degree comparison analysis results, it is determined which known base the unknown base is.

[0012] Furthermore:

[0013] The comparative analysis based on the fluorescence spectrum curves of molybdenum disulfide obtained by fitting includes the position and shape of the fluorescence peaks.

[0014] In steps S2 and S4, peak fitting of the fluorescence spectrum yields B, A, and A. - In step S5, the fluorescence spectra of molybdenum disulfide under the influence of unknown bases are compared and analyzed with those of molybdenum disulfide under the influence of known bases, specifically peak types B, A, and A. - Three peak types were used to determine which fluorescence spectrum of molybdenum disulfide under the influence of the unknown base best matched the fluorescence spectrum of molybdenum disulfide under the influence of the known base, thereby identifying the corresponding base.

[0015] Step S5 includes:

[0016] A was extracted from the fluorescence spectrum of molybdenum disulfide under the influence of the known bases. - Spectral characteristic parameters, calculate A - The proportion of the spectrum in the fluorescence spectrum of molybdenum disulfide under the influence of the known bases;

[0017] A was extracted from the fluorescence spectrum of molybdenum disulfide under the influence of the unknown base. - Spectral characteristic parameters, calculate A -The proportion of the spectrum in the fluorescence spectrum of molybdenum disulfide under the influence of the unknown base;

[0018] Comparison and analysis of A - The proportion of molybdenum disulfide under the influence of known bases and the proportion of molybdenum disulfide under the influence of unknown bases are used to determine which known base the unknown base is based on the degree of similarity between the two.

[0019] A is calculated by measuring the ratio of the areas formed by the curves. - The proportion of the spectrum in the total spectrum.

[0020] The known different bases include four bases: A, C, G, and T.

[0021] The molybdenum disulfide film is a molybdenum disulfide film grown on silicon dioxide.

[0022] In steps S1 and S3, 1-10 μl of DNA solution is dropped onto a molybdenum disulfide film, allowed to stand for 3-10 minutes, rinsed, and then detected using a fluorescence spectrometer.

[0023] After waiting 3-10 minutes, rinse the molybdenum disulfide film with ultrapure water.

[0024] In steps S1 and S3, the concentration of the DNA solution shall not exceed 100 ng / μl.

[0025] The present invention has the following beneficial effects:

[0026] This invention provides a method for identifying bases. A DNA test device is prepared by dropping a DNA solution onto a molybdenum disulfide (MoS2) thin film. A fluorescence spectrometer is used to detect the fluorescence spectrum data of MoS2 under the influence of bases. A feature matching analysis is performed between the fluorescence spectrum curves of MoS2 under the influence of unknown bases and those under the influence of known bases. Based on the matching analysis results, the unknown base is identified as a known base. This invention utilizes the physical adsorption and charge transfer mechanism of DNA and MoS2 to effectively identify bases. It also has low requirements for the concentration of the DNA solution used for testing and leverages the low cost and simple preparation of MoS2. Compared with existing technologies, this invention has advantages such as effective identification of low-concentration trace amounts of DNA bases, simple device structure, simple detection process, and low cost.

[0027] Other beneficial effects of the embodiments of the present invention will be further described below. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the device used in an embodiment of the present invention.

[0029] Figure 2 The fluorescence spectrum of molybdenum disulfide is shown in an embodiment of the present invention.

[0030] Figure 3 A is an embodiment of the present invention. - Percentage of the spectrum. Detailed Implementation

[0031] The embodiments of the present invention will be described in detail below. It should be emphasized that the following description is merely exemplary and not intended to limit the scope and application of the present invention.

[0032] This invention provides a method for identifying bases, comprising the following steps:

[0033] S1. A DNA testing device is prepared by dropping a DNA solution containing known different bases onto a molybdenum disulfide film, as shown in the figure. Figure 1 As shown, the device includes DNA1 and a molybdenum disulfide film 2, preferably formed on silicon dioxide 3; fluorescence spectroscopy data of molybdenum disulfide under the influence of known bases are detected using a fluorescence spectrometer;

[0034] S2. Perform curve fitting analysis on the fluorescence spectrum data of molybdenum disulfide under the influence of known bases to obtain the fluorescence spectrum curve of molybdenum disulfide under the influence of known bases.

[0035] S3. The DNA solution containing unknown bases is dropped onto a molybdenum disulfide film to prepare the target DNA testing device, the structure of which is as follows: Figure 1 As shown, the fluorescence spectrum of molybdenum disulfide in the target DNA testing device was detected using a fluorescence spectrometer;

[0036] S4. Perform curve fitting analysis on the fluorescence spectrum data of molybdenum disulfide obtained in step S3 to obtain the fluorescence spectrum curve of molybdenum disulfide under the influence of the unknown base.

[0037] S5. Based on the fluorescence spectrum curve of molybdenum disulfide under the influence of the unknown base, a feature matching degree comparison analysis is performed with the fluorescence spectrum curve of molybdenum disulfide under the influence of the known base. Based on the matching degree comparison analysis results, it is determined which known base the unknown base is.

[0038] Molybdenum disulfide (MoD) itself produces fluorescence upon photoexcitation. Electrons gaining light energy transition to a high-energy state; due to instability, they return to a low-energy state, emitting photons in the process – the basic process of fluorescence generation. Different bases have different chemical properties and structures, thus affecting the band structure and charge transfer of MoD, including influencing energy levels and electronic transitions, and quenching or enhancing fluorescence. These effects are reflected in the fluorescence spectrum of MoD; therefore, by analyzing the fluorescence spectrum, the influence of different bases on MoD can be determined. This invention, through analysis and calculation of fluorescence spectra, derives the influence of different bases on MoD and utilizes these principles to identify different bases.

[0039] Curve fitting analysis is a mathematical method used to analyze and fit experimental data, thereby deriving the data distribution patterns and fitted curves. In fluorescence spectroscopy, curve fitting analysis can be used to analyze the shape, peaks, and spectral distribution of fluorescence spectra, thus determining the quantitative relationship between the fluorescence spectrum and the analyte. In the fluorescence spectroscopy analysis of molybdenum disulfide, curve fitting analysis can be used to analyze the shape and peaks of the fluorescence spectrum, thereby determining the fluorescence emission properties and spectral distribution characteristics of molybdenum disulfide. The fitted curves can reflect the details and changes in the fluorescence spectrum, thus accurately identifying the influence of different bases on molybdenum disulfide. Furthermore, curve fitting analysis can also be used to calculate the proportion and degree of influence of different bases on molybdenum disulfide, thereby deriving the influence patterns of different bases on molybdenum disulfide. These patterns can be used for further analysis and identification, for example, to identify different bases.

[0040] The method for identifying bases provided in this invention involves dropping a DNA solution onto a molybdenum disulfide (MoS2) thin film to prepare a DNA testing device. A fluorescence spectrometer is used to detect the fluorescence spectrum data of MoS2 under the influence of bases. A feature matching analysis is performed between the fluorescence spectrum curves of MoS2 under the influence of unknown bases and those under the influence of known bases. Based on the matching analysis results, the unknown base is determined to be a known base. This method utilizes the physical adsorption and charge transfer mechanism of DNA and MoS2 to effectively identify bases. Furthermore, it has low requirements for the concentration of the DNA solution used for testing and fully leverages the advantages of low cost and simple preparation of MoS2. Compared with existing technologies, this invention has advantages such as effective identification of low-concentration trace amounts of DNA bases, simple device structure, simple detection process, and low cost.

[0041] In a preferred embodiment, the features of the comparative analysis based on the fitted fluorescence spectrum curve of molybdenum disulfide include the position and shape of the fluorescence peaks.

[0042] In a preferred embodiment, in steps S2 and S4, B, A, and A are obtained by peak fitting of the fluorescence spectrum. - In step S5, the fluorescence spectra of molybdenum disulfide under the influence of unknown bases are compared and analyzed with those of molybdenum disulfide under the influence of known bases, specifically peak types B, A, and A. - Three peak types were used to determine which fluorescence spectrum of molybdenum disulfide under the influence of the unknown base best matched the fluorescence spectrum of molybdenum disulfide under the influence of the known base, thereby identifying the corresponding base.

[0043] Fluorescence spectral data of molybdenum disulfide affected by unknown bases were collected and curve-fitted. Peak fitting of the fluorescence spectra yielded the values ​​of B, A, and A. - Three peak shapes were identified, and the fitted fluorescence spectrum was then compared and analyzed with fluorescence spectrum curves showing the effects of different bases. Fluorescence spectrum curves showing the effects of known bases were obtained based on the fluorescence spectral characteristics of molybdenum disulfide with known specific base effects. These characteristic parameters directly correspond to certain features of the curve, such as the position, shape, and intensity of the fluorescence peaks. The comparison and analysis of the B, A, and A peak shapes of the fluorescence spectrum curves were then performed. - The three peak types can determine which curve best matches the collected molybdenum disulfide fluorescence spectral data affected by unknown bases, thereby identifying the corresponding bases.

[0044] In a preferred embodiment, step S5 includes:

[0045] A was extracted from the fluorescence spectrum of molybdenum disulfide under the influence of the known bases. - Spectral characteristic parameters, calculate A - The proportion of the spectrum in the fluorescence spectrum of molybdenum disulfide under the influence of the known bases;

[0046] A was extracted from the fluorescence spectrum of molybdenum disulfide under the influence of the unknown base. - Spectral characteristic parameters, calculate A - The proportion of the spectrum in the fluorescence spectrum of molybdenum disulfide under the influence of the unknown base;

[0047] Comparison and analysis of A - The proportion of molybdenum disulfide under the influence of known bases and the proportion of molybdenum disulfide under the influence of unknown bases are used to determine which known base the unknown base is based on the degree of similarity between the two.

[0048] In a preferred embodiment, A is calculated by measuring the ratio of the areas formed by the curves. - The proportion of the spectrum in the total spectrum.

[0049] For A- Calculating the proportion of the spectrum in the total spectrum can better reveal the influence of different bases on molybdenum disulfide. A - It is a bound state composed of electrons and holes, and its spectral characteristics are related to the energy level structure and electronic transport properties of molybdenum disulfide. In the process of identifying bases using curve fitting analysis, A... - Calculating the proportions of the spectrum helps to better identify the effects of different bases on molybdenum disulfide. Specifically, the electron cloud density and distribution of different bases affect the energy level structure, electronic transport properties, and charge transfer of molybdenum disulfide, which further affects A. - The characteristics of the spectrum and its proportion in the total spectrum. By calculating the proportion of trion exciton spectra, the differences in the influence of different bases on molybdenum disulfide can be revealed, thus providing a basis for base identification and providing discriminative information for identifying bases using curve fitting analysis.

[0050] In a preferred embodiment, the known different bases include four bases: A, C, G, and T.

[0051] See Figure 1 In a preferred embodiment, the monolayer molybdenum disulfide film 2 is a molybdenum disulfide film grown on silicon dioxide 3. The monolayer molybdenum disulfide film 2 is preferably triangular with a side length greater than 10 μm, ensuring that water cannot wash away the molybdenum disulfide film.

[0052] In a preferred embodiment, in steps S1 and S3, the DNA solution is dropped onto a molybdenum disulfide film, with a DNA volume of 1–10 μl. After waiting for 3–30 minutes, the solution is rinsed and then detected using a fluorescence spectrometer.

[0053] In a preferred embodiment, after waiting 3-30 minutes, the molybdenum disulfide film is rinsed with ultrapure water.

[0054] In a preferred embodiment, the concentration of the DNA solution in steps S1 and S3 does not exceed 100 ng / μl. For example, it can be 50 ng / μl, 60 ng / μl, 70 ng / μl, 80 ng / μl, 90 ng / μl, or 100 μl.

[0055] The method of this invention for base identification has lower requirements for the concentration of the DNA solution to be tested. It can effectively identify trace amounts of DNA bases using a solution concentration of no more than 100 ng / μl, which has significant advantages over existing technologies.

[0056] The following describes specific embodiments of the present invention.

[0057] The apparatus used in this invention mainly includes a DNA testing device, which comprises a DNA solution to be tested (a solution with different bases A, G, T, and C) and a monolayer molybdenum disulfide film grown on silica. The DNA solution consists of a DNA sample and pure water, with a concentration of 50-100 ng / µl. The DNA solution to be tested is mixed with ultrapure water to avoid the influence of ionic impurities on the experimental results. Then, the DNA solution is dropped onto the molybdenum disulfide film, and after waiting for 3-10 minutes, it is rinsed with ultrapure water, thus preparing the DNA testing device.

[0058] The prepared DNA testing device was tested using a fluorescence spectrometer to measure the fluorescence spectrum of molybdenum disulfide, such as... Figure 2 As shown. B, A, and A can be obtained by fitting the fluorescence spectrum peaks. - Three peak shapes are observed, each corresponding to a different electronic transition process in molybdenum disulfide, influenced by spin orbitals and triexcitons. Calculations are performed for different base A peaks. - The proportions of the spectra reveal corresponding patterns. Since different bases affect the band structure and charge transfer of molybdenum disulfide, curve fitting analysis of the obtained fluorescence spectra can determine the exciton peaks of molybdenum disulfide under different bases. For example... Figure 3 As shown, A is calculated by fitting the obtained spectrum. - The proportion of C bases in the total spectrum reveals the following pattern: C bases have the greatest impact on molybdenum disulfide, followed by A bases, with T bases having the smallest impact. A higher proportion indicates that more negatively charged DNA electrons are transferred to molybdenum disulfide. Since T bases contain electrons from their double oxygen bonds, this leads to a greater impact on A bases. - It has minor variations. Figure 3 In the text, PW (pure water) indicates pure water, which is used as a solvent in DNA solutions.

[0059] like Figure 2 As shown, the fluorescence spectra of different bases bound to molybdenum disulfide and the fitted results are presented. The three fitted peaks, from left to right, represent B, A, and A, respectively. - Peaks for the three excitons. The DNA-MoS2 fluorescence spectrum changes to varying degrees under different base pairs, especially when fitting the spectral curves. The rightmost curve in the fitted curve represents A. - Fitting peak, Figure 3 By calculating the proportion of this peak in the total spectrum, different bases show a linear relationship, so different bases can be distinguished based on the numerical changes. Figure 2 By constructing A corresponding to different bases - Using the pattern of percentage changes as a reference sample, the determination of bases in unknown samples can be achieved.

[0060] The background section of this invention may include background information about the problems or environment in which the invention is being developed, and is not necessarily a description of prior art. Therefore, the content included in the background section does not constitute an admission of prior art by the applicant.

[0061] The above description provides a further detailed explanation of the present invention in conjunction with specific / preferred embodiments, and it should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various substitutions or modifications can be made to these described embodiments without departing from the concept of the present invention, and all such substitutions or modifications should be considered within the scope of protection of the present invention. In the description of this specification, the reference to terms such as "an embodiment," "some embodiments," "preferred embodiment," "example," "specific example," or "some examples," etc., indicates that the specific features, structures, materials, or characteristics described in connection with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in any suitable manner in one or more embodiments or examples. Without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification and the features of different embodiments or examples. Although the embodiments of the present invention and their advantages have been described in detail, it should be understood that various changes, substitutions, and modifications can be made herein without departing from the scope of protection of the patent application.

Claims

1. A method for identifying bases, characterized in that, Includes the following steps: S1. A DNA test device is prepared by dropping a DNA solution containing known different bases onto a molybdenum disulfide film. The fluorescence spectrum data of molybdenum disulfide under the influence of known bases is detected by a fluorescence spectrometer. S2. Perform curve fitting analysis on the fluorescence spectrum data of molybdenum disulfide under the influence of known bases to obtain the fluorescence spectrum curve of molybdenum disulfide under the influence of known bases. S3. Drop the DNA solution containing unknown bases onto a molybdenum disulfide film to prepare a target DNA testing device. Use a fluorescence spectrometer to detect the fluorescence spectrum of the molybdenum disulfide in the target DNA testing device. S4. Perform curve fitting analysis on the fluorescence spectrum data of molybdenum disulfide obtained in step S3 to obtain the fluorescence spectrum curve of molybdenum disulfide under the influence of the unknown base. S5. Based on the fluorescence spectrum curve of molybdenum disulfide under the influence of the unknown base, a feature matching degree comparison analysis is performed with the fluorescence spectrum curve of molybdenum disulfide under the influence of the known base. Based on the matching degree comparison analysis results, it is determined which known base the unknown base is.

2. The method for identifying bases as described in claim 1, characterized in that, The comparative analysis based on the fluorescence spectrum curves of molybdenum disulfide obtained by fitting includes the position and shape of the fluorescence peaks.

3. The method for identifying bases as described in claim 2, characterized in that, In steps S2 and S4, peak fitting of the fluorescence spectrum yields B, A, and A. - In step S5, the fluorescence spectra of molybdenum disulfide under the influence of unknown bases are compared and analyzed with those of molybdenum disulfide under the influence of known bases, specifically peak types B, A, and A. - Three peak types were used to determine which fluorescence spectrum of molybdenum disulfide under the influence of the unknown base best matched the fluorescence spectrum of molybdenum disulfide under the influence of the known base, thereby identifying the corresponding base.

4. The method for identifying bases as described in any one of claims 1 to 3, characterized in that, Step S5 includes: A was extracted from the fluorescence spectrum of molybdenum disulfide under the influence of the known bases. - Spectral characteristic parameters, calculate A - The proportion of the spectrum in the fluorescence spectrum of molybdenum disulfide under the influence of the known bases; A was extracted from the fluorescence spectrum of molybdenum disulfide under the influence of the unknown base. - Spectral characteristic parameters, calculate A - The proportion of the spectrum in the fluorescence spectrum of molybdenum disulfide under the influence of the unknown base; Comparison and analysis of A - The proportion of molybdenum disulfide under the influence of known bases and the proportion of molybdenum disulfide under the influence of unknown bases are used to determine which known base the unknown base is based on the degree of similarity between the two.

5. The method for identifying bases as described in claim 4, characterized in that, A is calculated by measuring the ratio of the areas formed by the curves. - The proportion of the spectrum in the total spectrum.

6. The method for identifying bases as described in any one of claims 1 to 5, characterized in that, The known different bases include four bases: A, C, G, and T.

7. The method for identifying bases as described in any one of claims 1 to 6, characterized in that, The molybdenum disulfide film is a single-layer molybdenum disulfide film grown on silicon dioxide.

8. The method for identifying bases as described in any one of claims 1 to 7, characterized in that, In steps S1 and S3, 1-10 μl of DNA solution is dropped onto a molybdenum disulfide film, allowed to stand for 3-30 minutes, rinsed, and then detected using a fluorescence spectrometer.

9. The method for identifying bases as described in claim 8, characterized in that, After waiting 3-30 minutes, rinse the molybdenum disulfide film with ultrapure water.

10. The method for identifying bases as described in any one of claims 1 to 9, characterized in that, In steps S1 and S3, the concentration of the DNA solution shall not exceed 100 ng / μl.

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