Base identification method based on semiconductor SERS (Surface Enhanced Raman Scattering) technology

Abstract

The invention discloses a base recognition method based on a semiconductor SERS (Surface Enhanced Raman Scattering) technology, and belongs to the technical field of biomedical detection. The specific method comprises the following steps: 1, synthesizing TiO2 nanoparticles; step 2, preparation of a TiO2 / 4-MBA system; and step three, identifying and detecting basic groups. According to the present invention, the scheme of DNA base identification by using the TiO2 / MBA system can achieve accurate and rapid DNA base identification, and the developed TiO2 / MBA system has characteristics of simple preparation, excellent base test performance, and high market application prospect. The invention not only provides a novel basic group detection means, but also has higher sensitivity and selectivity, can identify and detect four basic groups and non-covalent interaction between the four basic groups and 4-MBA in two pH environments, and has a simple, convenient and rapid operation process. The invention not only provides a new method for base analysis, but also provides technical support for early diagnosis of DNA-related diseases, genomics research and development of molecular sensors, and has wide application prospects.

Description

Technical Field

[0001] The present invention relates to a method for base recognition, in particular to a method for base recognition based on semiconductor SERS technology, belonging to the technical field of biomedical detection. Background Art

[0002] Ribonucleic acid molecules are composed of a monosaccharide (deoxyribose), a phosphate group, and four nitrogenous nucleobases, namely adenine (A), cytosine (C), guanine (G), and thymine (T). Exploring the laws and internal mechanisms of the interactions between different nucleic acid bases and probe molecules is an important scientific issue in base molecular recognition. Molecular recognition is mainly achieved by non-covalent interactions that bind two or more molecules and analyze the binding sites. The applications of non-covalent interactions in the fields of molecular recognition, drug design, nanomaterials, catalytic reactions, etc. are becoming increasingly widespread. These interactions include weak intermolecular forces such as hydrogen bonds, van der Waals forces, electrostatic interactions, and hydrophobic interactions. Through reasonable molecular design and optimization, these interactions can have a significant impact on molecular recognition, catalytic activity, and material functionalization. Through efficient and accurate DNA base recognition, the detection of gene mutations, early diagnosis of genetic diseases, and formulation of personalized treatment plans can be achieved. Traditional DNA base recognition methods such as polymerase chain reaction (PCR), gene chips, and DNA probe technology have achieved remarkable results in molecular diagnosis. However, these methods are often limited by problems such as complex operation, high cost, and long detection time. Therefore, developing a base recognition method with high efficiency, speed, low cost, and high sensitivity has become one of the current research hotspots.

[0003] In recent years, due to its characteristics of high sensitivity, high selectivity, and non-destructiveness, surface-enhanced Raman scattering (SERS) technology has become an important tool in the field of DNA detection. In SERS detection, the surface plasmon resonance effect and charge transfer chemical enhancement can greatly enhance the molecular signal, thus achieving high-sensitivity detection of base molecules. Especially in the research using semiconductor materials as the enhancement substrate, titanium dioxide (TiO 2 ) materials have become candidate materials that have received much attention due to their good chemical stability, non-toxicity, and adjustable surface properties. By combining appropriate molecular ligands, such as 4-mercaptobenzoic acid (4-MBA), the SERS signal can be significantly enhanced, and the recognition ability for base molecules can be improved.

[0004] Developing a method for studying the internal interaction mechanism between various molecules and nucleobases and its recognition method based on semiconductor SERS technology is of great significance to chemical biology and medicinal chemistry. Summary of the Invention

[0005] In order to solve the problems of low efficiency, slow speed, high cost, low sensitivity and low selectivity of existing base recognition methods, the present invention further provides a base recognition method based on semiconductor SERS technology.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] A base recognition method based on semiconductor SERS technology, the base recognition method is realized through the following steps:

[0008] S1: Synthesis of TiO 2 nanoparticles; Take analytical grade anhydrous ethanol and place it in a container. Add mercaptoacetic acid with a mass fraction of 99% thereto. After stirring at room temperature, add analytical grade tetrabutyl titanate to the container. Immediately after adding, evacuate the container to a vacuum state and continue stirring at room temperature. Then add deionized water to the reaction solution and continue stirring at normal temperature. Collect the reaction product by centrifugation, wash it with anhydrous ethanol multiple times to remove impurities, and finally dry it to obtain TiO 2 nanoparticles;

[0009] S2: Preparation of the TiO 2 / 4-MBA system; Disperse the TiO 2 nanoparticles prepared in S1 in an ethanol solution of 4-mercaptobenzoic acid with a concentration of 10 -3 M to obtain the TiO 2 / 4-MBA sample and then stir. Subsequently, rinse with ethanol to remove the free 4-MBA molecules on the surface of the TiO 2 particles to obtain a purified TiO 2 / 4-MBA system;

[0010] S3: Base recognition detection; Disperse the purified TiO 2 / 4-MBA system in an ethanol solution, then take the dispersed sample and drop it into the lid of a centrifuge tube. After the ethanol evaporates, form a TiO 2 / 4-MBA substrate. Then, take a solution with a pH of 2 without a base, a solution with a pH of 10 without a base, a base solution with a concentration of 10 -5 M and a pH of 2, and a base solution with a concentration of 10 - 5 M and a pH of 10 and drop them into the lid of the centrifuge tube respectively. After soaking, measure the SERS spectrum and analyze the changes in the characteristic peaks of the SERS spectrum to achieve the recognition of base molecules.

[0011] Further, the dosages of each substance in S1 are in the following proportions: 0.5 mL of analytical grade tetrabutyl titanate; 10 μL of mercaptoacetic acid with a mass fraction of 99%; 50 mL of analytical grade anhydrous ethanol; 1 mL of deionized water.

[0012] Further, the stirring times in S1 are 20 minutes, 6 hours, and 6 hours respectively.

[0013] Further, in S2, the proportion of TiO 2 nanoparticles dispersed in the ethanol solution of 4-mercaptobenzoic acid is that 1 mg of TiO 2 particles are dispersed in 1 mL of the ethanol solution of 4-MBA with a concentration of 10 -3 M.

[0014] Further, the stirring time in S2 is 2 hours.

[0015] Further, in S2, before the TiO 2 nanoparticles are dispersed in the ethanol solution of 4-mercaptobenzoic acid with a concentration of 10 -3 M, the TiO 2 nanoparticles are ground sufficiently.

[0016] Further, the ratio of the usage amount of the dispersed sample to the usage amount of different base solutions in S3 is 1:3.

[0017] Further, the specific method of dispersing the purified TiO 2 / 4-MBA system in the ethanol solution is to take TiO 2 powder and disperse it into the ethanol solution of 4-MBA and then mix evenly. The ratio of TiO 2 powder to the ethanol solution of 4-MBA is that 1 mg of TiO 2 powder is dispersed into 1 mL of the ethanol solution of 4-MBA.

[0018] Further, the soaking time in S3 is 2 hours.

[0019] Further, the data acquisition time of the SERS spectrum measurement in S3 is 10 s for each accumulation, and the laser power is 30 mW.

[0020] The beneficial effects of the present invention are as follows:

[0021] 1. The present invention develops a base recognition method based on semiconductor SERS technology. This method is based on the SERS detection of the TiO 2 / MBA composite system and can efficiently distinguish four typical bases in DNA: adenine, thymine, cytosine, and guanine. Under different pH conditions, the protonation or deprotonation state of DNA bases will affect TiO 2The interaction between the surface and 4-mercaptobenzoic acid has a significant impact, leading to changes in the SERS spectrum. By analyzing these spectral differences, accurate identification of the bases and their non-covalent interactions with 4-MBA can be achieved.

[0022] 2. The semiconductor SERS active substrate of the present invention can directly detect and identify base solutions without other pretreatment, with simple and efficient operation.

[0023] 3. The substrate TiO 2 / 4-MBA sensing system used in the present invention not only has high SERS activity but also has interface charge transfer sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the scanning electron micrograph of the TiO 2 nanoparticles of the present invention;

[0025] Figure 2 is the transmission image of the TiO 2 nanoparticles of the present invention;

[0026] Figure 3 is the SERS spectra of TiO 2 / 4-MBA and TiO 2 / 4-MBA / four bases obtained under pH 2 conditions of the present invention;

[0027] Figure 4 is the SERS spectra of TiO 2 / 4-MBA and TiO 2 / 4-MBA / four bases obtained under pH 10 conditions of the present invention;

[0028] Figure 5 is Figure 3 the partial enlarged view of the peak change at 1074 cm -1 ;

[0029] Figure 6 is Figure 3 the partial enlarged view of the peak change at 1593 cm -1 ;

[0030] Figure 7 is Figure 4 the partial enlarged view of the peak change at 1074 cm -1 ;

[0031] Figure 8 is Figure 4 the partial enlarged view of the peak change at 1593 cm -1 ;

[0032] Figure 9This is the SERS spectrum of the recognition of four bases by TiO 2 / 4-MBA under the condition of pH 2;

[0033] Figure 10 This is the SERS spectrum of the recognition of four bases by TiO 2 / 4-MBA under the condition of pH 10. Detailed implementation manners

[0034] Detailed implementation manner 1: Combining Figure 1-10 to illustrate this implementation manner. The base recognition method based on semiconductor SERS technology described in this implementation manner is realized through the following steps:

[0035] S1: Synthesis of TiO 2 nanoparticles; Take analytical grade anhydrous ethanol and place it in a container. Add mercaptoacetic acid with a mass fraction of 99% to it. After stirring at room temperature, add analytical grade tetrabutyl titanate to the container. Immediately after adding, evacuate the container to a vacuum state and continue stirring at room temperature. Then add deionized water to the reaction solution and continue stirring at normal temperature. Collect the reaction product by centrifugation, wash it with anhydrous ethanol multiple times to remove impurities, and finally dry to obtain TiO 2 nanoparticles. The dosage of each substance is carried out according to the following ratio: 0.5 mL of analytical grade tetrabutyl titanate; 10 μL of mercaptoacetic acid with a mass fraction of 99%; 50 mL of analytical grade anhydrous ethanol; 1 mL of deionized water. Preferably, the stirring times are 20 minutes, 6 hours, and 6 hours respectively, and the container is preferably a three-necked flask. As Figure 1 and Figure 2 shown, the scanning electron microscope image and transmission image of the synthesized TiO 2 nanoparticles.

[0036] S2: Preparation of TiO 2 / 4-MBA system; Disperse the TiO 2 nanoparticles prepared in S1 in an ethanol solution of 4-mercaptobenzoic acid with a concentration of 10 -3 M to obtain TiO 2 / 4-MBA sample and then stir. Preferably, before dispersing the TiO 2 particles in an ethanol solution of 4-MBA with a concentration of 10-3 M, the TiO 2 particle powder needs to be ground sufficiently and then taken and dispersed in the ethanol solution of 4-MBA. If it is not ground sufficiently, it will lead to uneven signals during SERS testing and poor repeatability. The ratio of TiO 2 nanoparticles dispersed in the ethanol solution of 4-mercaptobenzoic acid is 1 mg of TiO 2 particles dispersed in 1 mL of an ethanol solution with a concentration of 10-3 In an ethanol solution of 4-MBA of M, the stirring time was 2 hours. Ensure that 4-MBA molecules are effectively adsorbed on TiO 2 surface, so that 4-MBA molecules are fully adsorbed on TiO 2 nanoparticle surface. Subsequently, rinse with ethanol to remove the free 4-MBA molecules on the surface of TiO 2 particles, and obtain a purified TiO 2 / 4-MBA system.

[0037] S3: Base recognition detection; Disperse the purified TiO 2 / 4-MBA system in S2 in an ethanol solution. The specific method is to take TiO 2 powder and disperse it into the ethanol solution of 4-MBA and then mix evenly. The ratio of TiO 2 powder to the ethanol solution of 4-MBA is 1 mg TiO 2 powder dispersed into 1 mL of the ethanol solution of 4-MBA. Then take the dispersed sample and drop it into the centrifuge tube cap (containing a silicon wafer with a diameter of 0.6 cm). After the ethanol evaporates, a TiO 2 / 4-MBA substrate is formed. Then, take a solution with a pH of 2 without a base, a solution with a pH of 10 without a base, a base solution with a concentration of 10 -5 M and a pH of 2, and a base solution with a concentration of 10 -5 M and a pH of 10 and drop them into the centrifuge tube cap, so that the solution is in full contact with the TiO 2 / 4-MBA sample on the silicon wafer. The ratio of the usage amount of the dispersed sample to the usage amount of different base solutions is 1:3. After soaking, measure the SERS spectrum. Preferably, the soaking time is 2 hours, and measure the SERS spectrum of the sample on the silicon wafer. That is, soak the TiO2 / 4-MBA substrate in different base solutions for 2 h and then perform SERS testing. The data acquisition time for the SERS spectrum measurement is 10 s for each accumulation, and the laser power is 30 mW. Analyze the changes in the characteristic peaks of the SERS spectrum to achieve the recognition of base molecules.

[0038] The present invention proposes a base recognition method based on semiconductor SERS technology, for TiO 2SERS detection of the MBA composite system, which can efficiently distinguish four typical bases in DNA: adenine (A), thymine (T), cytosine (C), and guanine (G). Under different pH conditions, the protonation or deprotonation state of DNA bases will significantly affect the interaction between the TiO2 surface and 4-mercaptobenzoic acid (4-MBA), resulting in changes in the SERS spectrum. By analyzing these spectral differences, precise recognition of the bases and their non-covalent interactions with 4-MBA can be achieved.

[0039] This method not only provides a novel means of base detection but also has high sensitivity and selectivity. It can identify and detect four bases and their non-covalent interactions with 4-MBA under two pH environments, and has a simple and rapid operation process.

[0040] Example

[0041] A base recognition method based on semiconductor SERS technology, which is realized through the following steps:

[0042] S1: Synthesis of TiO 2 Nanoparticles

[0043] Take 50 mL of anhydrous ethanol in a three-necked flask, add 10 μL of mercaptoacetic acid, stir at room temperature for 20 min, then add 500 μL of tetrabutyl titanate to the three-necked flask. Immediately after adding, evacuate the three-necked flask to vacuum, stir at room temperature for 6 hours, add a certain amount of deionized water to the reaction solution system, stir at room temperature for 6 hours, wash with anhydrous ethanol several times after centrifugation, and dry to obtain TiO 2 Nanoparticles. Figure 1 For the SEM image of TiO 2 Nanoparticles, it can be seen that the material is uniformly prepared and has a good morphology; Figure 2 For the TEM image of TiO 2 Nanoparticles, the image further reveals that TiO 2 Nanospheres are assembled by small nanocrystals in an orderly manner.

[0044] S2: Preparation of the TiO 2 / 4-MBA system

[0045] After grinding the TiO 2 powder sufficiently, take 1 mg of TiO 2 particles and disperse them in 1 mL of an ethanol solution of 4-MBA with a concentration of 10 -3 M. Stir the obtained TiO 2 / 4-MBA sample for 2 h, and then rinse with ethanol to remove the free 4-MBA molecules on the surface of the TiO 2 particles. After purification, the TiO2 The 1 / 4-MBA was redispersed in 1 mL of ethanol solution, and then 20 μL of the dispersed sample was dropped onto a silicon wafer with a diameter of 0.6 cm. After the ethanol evaporated, SERS spectra were measured on it.

[0046] S3: Base recognition detection

[0047] In the TiO prepared in the above S2 2 The 1 / 4-MBA was dispersed in 1 mL of ethanol solution, and 1 mg of TiO 2 powder was dispersed in 1 mL of ethanol solution of 1 / 4-MBA, mixed evenly with a vortex mixer, and then 20 μL of the dispersed sample was dropped into the centrifuge tube lid (containing a silicon wafer with a diameter of 0.6 cm). After the ethanol evaporated, 60 μL of pH 2 and pH 10 solutions without bases and different base solutions with a concentration of 10 - 5 M and pH values of 2 and 10 were respectively dropped into the centrifuge tube lid, and after soaking for two hours, SERS spectra were measured on it.

[0048] The SERS spectra were obtained by a confocal micro-Raman spectrometer of the RTS2-301-DL model with an excitation wavelength of 532 nm. A 50× objective lens was used, the data acquisition time was 10 s, and the laser power was 30 mW. For each analyte, at least 3 SERS imaging experiments were performed to ensure signal reproducibility. All SERS spectra were obtained by averaging at least 10 individual spectra of each Raman image, and a single-crystal silicon wafer was used for system calibration (Raman band is 520.7 cm -1 ) before testing each sample.

[0049] As Figure 3 and Figure 4 shown, the SERS spectra of TiO 2 / 1 / 4-MBA and TiO 2 / 1 / 4-MBA / 4 bases under pH 2 and pH 10 conditions are respectively shown. All nucleobases are protonated at pH 2, and these nucleobases interact with 1 / 4-MBA through a combination of hydrogen bonding and ion-dipole interactions. After introducing the four bases in an environment with a pH value of 2, the characteristic peaks of 1 / 4-MBA show a shift to lower wavenumbers at 1074 and 1593 cm -1 ; in an environment with a pH value of 10, the nucleobases are deprotonated at pH 10. When the four bases are added separately, the 1 / 4-MBA peaks at 1074 and 1593 cm -1 shift to higher wavenumbers, which can be attributed to the ionic interaction between COO - and NH 3+ . As Figure 5 and Figure 6As shown, they are respectively the enlarged local views of the peak changes of the SERS spectra obtained from TiO 2 / 4-MBA and TiO 2 / 4-MBA / 4 bases at 1074 cm -1 and 1593 cm -1 ; the peak at 1074 cm -1 , and the peak at 1593 cm -1 are indexed as the ring-breathing β(C=C) and symmetric stretching ν(C=C) modes. The peak at 1074 cm -1 shifts to 1069 cm -1 , and the peak at 1593 cm -1 shifts to 1588 cm -1 . As Figure 7 and Figure 8 shown, they are respectively the enlarged local views of the peak changes of the SERS spectra obtained from TiO 2 / 4-MBA and TiO 2 / 4-MBA / 4 bases at 1074 cm -1 and 1593 cm -1 ; the peak at 1074 cm -1 shifts to 1077 cm -1 , and the peak at 1593 cm -1 shifts to 1597 cm -1 . As Figure 9 and Figure 10 shown, they are respectively the SERS spectra of TiO 2 / 4-MBA / 4 bases recognition under pH 2 and pH 10 conditions; the stacked SERS spectra shown further indicate that the four bases can also be recognized by the peaks near 1300 and 1400 cm -1 .

[0050] This method specifically involves the development of a detection system based on semiconductor surface-enhanced Raman scattering technology, which is a SERS detection system for base recognition. Using the TiO 2 / MBA system for DNA base recognition can achieve accurate and rapid DNA base recognition. The developed TiO 2 / MBA system is simple to prepare, has excellent performance for base testing, and has high market application prospects. Moreover, it has high sensitivity and selectivity, can detect DNA bases at low concentrations, and has a simple and rapid operation process.

[0051] The above are only the preferred embodiments of the present invention, and do not impose any form of limitation on the present invention. Although the present invention has been disclosed above with the preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some changes or modifications to equivalent embodiments of equivalent changes within the scope of the technical solution of the present invention by using the above-disclosed technical content. However, as long as it does not depart from the technical solution content of the present invention and is based on the technical essence of the present invention, any simple modification, equivalent replacement, and improvement made to the above embodiments still fall within the protection scope of the technical solution of the present invention.

Claims

1. A base recognition method based on semiconductor SERS technology, characterized in that: The base recognition method is achieved by the following steps: S1: Synthesis of TiO2 nanoparticles; analytical grade anhydrous ethanol is placed in a container, 99% by mass of thioglycolic acid is added thereto, and after stirring at room temperature, analytical grade tetrabutyl titanate is added to the container, and immediately after the addition, the container is evacuated to a vacuum state, and stirring is continued at room temperature, and then deionized water is added to the reaction solution, and stirring is continued at room temperature, and the reaction product is collected by centrifugation, washed with anhydrous ethanol for multiple times to remove impurities, and finally dried to obtain TiO2 nanoparticles; S2: Preparation of TiO2 / 4-MBA system; The TiO2 nanoparticles prepared in S1 were dispersed in a 10 -3 The TiO2 / 4-MBA sample was obtained from an ethanol solution of 4-mercaptobenzoic acid at 400 M and then stirred, and then rinsed with ethanol to remove the free 4-MBA molecules on the surface of TiO2 particles to obtain a purified TiO2 / 4-MBA system; S3: Base recognition detection; the purified TiO2 / 4-MBA system in S2 was dispersed in an ethanol solution, and then the dispersed sample was dripped into the centrifuge tube cap. After the ethanol evaporated, a TiO2 / 4-MBA substrate was formed. Then, a solution with a pH value of 2 without base, a solution with a pH value of 10 without base, and a solution with a concentration of 10 were respectively taken. -5 M, a base solution with a pH of 2 and a concentration of 10 -5 M, a base solution with a pH value of 10 is dripped into the centrifuge tube cap, and the SERS spectrum is measured after soaking, and the changes in the characteristic peaks of the SERS spectrum are analyzed to achieve the identification of the base molecules.

2. The base recognition method based on semiconductor SERS technology according to claim 1, characterized in that: The amounts of the substances in S1 are in the following proportions: 0.5 mL of analytical grade tetrabutyl titanate; 10 μL of 99% by mass thioglycolic acid; 50 mL of analytical grade anhydrous ethanol; and 1 mL of deionized water.

3. The base recognition method based on semiconductor SERS technology according to claim 2, characterized in that: The stirring times in S1 were 30 minutes, 6 hours and 6 hours respectively.

4. The base recognition method based on semiconductor SERS technology according to claim 1, characterized in that: The ratio of TiO2 nanoparticles dispersed in ethanol solution of 4-mercaptobenzoic acid in S2 is 1 mg of TiO2 particles dispersed in 1 mL of 10 -3 M of 4-MBA in ethanol.

5. The base recognition method based on semiconductor SERS technology according to claim 4, characterized in that: The stirring time in S2 was 2 hours.

6. The base recognition method based on semiconductor SERS technology according to claim 5, characterized in that: TiO2 nanoparticles are dispersed in S2 at a concentration of 10 -3 The TiO2 nanoparticles were thoroughly ground before being added to a 100 M 4-mercaptobenzoic acid ethanol solution.

7. The base recognition method based on semiconductor SERS technology according to claim 1, characterized in that: The ratio of the amount of the dispersed sample used in S3 to the amount of the different base solutions used is 1:

3.

8. The base recognition method based on semiconductor SERS technology according to claim 7, characterized in that: The specific method of dispersing the purified TiO2 / 4-MBA system in the ethanol solution described in S3 is to disperse the TiO2 powder into the ethanol solution of 4-MBA and mix them evenly. The ratio of TiO2 powder to the ethanol solution of 4-MBA is 1 mg TiO2 powder dispersed in 1 mL 4-MBA ethanol solution.

9. The base recognition method based on semiconductor SERS technology according to claim 8, characterized in that: The soaking time in S3 is 2 hours.

10. The base recognition method based on semiconductor SERS technology according to claim 9, characterized in that: The data collection time for the SERS spectroscopy measurement described in S3 was 10 s for 1 accumulation, and the laser power was 30 mW.

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