A base recognition method based on semiconductor SERS technology

Abstract

The application discloses a base recognition method based on semiconductor SERS technology and belongs to the technical field of biomedical detection. The specific method comprises the following steps: step one, synthesis of TiO2 nanoparticles; step two, preparation of a TiO2 / 4-MBA system; and step three, base recognition detection. The TiO2 / MBA system can be used for accurate and rapid DNA base recognition, and the developed TiO2 / MBA system is simple to prepare and has excellent base test performance, and has a high market application prospect. The application not only provides a novel base detection method, but also has high sensitivity and selectivity, can recognize and detect four bases and the non-covalent interaction between the four bases and 4-MBA in two pH environments, and has a simple and rapid operation process. The application 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 a wide application prospect.

Description

Technical Field

[0001] This invention relates to a base identification method, specifically a base identification method based on semiconductor SERS technology, belonging to the field of biomedical detection technology. Background Technology

[0002] RNA molecules consist of a monosaccharide (deoxyribose), a phosphate group, and four nitrogenous nucleotide bases: adenine (A), cytosine (C), guanine (G), and thymine (T). Exploring the patterns and underlying mechanisms of interactions between different nucleic acid bases and probe molecules is a crucial scientific question in base molecular recognition. Molecular recognition is primarily achieved through non-covalent interactions that bind two or more molecules and analyze the binding sites. Non-covalent interactions are increasingly widely used in molecular recognition, drug design, nanomaterials, and catalytic reactions. These interactions include weak forces such as hydrogen bonds, van der Waals forces, electrostatic interactions, and hydrophobic interactions. Through rational molecular design and optimization, these interactions can significantly impact molecular recognition, catalytic activity, and material functionalization. Efficient and accurate DNA base recognition enables the detection of gene mutations, early diagnosis of genetic diseases, and the development of personalized treatment plans. Traditional DNA base recognition methods, such as polymerase chain reaction (PCR), gene chips, and DNA probe technology, have achieved significant results in molecular diagnostics. However, these methods are often limited by operational complexity, high cost, and long detection times. Therefore, developing an efficient, fast, low-cost, and highly sensitive base recognition method has become one of the current research hotspots.

[0003] In recent years, surface-enhanced Raman scattering (SERS) technology has become an important tool in the field of DNA detection due to its high sensitivity, high selectivity, and non-destructive nature. In SERS detection, surface plasmon resonance and charge-transfer chemical enhancement can significantly amplify molecular signals, thereby achieving highly sensitive detection of base molecules. Particularly in research utilizing semiconductor materials as enhancement substrates, titanium dioxide (TiO2) has become a highly anticipated candidate material due to its excellent chemical stability, non-toxicity, and tunable surface properties. By combining suitable molecular ligands, such as 4-mercaptobenzoic acid (4-MBA), the SERS signal can be significantly enhanced, and the recognition ability of base molecules can be improved.

[0004] Developing a semiconductor SERS-based method for studying the intrinsic interaction mechanisms between various molecules and nucleobases and their identification 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 and low sensitivity and selectivity of existing base identification methods, this invention proposes a base identification method based on semiconductor SERS technology.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A base identification method based on semiconductor SERS technology, wherein the base identification method is implemented through the following steps:

[0008] S1: Synthesis of TiO2 nanoparticles; Analytical grade anhydrous ethanol was placed in a container, and 99% mercaptoacetic acid was added to it. After stirring at room temperature, analytical grade tetrabutyl titanate was added to the container. Immediately after addition, the container was evacuated to a vacuum state, and stirring was continued at room temperature. Then, deionized water was added to the reaction solution, and stirring was continued at room temperature. The reaction product was collected by centrifugation, washed multiple times with anhydrous ethanol to remove impurities, and finally dried to obtain TiO2 nanoparticles.

[0009] S2: Preparation of the TiO2 / 4-MBA system; the TiO2 nanoparticles obtained in S1 were dispersed in a solution with a concentration of 10... -3 After obtaining the TiO2 / 4-MBA sample from the ethanol solution of M's 4-mercaptobenzoic acid, the mixture was stirred and then washed with ethanol to remove free 4-MBA molecules on the surface of TiO2 particles, resulting in a purified TiO2 / 4-MBA system.

[0010] S3: Base Recognition Detection; The purified TiO2 / 4-MBA system from S2 was dispersed in an ethanol solution. The dispersed sample was then dropped into a centrifuge tube cap. After the ethanol evaporated, a TiO2 / 4-MBA substrate was formed. Subsequently, solutions with a pH of 2 (no bases), a pH of 10 (no bases), and a concentration of 10 were taken respectively. -5 M, a basic solution with a pH of 2, and a concentration of 10 - 5 A base solution with pH 10 is added dropwise to the centrifuge tube cap. After soaking, the SERS spectrum is measured, and the changes in the characteristic peaks of the SERS spectrum are analyzed to identify the base molecules.

[0011] Furthermore, the amounts of each substance in S1 are as follows: 0.5 mL of analytical grade tetrabutyl titanate; 10 μL of 99% mercaptoacetic acid; 50 mL of analytical grade anhydrous ethanol; and 1 mL of deionized water.

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

[0013] Furthermore, in S2, the proportion of TiO2 nanoparticles dispersed in an ethanol solution of 4-mercaptobenzoic acid was 1 mg of TiO2 particles dispersed in 1 mL of a 10% concentration. -3 M in an ethanol solution of 4-MBA.

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

[0015] Furthermore, the TiO2 nanoparticles in S2 are dispersed in a solution with a concentration of 10 -3 TiO2 nanoparticles were thoroughly ground in an ethanol solution of 4-mercaptobenzoic acid.

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

[0017] Furthermore, the specific method for dispersing the purified TiO2 / 4-MBA system in an ethanol solution as described in S3 is to take TiO2 powder, disperse it in an ethanol solution of 4-MBA, and then mix it evenly. The ratio of TiO2 powder to 4-MBA ethanol solution is 1 mg TiO2 powder dispersed in 1 mL 4-MBA ethanol solution.

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

[0019] Furthermore, the data acquisition time for the SERS spectral measurement described in S3 is 10 seconds per measurement, and the laser power is 30mW.

[0020] The beneficial effects of this invention are:

[0021] 1. This invention develops a base recognition method based on semiconductor SERS technology. This method, based on SERS detection of a TiO2 / MBA composite system, can efficiently distinguish four typical DNA bases: adenine, thymine, cytosine, and guanine. Under different pH conditions, the protonated or deprotonated state of DNA bases significantly affects the interaction between the TiO2 surface and 4-mercaptobenzoic acid, thus leading to changes in the SERS spectrum. By analyzing these spectral differences, accurate identification of bases and their non-covalent interactions with 4-MBA can be achieved.

[0022] 2. The semiconductor SERS active substrate of this invention can directly detect and identify basic solutions without the need for other pretreatment, making the operation simple and efficient.

[0023] 3. The TiO2 / 4-MBA sensing system used in this invention has both high SERS activity and interfacial charge transfer sensitivity. Attached Figure Description

[0024] Figure 1 This is a scanning electron microscope image of the TiO2 nanoparticles of the present invention;

[0025] Figure 2 This is a transmission image of the TiO2 nanoparticles of the present invention;

[0026] Figure 3 This is the SERS spectrum obtained by the present invention under pH 2 conditions for TiO2 / 4-MBA and TiO2 / 4-MBA / four bases;

[0027] Figure 4 This is the SERS spectrum obtained by the present invention under pH 10 conditions for TiO2 / 4-MBA and TiO2 / 4-MBA / four bases;

[0028] Figure 5 yes Figure 3 At 1074cm -1 A magnified view of the peak value variation;

[0029] Figure 6 yes Figure 3 At 1593cm -1 A magnified view of the peak value variation;

[0030] Figure 7 yes Figure 4 At 1074cm -1 A magnified view of the peak value variation;

[0031] Figure 8 yes Figure 4 At 1593cm -1 A magnified view of the peak value variation;

[0032] Figure 9 This is the SERS spectrum of TiO2 / 4-MBA / four base recognition under pH 2 conditions according to the present invention;

[0033] Figure 10 This is the SERS spectrum of TiO2 / 4-MBA / four base recognition under pH 10 conditions according to the present invention. Detailed Implementation

[0034] Specific implementation method one: Combining Figure 1-10 This embodiment describes a base identification method based on semiconductor SERS technology, which is implemented through the following steps:

[0035] S1: Synthesis of TiO2 nanoparticles; Analytical grade anhydrous ethanol was placed in a container, and 99% mercaptoacetic acid was added. After stirring at room temperature, analytical grade tetrabutyl titanate was added to the container. Immediately after addition, the container was evacuated to a vacuum state, and stirring continued at room temperature. Then, deionized water was added to the reaction solution, and stirring continued at room temperature. The reaction product was collected by centrifugation, washed multiple times with anhydrous ethanol to remove impurities, and finally dried to obtain TiO2 nanoparticles. The amounts of each substance were as follows: 0.5 mL analytical grade tetrabutyl titanate; 10 μL 99% mercaptoacetic acid; 50 mL analytical grade anhydrous ethanol; 1 mL deionized water. Preferably, the stirring times for the three stages were 20 minutes, 6 hours, and 6 hours, respectively, and the container was preferably a three-hole flask. Figure 1 and Figure 2 The image shown is a scanning electron microscope (SEM) image and a transmission electron microscope (TEM) image of the synthesized TiO2 nanoparticles.

[0036] S2: Preparation of the TiO2 / 4-MBA system; the TiO2 nanoparticles obtained in S1 were dispersed in a solution with a concentration of 10... -3 After obtaining the TiO2 / 4-MBA sample in an ethanol solution of 4-mercaptobenzoic acid (M), the mixture is stirred. Preferably, before dispersing the TiO2 particles in the 10⁻³ M 4-MBA ethanol solution, the TiO2 particle powder needs to be thoroughly ground before being dispersed in the 4-MBA ethanol solution. Insufficient grinding will result in uneven signal and poor repeatability during SERS testing. The ratio of TiO2 nanoparticles dispersed in the 4-mercaptobenzoic acid ethanol solution is 1 mg of TiO2 particles dispersed in 1 mL of a 10⁻³ M 4-MBA ethanol solution. -3 The 4-MBA solution of M was stirred for 2 hours to ensure effective adsorption of 4-MBA molecules onto the TiO2 surface, allowing for full adsorption of 4-MBA molecules onto the TiO2 nanoparticle surface. Subsequently, the TiO2 nanoparticles were rinsed with ethanol to remove free 4-MBA molecules from the TiO2 particle surface, yielding a purified TiO2 / 4-MBA system.

[0037] S3: Base Recognition Detection; The purified TiO2 / 4-MBA system from S2 was dispersed in an ethanol solution. Specifically, TiO2 powder was dispersed into an ethanol solution of 4-MBA and mixed thoroughly. The ratio of TiO2 powder to 4-MBA ethanol solution was 1 mg TiO2 powder to 1 mL 4-MBA ethanol solution. The dispersed sample was then dropped into a centrifuge tube cap (containing a silicon wafer with a diameter of 0.6 cm). After the ethanol evaporated, a TiO2 / 4-MBA substrate was formed. Subsequently, solutions with a pH of 2 (no bases), a pH of 10 (no bases), and a concentration of 10 were taken respectively. -5 M, a basic solution with a pH of 2, and a concentration of 10 -5A base solution with pH 10 (M) is added dropwise to the centrifuge tube cap to ensure thorough contact between the solution and the TiO2 / 4-MBA sample on the silicon wafer. The ratio of the dispersed sample volume to the different base solutions used is 1:3. SERS spectroscopy is measured after immersion, preferably for 2 hours. Specifically, the TiO2 / 4-MBA substrate is immersed in different base solutions for 2 hours before SERS testing. Data acquisition for SERS spectroscopy is performed at 10-second intervals, with a laser power of 30mW. Changes in the characteristic peaks of the SERS spectrum are analyzed to identify the base molecules.

[0038] This invention proposes a base recognition method based on semiconductor SERS technology, specifically a TiO2 / MBA composite system for SERS detection. This method can efficiently distinguish four typical DNA bases: adenine (A), thymine (T), cytosine (C), and guanine (G). Under different pH conditions, the protonated or deprotonated state of DNA bases significantly affects the interaction between the TiO2 surface and 4-mercaptobenzoic acid (4-MBA), leading to changes in the SERS spectrum. By analyzing these spectral differences, accurate identification of 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 procedure.

[0040] Example

[0041] A base identification method based on semiconductor SERS technology, wherein the base identification method is implemented through the following steps:

[0042] S1: Synthesis of TiO2 nanoparticles

[0043] Take 50 mL of anhydrous ethanol into a three-hole flask, add 10 μL of mercaptoacetic acid, stir at room temperature for 20 min, then add 500 μL of tetrabutyl titanate to the three-hole flask. Immediately after adding, evacuate the three-hole flask to a vacuum. After stirring at room temperature for 6 hours, add a certain amount of deionized water to the reaction system, stir at room temperature for 6 hours, centrifuge, wash several times with anhydrous ethanol, and dry to obtain TiO2 nanoparticles. Figure 1 The image shows a scanning electron microscope (SEM) image of TiO2 nanoparticles, which indicates that the material is uniformly prepared and has a good morphology. Figure 2 The image shows a transmission electron microscope (TEM) image of TiO2 nanoparticles, which further reveals that TiO2 nanospheres are composed of ordered small nanocrystals.

[0044] S2: Preparation of the TiO2 / 4-MBA system

[0045] After thoroughly grinding the TiO2 powder, take 1 mg of TiO2 particles and disperse them in 1 mL of a 10% concentration solution. -3 The obtained TiO2 / 4-MBA sample was stirred for 2 hours in an ethanol solution of 4-MBA in M, and then rinsed with ethanol to remove free 4-MBA molecules from the surface of TiO2 particles. The purified TiO2 / 4-MBA was then redispersed in 1 mL of ethanol solution, and 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.

[0046] S3: Base Recognition and Detection

[0047] The TiO2 / 4-MBA prepared in S2 above was dispersed in 1 mL of ethanol solution. 1 mg of TiO2 powder was dispersed in 1 mL of 4-MBA ethanol solution and mixed thoroughly using a vortex mixer. Then, 20 μL of the dispersed sample was dropped into a centrifuge tube cap (containing a silicon wafer with a diameter of 0.6 cm). After the ethanol evaporated, 60 μL of base-free pH 2 and pH 10 solutions, and a 10% concentration solution were respectively taken. - 5 M, different base solutions with pH values ​​of 2 and 10 were added to the centrifuge tube cap, and after soaking for two hours, their SERS spectra were measured.

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

[0049] like Figure 3 and Figure 4 The figures show the SERS spectra of TiO2 / 4-MBA and TiO2 / 4-MBA / 4 bases obtained under pH 2 and pH 10 conditions, respectively. All nucleotide bases are protonated at pH 2. These nucleobases interact with 4-MBA through a combination of hydrogen bonds and ionic dipole interactions. After introducing four bases at pH 2, the characteristic peaks of 4-MBA are at 1074 and 1593 cm⁻¹. -1 The wavenumbers shifted to lower values; in an environment with a pH of 10, the nucleotide bases were deprotonated at pH 10, and when the four bases were added respectively, the wavenumbers shifted to lower values ​​at 1074 and 1593 cm⁻¹.-1 The 4-MBA peak shifts to higher wavenumbers, which can be attributed to COO. - and NH 3+ The interactions between ions. For example... Figure 5 and Figure 6 The figures shown are the SERS spectra of TiO2 / 4-MBA and TiO2 / 4-MBA / 4 bases obtained under pH 2 conditions at 1074 cm⁻¹. -1 and 1593cm -1 A magnified view of the peak value variation at 1074cm. -1 1593cm -1 The index is for circumferential respiration β (C=C), symmetrical stretching ν (C=C) pattern 1074cm -1 Offset to 1069cm -1 Location: 1593cm -1 Offset to 1588cm -1 Place. For example Figure 7 and Figure 8 The figures show the SERS spectra of TiO2 / 4-MBA and TiO2 / 4-MBA / 4 bases at pH 10, respectively, at 1074 cm⁻¹. -1 and 1593cm -1 A magnified view of the peak value variation at 1074cm. -1 Offset to 1077cm -1 Location: 1593cm -1 Offset to 1597cm -1 Place. For example Figure 9 and Figure 10 The figures show the SERS spectra of TiO2 / 4-MBA / 4-base recognition under pH 2 and pH 10 conditions, respectively; the stacked SERS spectra shown further indicate that at 1300 and 1400 cm⁻¹, the SERS spectrum is significantly different. -1 The nearby peaks can also identify four bases.

[0050] This method specifically involves developing a detection system based on semiconductor surface-enhanced Raman scattering (SERS) technology, specifically a SERS detection system for base identification. The scheme utilizing the TiO2 / MBA system for DNA base identification achieves accurate and rapid DNA base recognition. The developed TiO2 / MBA system is simple to prepare, exhibits excellent base detection performance, and has high market application prospects. Furthermore, it possesses high sensitivity and selectivity, enabling the detection of DNA bases at low concentrations, and features a simple and rapid operating procedure.

[0051] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent substitutions, and improvements made to the above embodiments without departing from the scope of the present invention, based on the technical essence of the present invention and within the spirit and principles of the present invention, shall still fall within the protection scope of the present invention.

Claims

1. A base recognition method based on semiconductor SERS technology, characterized in that: The base recognition method is implemented through the following steps: S1: Synthesis of TiO2 nanoparticles; Analytical grade anhydrous ethanol was placed in a container, and 99% mercaptoacetic acid was added to it. After stirring at room temperature, analytical grade tetrabutyl titanate was added to the container. Immediately after addition, the container was evacuated to a vacuum state, and stirring was continued at room temperature. Then, deionized water was added to the reaction solution, and stirring was continued at room temperature. The reaction product was collected by centrifugation, washed multiple times with anhydrous ethanol to remove impurities, and finally dried to obtain TiO2 nanoparticles. S2: Preparation of the TiO2 / 4-MBA system; the TiO2 nanoparticles obtained in S1 were dispersed in a solution with a concentration of 10... -3 After obtaining the TiO2 / 4-MBA sample from the ethanol solution of M's 4-mercaptobenzoic acid, the mixture was stirred and then washed with ethanol to remove free 4-MBA molecules from the surface of the TiO2 particles, thus obtaining the purified TiO2 / 4-MBA system. S3: Base recognition detection; The purified TiO2 / 4-MBA system from S2 was dispersed in an ethanol solution. The dispersed sample was then dropped into a centrifuge tube cap. After the ethanol evaporated, a TiO2 / 4-MBA substrate was formed. Subsequently, solutions with a pH of 2 (no bases), a pH of 10 (no bases), and a concentration of 10 were taken respectively. -5 M, a basic solution with a pH of 2 and a concentration of 10 -5 M, a base solution with a pH of 10, is added to the centrifuge tube cap. After soaking, SERS spectra are measured. Under different pH conditions, the protonated or deprotonated state of DNA bases will significantly affect the interaction between the TiO2 surface and 4-MBA, thus causing changes in the SERS spectrum. By analyzing the changes in the characteristic peaks of the SERS spectrum, the base molecules can be identified.

2. The base recognition method based on semiconductor SERS technology according to claim 1, characterized in that: The amounts of each substance in S1 are as follows: 0.5 mL of analytical grade tetrabutyl titanate; 10 μL of 99% mercaptoacetic 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 20 minutes, 6 hours, and 6 hours, respectively.

4. The base recognition method based on semiconductor SERS technology according to claim 1, characterized in that: In S2, the proportion of TiO2 nanoparticles dispersed in an ethanol solution of 4-mercaptobenzoic acid was 1 mg of TiO2 particles dispersed in 1 mL of a 10% concentration. -3 M in an ethanol solution of 4-MBA.

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

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

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

3.

8. The base recognition method based on semiconductor SERS technology according to claim 7, characterized in that: The specific method for dispersing the purified TiO2 / 4-MBA system in an ethanol solution as described in S3 is to take TiO2 powder, disperse it in an ethanol solution of 4-MBA, and mix it evenly. The ratio of TiO2 powder to 4-MBA ethanol solution 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 SERS spectral measurement described in S3 uses a data acquisition time of 10 seconds for one acquisition, and a laser power of 30mW.

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