A SERS substrate for amplifying and enhancing low-frequency Raman signals of biomass molecules

Abstract

The present invention relates to the field of spectroscopy and molecular recognition technology, and more specifically to a method based on SERS (surface enhanced Raman scattering spectroscopy) technology. The present invention provides a SERS substrate for detecting a variety of biomass molecules, using a chain polymer as a soft template to form a "head-to-head" localized self-assembly structure of metal single atoms on the carrier surface. This structure not only promotes the mutual proximity of metal single atoms, but also physically blocks the aggregation of metal atoms into cluster structures by passivating the carrier surface. The charge redistribution between neighboring metal single atoms regulates the electronic state of the entire semiconductor substrate surface, thereby inducing efficient photoinduced charge transfer, thereby amplifying the low-frequency Raman signal of the biomass molecules.

Description

Technical Field

[0001] The present invention relates to the field of spectroscopy and molecular recognition technology, and more specifically to a method based on SERS (surface enhanced Raman scattering spectroscopy) technology, in which single-atom tungsten-modified titanate nanotubes are prepared as SERS substrates to amplify and enhance the low-frequency Raman signals of various biomass molecules (glucose, uric acid, dopamine, triglycerides and phenylalanine) to achieve selective testing. Background Art

[0002] Biomass molecules (such as carbohydrates, lipids, amino acids, and neurotransmitters) are core substances that sustain human life. Their types, concentrations, and metabolic dynamics are directly related to human health. For example, high blood sugar levels indicate diabetes, elevated uric acid levels are a precursor to gout, abnormal dopamine levels can induce psychological problems such as depression, elevated triglycerides increase the risk of hyperlipidemia, and abnormal phenylalanine levels indicate intellectual disability. Therefore, achieving precise detection and sensing of these biomass molecules has profound implications for medicine, health management, and biological research.

[0003] Currently, the main detection methods for biomass molecules include enzyme-linked immunosorbent assay (ELISA), infrared spectroscopy, liquid chromatography, and tandem mass spectrometry. GB 5009.124-2016 uses chromatographic column separation of phenylalanine and ultraviolet (UV) detection for quantitative analysis. This method consumes a large amount of reagent per test, has poor accuracy, and incurs high instrument maintenance costs.

[0004] Surface enhanced Raman spectroscopy (SERS) is an ultra-sensitive and rapid surface analysis technique. The Raman signal of biomass molecules is in the low frequency part (usually the wave number range < 200cm -1 ) is more obvious, so low-frequency Raman signal detection has unique advantages in biomass molecular detection. By capturing intermolecular interactions and large-scale vibration information, it makes up for the shortcomings of traditional detection methods in specificity, dynamic monitoring and label-free analysis, especially showing unique potential in complex biological systems, real-time physiological process research and precision medicine.

[0005] However, existing commercial SERS substrates are mostly based on nanostructures of precious metal materials such as gold and silver. Currently available SERS-based methods for detecting biomass molecules typically use gold sol as a substrate. Due to the indiscriminate amplification of the Raman signals of the substrate components by the electromagnetic enhancement effect of the precious metal substrate, there is a strong interference signal when testing the low-frequency portion. In addition, precious metal SERS detection often requires a complex pre-treatment process, facing problems such as long time consumption, high cost, poor selectivity, and poor stability, which has limitations in practical application. Therefore, it is of great significance to develop a method that is fast, efficient, simple to operate, low-cost, and can selectively and effectively identify the low-frequency Raman signals of biomass molecules. Summary of the Invention

[0006] The main purpose of the present invention is to provide a highly localized aggregation of single-atom tungsten modified titanate nanotubes for use in amplifying and enhancing the low-frequency Raman signals of various biomass molecules (glucose, uric acid, dopamine, triglycerides and phenylalanine), thereby achieving selective detection and overcoming the shortcomings of the existing technology.

[0007] The present invention provides a SERS substrate for amplifying and enhancing low-frequency Raman signals of biomass molecules. The SERS substrate is a titanate nanotube modified with single-atom tungsten.

[0008] The present invention also provides a method for preparing a SERS substrate for amplifying and enhancing low-frequency Raman signals of biomass molecules, which comprises the following steps:

[0009] Step (1): reacting a mixed reaction system comprising anatase titanium dioxide powder, alkali solution and water at 120-160° C. for 10-30 hours to obtain a titanate precipitate;

[0010] Step (2): acidifying the titanate precipitate with nitric acid for at least 24 hours to obtain titanate nanotubes;

[0011] Step (3): In a sodium tungstate solution, tungstate ions are captured by the end of a polyethylene glycol soft template to form a tungsten-containing precursor, which is then mixed with the titanate nanotubes and heated and stirred. A large number of metal single-atom "head-to-head" localized self-assembly structures are formed on the surface of the titanate nanotube carrier by utilizing the hydrogen bonding between the soft template and the titanate nanotubes, thereby obtaining a highly locally aggregated metal single-atom SERS substrate.

[0012] Preferably, in step (1), the alkali solution is sodium hydroxide, and the mass ratio of the titanium dioxide powder, sodium hydroxide and water is 3.6:10-30:40-60.

[0013] Preferably, before step (2), the titanate precipitate is washed with deionized water to a pH of 7; after step (2), the titanate nanotubes are washed with deionized water to a pH of 7.

[0014] Preferably, in step (3), the molecular weight of the polyethylene glycol is 500-1000, and the molar ratio of the sodium tungstate to the polyethylene glycol is 1-2:1-2.

[0015] Preferably, in step (3), the tungsten-containing precursor needs to be adjusted to pH 4.5-5.5 using acid, and then stirred continuously for 2-20 hours in a light-proof environment.

[0016] Preferably, in step (3), the titanate nanotubes are first added to an ethylene glycol / water mixed solvent to form a dispersion, and then the tungsten-containing precursor is added to the dispersion to form a mixture, and the mixture is stirred, centrifuged, washed, dried and then calcined to obtain the highly localized metal single atom SERS substrate.

[0017] Preferably, the volume ratio of ethylene glycol to water in the mixed solvent is 1:5-20, the ratio of titanate nanotubes to the mixed solvent is 1g:100-300ml; and the mass ratio of titanate nanotubes to tungsten-containing precursor in the mixture is 1:10-20.

[0018] Preferably, the stirring is carried out at 60-80° C. for 2-6 hours; and the calcination is carried out at 180-250° C. in an argon atmosphere for 4-8 hours.

[0019] The SERS substrate prepared by the present invention can be used in the selective enhancement of low-frequency Raman signals of biomass molecules, and the biomass molecular sieve is one of glucose, uric acid, dopamine, triglyceride and phenylalanine.

[0020] Compared with the existing technology, the advantages of the present invention are:

[0021] (1) The present invention provides a method for preparing a SERS substrate for detecting various biomass molecules. A chain polymer is used as a soft template to form a localized self-assembled structure of metal single atoms in a head-to-head manner on the carrier surface. This structure not only promotes the proximity of metal single atoms, but also physically blocks the aggregation of metal atoms into cluster structures by passivating the carrier surface. The charge redistribution between neighboring metal single atoms regulates the electronic state of the entire semiconductor substrate surface, thereby inducing efficient photoinduced charge transfer, thereby amplifying the low-frequency Raman signal of biomass molecules.

[0022] (2) The preparation process of the SERS active substrate material for biomass molecule detection in the present invention is simple, low-cost, easy to operate, fast and effective; most importantly, it can realize selective low-frequency SERS detection of various types of biomass molecules (glucose, uric acid, dopamine, triglycerides and phenylalanine), which can reveal large-scale vibration information in the molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a scanning transmission electron micrograph of highly localized aggregated single-atom tungsten modified titanate nanotubes in Example 1;

[0024] Figure 2 This is a high-angle annular dark-field scanning transmission electron microscopy image of highly localized aggregated single-atom tungsten-modified titanate nanotubes in Example 1;

[0025] Figure 3 1 is a comparison diagram of the enhanced Raman spectrum of glucose obtained by using the SERS substrate prepared in Example 1 in Example 2;

[0026] Figure 4 This is a comparison diagram of the enhanced Raman spectrum of uric acid obtained by using the SERS substrate prepared in Example 1 in Example 3;

[0027] Figure 5 4 is a comparison diagram of the enhanced Raman spectrum of dopamine produced by the SERS substrate prepared in Example 1;

[0028] Figure 6 This is a comparison diagram of the enhanced Raman spectrum of triglyceride obtained by the SERS substrate prepared in Example 1 in Example 5;

[0029] Figure 7 This is a comparison diagram of the enhanced Raman spectrum of phenylalanine obtained by using the SERS substrate prepared in Example 1 in Example 6;

[0030] Figure 8 1 is a comparison diagram of the enhanced Raman spectra of histamine and norepinephrine obtained by using the SERS substrate prepared in Example 1 in Example 7. DETAILED DESCRIPTION

[0031] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0032] Unless otherwise specified, the experimental materials used in the examples can be purchased from conventional biochemical reagent companies.

[0033] Example 1

[0034] A method for preparing a single-atom SERS substrate for highly localized aggregation comprises the following steps:

[0035] 3.6 g of titanium dioxide powder was mixed with 50 ml of 10 M sodium hydroxide solution. The mixture was then hydrothermally treated at 150°C for 20 hours in a 100 ml Teflon-lined autoclave. After cooling to room temperature, the titanate precipitate was washed with deionized water to a pH of 7. Finally, the solution was acidified with 0.1 M nitric acid for over 24 hours under continuous stirring and then thoroughly washed with deionized water to a pH of 7, yielding titanate nanotubes. 1.3 mmol of sodium tungstate dihydrate and 1.3 mmol of PEG (molecular weight 1000) were dissolved in 15 ml of deionized water. After stirring at 20°C for one hour, 0.1 M sulfuric acid was added dropwise to the mixture until the pH reached 5. Deionized water was added to bring the solution volume to 20 ml, and the solution was stirred in the dark for 6 hours to yield the single-atom precursor. 0.1g of titanate nanotubes was dispersed in 20ml of ethylene glycol / water (1:10 by volume) solution, and 1.5ml of the single-atom precursor was then added dropwise to the dispersion. The mixture was sealed, stirred continuously at 75°C for 4 hours, stored at room temperature for 12 hours, and then centrifuged to obtain the product. The product was washed with deionized water and then ethanol until the pH value reached approximately 7, then dried in a vacuum oven at 60°C and calcined under argon at 200°C for 6 hours to obtain highly localized, aggregated single-atom tungsten-modified titanate nanotubes.

[0036] like Figure 1 As shown in the scanning transmission electron microscope image, the morphology of the nanotubes remains unchanged after modification with single atomic tungsten, and there are no metal clusters on the surface. Figure 2 As shown, high-angle annular dark field-scanning transmission electron microscopy can show that single atomic tungsten presents a highly localized distribution form, and the red circle in the figure is a typical local aggregation state.

[0037] Example 2

[0038] SERS test on glucose:

[0039] Prepare 800 μl of a 10 -4 M glucose aqueous solution and 200 microliters of 0.4 mg / ml single-atom tungsten modified titanate nanotube dispersion, then mixed the two and left them in the dark for 3 hours to allow the glucose molecules to reach adsorption-desorption equilibrium with the substrate. Finally, 20 microliters of the mixed suspension was dropped onto a clean silicon wafer and dried at 60 ° C for 2 hours. In order to show the SERS effect, 20 microliters of 10 -4 A 100 M glucose aqueous solution was dropped onto a clean silicon wafer and dried at 60°C for 2 hours. The Raman test conditions were fixed, with a laser spot diameter of 1 μm, a 50 × L objective lens, a collection time of 30 seconds, and a laser intensity of 5%. The Raman spectrum comparison is shown in the figure below. Figure 3 shown.

[0040] Example 3

[0041] SERS test on uric acid:

[0042] Prepare 800 μl of a 10 -4 M uric acid aqueous solution and 200 microliters of 0.6 mg / ml single-atom tungsten modified titanate nanotube dispersion, then mixed the two and let it stand in the dark for 3 hours to allow the adsorption-desorption equilibrium between the uric acid molecules and the substrate to be reached. Finally, 20 microliters of the mixed suspension was dropped onto a clean silicon wafer and dried at 60 ° C for 2 hours. In order to show the SERS effect, 20 microliters of 10 -4 A 100-μm uric acid aqueous solution was dropped onto a clean silicon wafer and dried at 60°C for 2 hours. The Raman test conditions were fixed: a laser spot diameter of 1 μm, a 50 × L objective lens, a collection time of 10 seconds, and a laser intensity of 1%. The Raman spectra are shown in the figure below. Figure 4 shown.

[0043] Example 4

[0044] SERS test on dopamine:

[0045] Prepare 800 μl of a 10 -4 M dopamine aqueous solution and 200 microliters of 0.8 mg / ml single-atom tungsten modified titanate nanotube dispersion, then mixed the two and let it stand in the dark for 3 hours to allow the adsorption-desorption equilibrium between the dopamine molecules and the substrate to be reached. Finally, 20 microliters of the mixed suspension was dropped onto a clean silicon wafer and dried at 60 ° C for 2 hours. In order to show the SERS effect, 20 microliters of 10 -4 A 100 M dopamine aqueous solution was dropped onto a clean silicon wafer and dried at 60°C for 2 hours. The Raman test conditions were fixed, with a laser spot diameter of 1 μm, a 50 × L objective lens, a collection time of 20 seconds, and a laser intensity of 1%. The Raman spectra are shown in the figure below. Figure 5 shown.

[0046] Example 5

[0047] SERS test on triglycerides:

[0048] Prepare 800 μl of a 10 -4M triglyceride ethanol solution and 200 microliters of single-atom tungsten modified titanate nanotube dispersion with a concentration of 0.8 mg / ml, then mixed the two and let it stand in the dark for 3 hours to allow the adsorption-desorption equilibrium between the triglyceride molecules and the substrate to be reached. Finally, 20 microliters of the mixed suspension was dropped onto a clean silicon wafer and dried at 60 ° C for 2 hours. In order to show the SERS effect, 20 microliters of 10 -4 A 100-M triglyceride ethanol solution was dropped onto a clean silicon wafer and dried at 60°C for 2 hours. Raman test conditions were fixed: a laser spot diameter of 1 μm, a 50 × L objective lens, a collection time of 50 seconds, and a laser intensity of 5%. The Raman spectrum comparison is shown in the figure below. Figure 6 shown.

[0049] Example 6

[0050] SERS test of phenylalanine:

[0051] Prepare 800 μl of a 10 -4 M phenylalanine aqueous solution and 200 microliters of 1 mg / ml single-atom tungsten modified titanate nanotube dispersion were mixed and left to stand in the dark for 3 hours to allow the adsorption-desorption equilibrium between the phenylalanine molecules and the substrate to be reached. Finally, 20 microliters of the mixed suspension was dropped onto a clean silicon wafer and dried at 60 ° C for 2 hours. In order to show the SERS effect, 20 microliters of 10 -4 A 100 M phenylalanine aqueous solution was dropped onto a clean silicon wafer and dried at 60°C for 2 hours. The Raman test conditions were fixed: a laser spot diameter of 1 μm, a 50 × L objective lens, a collection time of 20 seconds, and a laser intensity of 5%. The Raman spectra are shown in the figure below. Figure 7 shown.

[0052] Example 7

[0053] SERS testing of histamine and norepinephrine:

[0054] Prepare 800 μl of a 10 -4 M histamine aqueous solution and 200 μl of 0.5 mg / ml single atom tungsten modified titanate nanotube dispersion, then mix the two and let them stand in the dark for 3 hours to allow the histamine molecules to reach adsorption-desorption equilibrium with the substrate. -4M norepinephrine aqueous solution and 200 microliters of 0.5mg / ml single-atom tungsten modified titanate nanotube dispersion, then mixed the two and let it stand in the dark for 3 hours to allow the adsorption-desorption equilibrium between the norepinephrine molecules and the substrate to be reached. Finally, 20 microliters of the mixed suspension was dropped onto a clean silicon wafer and dried at 60 ° C for 2 hours. In order to show the SERS effect, 20 microliters of 10-4M histamine aqueous solution and 20 microliters of 10-4M histamine aqueous solution were mixed. -4 The norepinephrine aqueous solution of 100 M was dropped onto a clean silicon wafer and dried at 60 °C for 2 hours. The Raman test conditions were fixed, with a laser spot diameter of 1 μm, a 50 × L objective lens, a collection time of 30 seconds, and a laser intensity of 10%. The Raman spectrum comparison is shown in the figure below. Figure 8 shown.

[0055] Combine Figure 3-8 As can be seen, the highly localized, aggregated single-atom tungsten-modified titanate nanotube SERS prepared in Example 1 amplified the low-frequency Raman signals of various biomass molecules (glucose, uric acid, dopamine, triglycerides, and phenylalanine), exhibiting some molecular-selective enhancement. However, no significant SERS enhancement was observed for histamine and norepinephrine, also biomass molecules.

[0056] Should be understood that, the order of each step or the order in which specific action is performed is not very important, as long as the present invention teachings remain operable.In addition, two or more steps or actions can be performed simultaneously.

Claims

1. A SERS substrate for amplifying and enhancing the low-frequency Raman signal of biomass molecules, characterized in that The SERS substrate is a titanate nanotube modified with single-atom tungsten.

2. A method for preparing a SERS substrate for amplifying and enhancing low-frequency Raman signals of biomass molecules, characterized in that: The steps include: Step (1): reacting a mixed reaction system comprising anatase titanium dioxide powder, alkali solution and water at 120-160° C. for 10-30 hours to obtain a titanate precipitate; Step (2): acidifying the titanate precipitate with nitric acid for at least 24 hours to obtain titanate nanotubes; Step (3): In a sodium tungstate solution, a tungstate ion is captured by the end of a polyethylene glycol soft template to form a tungsten-containing precursor, which is then mixed with the titanate nanotubes and heated and stirred. By utilizing the hydrogen bonding between the soft template and the titanate nanotubes, a large number of metal single-atom "head-to-head" localized self-assembly structures are formed on the surface of the titanate nanotube carrier, thereby obtaining a highly locally aggregated metal single-atom SERS substrate.

3. The preparation method according to claim 2, characterized in that In step (1), the alkali solution is sodium hydroxide, and the mass ratio of the titanium dioxide powder, sodium hydroxide and water is 3.6:10-30:40-60.

4. The preparation method according to claim 2, characterized in that Before step (2), the titanate precipitate is washed with deionized water to a pH of 7; after step (2), the titanate nanotubes are washed with deionized water to a pH of 7.

5. The preparation method according to claim 2, characterized in that In step (3), the molecular weight of the polyethylene glycol is 500-1000, and the molar ratio of the sodium tungstate to the polyethylene glycol is 1-2:1-2.

6. The preparation method according to claim 2, characterized in that In step (3), the tungsten-containing precursor needs to be adjusted to pH 4.5-5.5 using acid, and then stirred continuously for 2-20 hours in a light-proof environment.

7. The preparation method according to claim 2, characterized in that In step (3), the titanate nanotubes are first added to an ethylene glycol / water mixed solvent to form a dispersion, and then the tungsten-containing precursor is added to the dispersion to form a mixture, and the mixture is stirred, centrifuged, washed, dried and then calcined to obtain the highly localized metal single atom SERS substrate.

8. The preparation method according to claim 7, characterized in that The volume ratio of ethylene glycol to water in the mixed solvent is 1:5-20, the ratio of titanate nanotubes to the mixed solvent is 1g:100-300ml; and the mass ratio of titanate nanotubes to tungsten-containing precursor in the mixture is 1:10-20.

9. The preparation method according to claim 7, characterized in that The stirring is carried out at 60-80° C. for 2-6 hours; and the calcination is carried out at 180-250° C. under an argon atmosphere for 4-8 hours.

10. Use of the SERS substrate according to claim 1 in selectively enhancing low-frequency Raman signals of biomass molecules, characterized in that: The biomass molecular sieve is one of glucose, uric acid, dopamine, triglyceride and phenylalanine.

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