Nanometer material detection method based on surface enhanced Raman spectroscopy

Abstract

The invention relates to the technical field of analysis and detection, in particular to a surface enhanced Raman spectroscopy (SERS)-based nano material detection method, which is characterized in that a heterojunction composite structure consisting of noble metal nano stars and semiconductor oxide nano particles is used as an SERS active substrate; and a specific recognition molecule carrying a Raman reporter molecule is used as a detection probe. During detection, the recognition molecules are firstly combined with the to-be-detected nano material, and then the compound is adsorbed on the surface of the substrate. The tip structure of the noble metal nanostar and a heterojunction interface jointly generate a synergistic effect of electromagnetic enhancement and chemical enhancement, and a reporter molecule signal is doubly amplified, so that high-sensitivity and high-selectivity detection on a to-be-detected nano material is realized. The method solves the problems of weak signal and poor selectivity of the traditional method, and is suitable for rapid and quantitative analysis of trace nano materials in the environment.

Description

Technical Field

[0001] This invention relates to the field of analytical detection technology, and in particular to a method for detecting nanomaterials based on surface-enhanced Raman spectroscopy. Background Technology

[0002] The widespread application of nanomaterials in energy, catalysis, biomedicine, and other fields has brought considerable attention to their release, migration, transformation, and potential ecological risks in the environment. Therefore, developing highly sensitive, rapid, and reliable nanomaterial detection technologies is crucial. Existing nanomaterial detection methods, such as transmission electron microscopy (TEM) and inductively coupled plasma mass spectrometry (ICP-MS), while providing accurate information, are often expensive, require complex sample pretreatment, and are difficult to implement for rapid on-site detection.

[0003] Surface-enhanced Raman spectroscopy (SERS) has become a highly attractive analytical tool due to its extremely high sensitivity (even enabling single-molecule detection), rich fingerprint spectral information, resistance to photobleaching, and potential for rapid detection. SERS enhancement mainly originates from the electromagnetic field enhancement mechanism generated by localized surface plasmon resonance (LSPR) on the surface of noble metal (such as gold and silver) nanostructures.

[0004] Currently, studies have attempted to use SERS for nanoparticle detection. Common strategies include directly adsorbing the nanoparticles to be tested onto traditional SERS substrates (such as aggregated gold or silver sols), or modifying the SERS substrate or nanoparticles with recognition molecules (such as antibodies or aptamers) to specifically bind the target nanoparticles to the enhanced "hot spot" region. However, these methods have the following limitations: (1) the enhancement ability of traditional noble metal SERS substrates is limited, and the distribution of "hot spots" is uneven, resulting in poor signal reproducibility; (2) for nanomaterials with weak Raman signals, direct detection sensitivity is insufficient; (3) in complex environmental matrices, non-specific adsorption may lead to severe background interference and false positive signals; (4) relying on recognition molecules to directly modify the SERS substrate may result in the shielding of active sites on the substrate or inactivation of recognition molecules.

[0005] Therefore, there is an urgent need to develop a novel SERS detection strategy that can simultaneously achieve ultra-high sensitivity, excellent selectivity, and good signal reproducibility to address the challenges of detecting trace nanomaterials in complex real-world samples. Summary of the Invention

[0006] To overcome the aforementioned deficiencies of the prior art, the present invention provides a method for detecting nanomaterials based on surface-enhanced Raman spectroscopy, thereby addressing the problems existing in the background art.

[0007] This invention provides the following technical solution: a method for detecting nanomaterials based on surface-enhanced Raman spectroscopy, comprising the following steps: A SERS-active substrate is provided, the substrate comprising a heterojunction composite structure formed by noble metal nanostars having at least one tip structure and semiconductor oxide nanoparticles; The nanomaterial sample to be tested is mixed with a solution containing specific recognition molecules to form a mixture. The specific recognition molecules can selectively bind to the surface of the nanomaterial to be tested and carry Raman reporter molecules. The mixture is dropped onto the surface of the SERS active substrate, so that the nanomaterial to be tested, which is bound with Raman reporter molecules, is either passed through the specific recognition molecules or directly adsorbed near the tip region of the heterojunction composite structure. The region on the SERS active substrate where the sample is adsorbed was detected using a Raman spectrometer to obtain an enhanced Raman spectral signal. The heterojunction interface between the noble metal nanostar and the semiconductor oxide nanoparticle, along with the tip structure, jointly generate a synergistic effect of electromagnetic and chemical enhancement, which is used to dually enhance the signal of the Raman reporter molecule.

[0008] Furthermore, the noble metal nanostars are gold nanostars or silver nanostars, and the semiconductor oxide nanoparticles are one of titanium dioxide, zinc oxide, or iron oxide nanoparticles.

[0009] Furthermore, the method for preparing the SERS active substrate includes: A dispersion of the semiconductor oxide nanoparticles is provided; A noble metal precursor salt and a morphology control agent are added to the dispersion, and the noble metal nanostars are grown in situ on the surface of the semiconductor oxide nanoparticles by chemical reduction to form the heterojunction composite structure. The resulting heterojunction composite structure dispersion is deposited on a solid support substrate to form the SERS active substrate.

[0010] Furthermore, the specific recognition molecule is an aptamer, antibody, or ligand molecule, one end of which is modified with a functional group capable of specifically binding to the surface of the nanomaterial to be tested, and the other end is modified with the Raman reporter molecule.

[0011] Furthermore, the Raman reporter molecule is a molecule with a unique Raman fingerprint peak, including but not limited to rhodamine dyes, crystal violet, p-mercaptobenzoic acid, or cyano compounds.

[0012] Furthermore, the nanomaterial to be tested is at least one of engineered nanomaterials, metal nanoparticles, oxide nanoparticles, quantum dots, or two-dimensional nanomaterials.

[0013] Furthermore, after acquiring the enhanced Raman spectral signal, the method also includes a quantitative analysis step: Select a characteristic Raman peak intensity value of the Raman reporter molecule; Based on a pre-established standard curve of the characteristic Raman peak intensity value and the concentration of the nanomaterial to be tested, the concentration of the nanomaterial in the sample to be tested is determined.

[0014] Furthermore, before adding the mixture to the surface of the SERS active substrate, the substrate is pretreated, including plasma cleaning or ultraviolet ozone treatment, to improve its hydrophilicity and adsorption capacity.

[0015] Furthermore, the method also includes a signal amplification step: introducing multiple Raman reporter molecules onto the specific recognition molecule, or using an enzyme-linked reaction system capable of catalyzing the generation of multiple Raman reporter molecules.

[0016] A SERS-active substrate for detecting nanomaterials, characterized in that it comprises a solid support substrate and a SERS-active layer loaded thereon, wherein the SERS-active layer comprises a heterojunction composite structure formed by noble metal nanostars and semiconductor oxide nanoparticles as described above.

[0017] The technical effects and advantages of this invention are as follows: Ultra-high sensitivity: By utilizing the synergistic effect of the strong electromagnetic "hot spot" generated at the tip of the noble metal nanostar and the charge transfer chemical enhancement induced by the heterojunction interface, the Raman reporter molecule signal is doubly amplified, and the detection limit is significantly reduced to the ng / L or even pg / L level.

[0018] High selectivity: By employing a strategy of pre-binding specific recognition molecules with target nanomaterials in the sample, contamination and non-specific adsorption caused by direct contact between complex matrices and SERS substrates are avoided. Recognition molecules bind to the target more efficiently in homogeneous solutions, resulting in better selectivity.

[0019] Stable signal and good reproducibility: The solid-state SERS substrate avoids the instability of sol agglomeration, the preparation of heterojunction composite structure is controllable, the hot spot distribution is relatively uniform, and the relative standard deviation (RSD) of Raman signal intensity can be less than 10%.

[0020] High versatility: By changing the recognition molecule and Raman reporter molecule, this method can be adapted to the detection of different types of nanomaterials with different surface properties, and has a wide range of applications.

[0021] Facilitates quantitative analysis: The signal intensity of Raman reporter molecules has a good correlation with the concentration of target nanomaterials, making it easy to establish standard curves and achieve quantitative detection.

[0022] The operation is relatively simple: the sample pretreatment steps are simplified and the detection speed is fast, which makes it promising for rapid on-site screening. Attached Figure Description

[0023] Figure 1 This is a flowchart of the method of the present invention; Detailed Implementation The technical solution of the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are only used to explain the present invention and are not intended to limit the scope of protection of the present invention.

[0024] Please see Figure 1 As shown, a method for detecting nanomaterials based on surface-enhanced Raman spectroscopy includes the following steps: A SERS-active substrate is provided, the substrate comprising a heterojunction composite structure formed by noble metal nanostars having at least one tip structure and semiconductor oxide nanoparticles; The nanomaterial sample to be tested is mixed with a solution containing specific recognition molecules to form a mixture. The specific recognition molecules can selectively bind to the surface of the nanomaterial to be tested and carry Raman reporter molecules. The mixture is dropped onto the surface of the SERS active substrate, so that the nanomaterial to be tested, which is bound with Raman reporter molecules, is either passed through the specific recognition molecules or directly adsorbed near the tip region of the heterojunction composite structure. The region on the SERS active substrate where the sample is adsorbed was detected using a Raman spectrometer to obtain an enhanced Raman spectral signal. The heterojunction interface between the noble metal nanostar and the semiconductor oxide nanoparticle, along with the tip structure, jointly generate a synergistic effect of electromagnetic and chemical enhancement, which is used to dually enhance the signal of the Raman reporter molecule.

[0025] You can follow these steps: First, a special substrate for enhancing Raman signals is prepared. This substrate is loaded with a composite nanomaterial, which consists of gold or silver nanoparticles with multiple spike-like structures combined with metal oxide nanoparticles such as titanium dioxide.

[0026] Next, the nanomaterial sample to be tested is processed. A certain amount of sample solution is taken and mixed with another solution containing a "recognition-reporter" unit. This "recognition-reporter" unit consists of two parts: one part is a molecule (such as a specific DNA sequence or an antibody) that can specifically bind to the surface of the target nanomaterial, like a key to a lock; the other part is a labeled molecule (i.e., a Raman reporter molecule) attached to it that can emit a specific Raman signal. After mixing, the two are incubated for a period of time to allow the recognition molecule to find and bind to the target nanomaterial.

[0027] Next, the mixed liquid is dropped onto a pre-prepared special substrate surface and left to stand for a period of time. During this process, the target nanomaterials that have already incorporated Raman reporter molecules are attracted and fixed near the spike-like structures of the composite nanomaterials on the substrate surface.

[0028] Finally, the substrate region where the sample was dropped was irradiated and scanned using a Raman spectrometer, and the obtained Raman spectral signals were collected. At this point, because the spikes of the gold / silver nanoparticles can generate a strong local electromagnetic field, and the interface between them and titanium dioxide can promote charge transfer, these two effects work synergistically to greatly enhance the signal of the Raman reporter molecules attached to the target, thus allowing it to be sensitively detected by the instrument.

[0029] Furthermore, the noble metal nanostars are gold nanostars or silver nanostars, and the semiconductor oxide nanoparticles are one of titanium dioxide, zinc oxide, or iron oxide nanoparticles.

[0030] In preparing the aforementioned special substrate, a preferred approach is to use gold to fabricate nanostars with a spiked structure. Simultaneously, titanium dioxide nanoparticles are chosen as the semiconductor material for composite preparation. Gold nanostars provide extremely strong electromagnetic field enhancement, while titanium dioxide is chemically stable and can generate effective chemical enhancement; the combination of the two yields excellent results.

[0031] Furthermore, the method for preparing the SERS active substrate includes: A dispersion of the semiconductor oxide nanoparticles is provided; A noble metal precursor salt and a morphology control agent are added to the dispersion, and the noble metal nanostars are grown in situ on the surface of the semiconductor oxide nanoparticles by chemical reduction to form the heterojunction composite structure. The resulting heterojunction composite structure dispersion is deposited on a solid support substrate to form the SERS active substrate.

[0032] The preparation of this special reinforced substrate can be carried out as follows: The first step is to prepare an ethanol dispersion of titanium dioxide nanoparticles with a concentration of approximately 1 mg per milliliter.

[0033] In the second step, a certain amount of the above dispersion was taken and mixed with an aqueous solution of hexadecyltrimethylammonium bromide. Then, aqueous solutions of chloroauric acid and ascorbic acid were added sequentially, and the mixture was gently shaken and allowed to stand in a water bath at 30 degrees Celsius for about 3 hours. During this process, ascorbic acid reduces gold ions to gold atoms, which preferentially grow on the surface of titanium dioxide particles, forming a gold nanostar structure with multiple spikes, ultimately yielding a composite of gold nanostars and titanium dioxide.

[0034] The third step involves centrifuging the reacted solution, collecting the solid product, washing it with deionized water and ethanol, and then redispersing it in ethanol. Finally, a certain amount of this dispersion is drop-coated onto a clean silicon wafer surface. After the solvent evaporates naturally and dries, a uniform active film is formed on the silicon wafer, which serves as the desired detection substrate.

[0035] Furthermore, the specific recognition molecule is an aptamer, antibody, or ligand molecule, one end of which is modified with a functional group capable of specifically binding to the surface of the nanomaterial to be tested, and the other end is modified with the Raman reporter molecule.

[0036] The "recognition-report" unit mentioned above can have a recognition component that is a specially designed DNA sequence (called an aptamer) capable of binding to specific nanomaterials with high affinity. For example, for silica nanoparticles, an aptamer that specifically binds to their surface hydroxyl groups can be selected. One end of the aptamer is chemically modified (e.g., by adding an amino group), and then a cross-linking agent (e.g., sulfonyl-SMCC) is used to chemically react with the thiol group on a Raman reporter molecule (e.g., p-mercaptobenzoic acid), thereby covalently linking the two together to form a complete probe molecule. Purification can be performed using chromatographic techniques before use.

[0037] Furthermore, the Raman reporter molecule is a molecule with a unique Raman fingerprint peak, including but not limited to rhodamine dyes, crystal violet, p-mercaptobenzoic acid, or cyano compounds.

[0038] The selected Raman reporter molecule needs to have strong and easily distinguishable Raman characteristic peaks. Commonly used molecules include: Rhodamine 6G (located at 1360, 1508, and...). The nearby peaks are very strong), crystal violet (located at 1175 and The peak is significant), or p-mercaptobenzoic acid (its peak is significant). The peaks at these points are often used for quantitative analysis. The Raman signals of these molecules are unique and not easily confused with background interference.

[0039] Furthermore, the nanomaterial to be tested is at least one of engineered nanomaterials, metal nanoparticles, oxide nanoparticles, quantum dots, or two-dimensional nanomaterials.

[0040] This method can detect a wide variety of nanomaterials. For example, it can detect artificially synthesized silica or polystyrene nanospheres; it can detect metal nanoparticles, such as gold and silver nanoparticles; it can detect metal oxide nanoparticles, such as cerium oxide and zinc oxide; it can also detect semiconductor quantum dots such as cadmium sulfide, or two-dimensional materials such as graphene oxide sheets. As long as its surface can be recognized and bound by corresponding recognition molecules (such as aptamers, antibodies, or specific ligands), this method can be applied.

[0041] Furthermore, after acquiring the enhanced Raman spectral signal, the method also includes a quantitative analysis step: Select a characteristic Raman peak intensity value of the Raman reporter molecule; Based on a pre-established standard curve of the characteristic Raman peak intensity value and the concentration of the nanomaterial to be tested, the concentration of the nanomaterial in the sample to be tested is determined.

[0042] To achieve quantitative detection, it is necessary to first establish the correlation between concentration and signal intensity. The specific procedure is as follows: prepare a series of target nanomaterial standard solutions with known concentrations, process and measure them one by one according to the detection steps described above (incubation, sample addition, detection). Record a fixed characteristic peak of the Raman reporter molecule at each concentration (e.g., for p-mercaptobenzoic acid). The intensity value of the Raman peak is used. A standard working curve is plotted with the logarithm of the nanomaterial concentration on the x-axis and the Raman peak intensity on the y-axis. When actually testing unknown samples, after measuring the Raman peak intensity, the concentration of the target nanomaterial in the sample can be calculated by substituting it into this standard curve.

[0043] Furthermore, before adding the mixture to the surface of the SERS active substrate, the substrate is pretreated, including plasma cleaning or ultraviolet ozone treatment, to improve its hydrophilicity and adsorption capacity.

[0044] Before adding the sample mixture to the substrate surface, the prepared solid substrate can be cleaned and activated. For example, the substrate can be placed in an oxygen plasma cleaner and treated for 60 seconds at 100 watts. This treatment effectively removes organic contaminants from the substrate surface and changes it from hydrophobic to hydrophilic. This allows the liquid to spread more evenly on the substrate surface when the sample solution is added, which is beneficial for more uniform adsorption of the target analyte and improves the reproducibility of the detection.

[0045] Furthermore, the method also includes a signal amplification step: introducing multiple Raman reporter molecules onto the specific recognition molecule, or using an enzyme-linked reaction system capable of catalyzing the generation of multiple Raman reporter molecules.

[0046] To further enhance detection sensitivity, signal amplification design can be implemented for the "recognition-report" unit. One approach is a "multi-label" strategy: instead of one, multiple Raman reporter molecules are linked to a single recognition molecule. For example, a dendritic polymer can be synthesized, Raman reporter molecules can be modified at the ends of its multiple branches, and then the recognition molecule can be linked to the core of the dendritic polymer. This allows a single recognition event to generate a significantly increased signal. Another approach is an "enzyme-linked amplification" strategy: the recognition molecule is linked to an enzyme (such as horseradish peroxidase). When a recognition binding event occurs, the enzyme substrate (such as 3,3',5,5'-tetramethylbenzidine TMB) is added. The enzyme catalyzes the production of a large amount of insoluble polymer precipitate, which itself, or through a coupling reaction, can generate a strong Raman signal, thus achieving cascaded signal amplification.

[0047] A SERS-active substrate for detecting nanomaterials, characterized in that it comprises a solid support substrate and a SERS-active layer loaded thereon, wherein the SERS-active layer comprises a heterojunction composite structure formed by noble metal nanostars and semiconductor oxide nanoparticles as described above.

[0048] This invention also provides a ready-made substrate product that can be directly used for detection. The product comprises a solid support sheet (such as a silicon wafer, glass plate, or plastic film) on which an active material is firmly loaded. This active material is a composite of gold nanostars and titanium dioxide nanoparticles prepared by the above method. This product can be pre-produced in batches, eliminating the need for users to synthesize nanomaterials themselves. After unpacking, it can be used for sample detection after simple pretreatment (such as plasma cleaning), offering convenience and speed. The above are merely specific embodiments of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A method for detecting nanomaterials based on surface-enhanced Raman spectroscopy (SERS), characterized in that, Includes the following steps: A SERS-active substrate is provided, the substrate comprising a heterojunction composite structure formed by noble metal nanostars having at least one tip structure and semiconductor oxide nanoparticles; The nanomaterial sample to be tested is mixed with a solution containing specific recognition molecules to form a mixture. The specific recognition molecules can selectively bind to the surface of the nanomaterial to be tested and carry Raman reporter molecules. The mixture is dropped onto the surface of the SERS active substrate, so that the nanomaterial to be tested, which is bound with Raman reporter molecules, is either passed through the specific recognition molecules or directly adsorbed near the tip region of the heterojunction composite structure. The region on the SERS active substrate where the sample is adsorbed was detected using a Raman spectrometer to obtain an enhanced Raman spectral signal. The heterojunction interface between the noble metal nanostar and the semiconductor oxide nanoparticle, along with the tip structure, jointly generate a synergistic effect of electromagnetic and chemical enhancement, which is used to dually enhance the signal of the Raman reporter molecule.

2. The nanomaterial detection method according to claim 1, characterized in that, The noble metal nanostars are gold nanostars or silver nanostars, and the semiconductor oxide nanoparticles are one of titanium dioxide, zinc oxide, or iron oxide nanoparticles.

3. The nanomaterial detection method according to claim 1, characterized in that, The method for preparing the SERS active substrate includes: A dispersion of the semiconductor oxide nanoparticles is provided; A noble metal precursor salt and a morphology control agent are added to the dispersion, and the noble metal nanostars are grown in situ on the surface of the semiconductor oxide nanoparticles by chemical reduction to form the heterojunction composite structure. The resulting heterojunction composite structure dispersion is deposited on a solid support substrate to form the SERS active substrate.

4. The nanomaterial detection method according to claim 1, characterized in that, The specific recognition molecule is an aptamer, antibody, or ligand molecule, one end of which is modified with a functional group that can specifically bind to the surface of the nanomaterial to be tested, and the other end is modified with the Raman reporter molecule.

5. The nanomaterial detection method according to claim 4, characterized in that, The Raman reporter molecule is a molecule with a unique Raman fingerprint peak, including but not limited to rhodamine dyes, crystal violet, p-mercaptobenzoic acid, or cyano compounds.

6. The nanomaterial detection method according to claim 1, characterized in that, The nanomaterial to be tested is at least one of engineered nanomaterials, metal nanoparticles, oxide nanoparticles, quantum dots, or two-dimensional nanomaterials.

7. The nanomaterial detection method according to claim 1, characterized in that, After acquiring the enhanced Raman spectral signal, the method further includes a quantitative analysis step: Select a characteristic Raman peak intensity value of the Raman reporter molecule; Based on a pre-established standard curve of the characteristic Raman peak intensity value and the concentration of the nanomaterial to be tested, the concentration of the nanomaterial in the sample to be tested is determined.

8. The method for detecting nanomaterials according to claim 1, characterized in that, Before the mixture is dropped onto the surface of the SERS active substrate, the substrate is pretreated, including plasma cleaning or ultraviolet ozone treatment, to improve its hydrophilicity and adsorption capacity.

9. The method for detecting nanomaterials according to claim 1, characterized in that, The method further includes a signal amplification step: introducing multiple Raman reporter molecules onto the specific recognition molecule, or using an enzyme-linked reaction system capable of catalyzing the generation of multiple Raman reporter molecules.

10. A SERS-active substrate for the detection of nanomaterials, characterized in that, It includes a solid support substrate and a SERS active layer loaded thereon, wherein the SERS active layer comprises a heterojunction composite structure formed by noble metal nanostars and semiconductor oxide nanoparticles as described in any one of claims 1-3.

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