A method for identifying and detecting carotenoids based on potential regulation SERS

Abstract

The application discloses a method for identifying and detecting carotenoids based on potential regulation SERS, which comprises the following steps: preparing a carbon-based microelectrode, preparing a nano-silver particle modified SERS active microelectrode, constructing a carotenoid detection system, qualitatively analyzing carotenoids, and quantitatively analyzing carotenoids. The application provides a method for screening different carotenoids by potential regulation, realizes rapid enrichment of fat-soluble carotenoids which are not easy to be adsorbed on a SERS active substrate, enhances the sensitivity of SERS detection of carotenoids, is simple to operate, low in cost, and is a new method and new technology urgently needed for food safety analysis and detection.

Description

Technical Field

[0001] This invention belongs to the field of food safety analysis and chemical detection sensing technology, specifically involving a method for identifying and detecting carotenoids based on potential-modulated SERS. Background Technology

[0002] Carotenoids are widely distributed and diverse in nature, playing important roles in humans and other mammals. For example, β-carotene is a precursor to vitamin A, which is a crucial component of photosensors in the visual process. Surface-enhanced Raman scattering (SERS) is a commonly used sensing technique. When molecules are adsorbed onto rough metal surfaces, such as silver or gold nanoparticles, the inelastic light scattering of the molecules is greatly enhanced. In aqueous solutions, only carotenoids with hydrophilic terminal groups readily adsorb onto silver or gold nanoparticles, while β-carotene, like other nonpolar carotenoids, has low affinity for the surface of silver nanoparticles. Studies have shown that modifying the substrate surface is an effective method for regulating SERS enhancement performance and analyte adsorption modes. For example, to obtain the SERS spectra of carotenoids, organic layers are often modified onto the surface of silver or gold nanoparticles to increase affinity. However, this also highlights the shortcomings of organic layers in interfering with or reducing the SERS signal of silver or gold nanoparticles.

[0003] This invention utilizes carbon fiber as an electrode carrier to fabricate microelectrodes, and modifies the carbon fiber surface with uniformly sized silver nanoparticles. By combining electrochemical sensing technology with the high sensitivity of SERS, a method for identifying and detecting two carotenoids through potential-controlled SERS was developed. This method possesses both qualitative and quantitative analytical capabilities. The optimal potential adjustment allows for rapid enrichment of carotenoid molecules on the electrode surface, enabling the simultaneous detection of at least two carotenoids. Summary of the Invention

[0004] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0005] In view of the problems existing in the above and / or existing technologies for identifying and detecting carotenoids, the present invention is proposed.

[0006] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a method for identifying and detecting carotenoids based on potential-modulated SERS.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for identifying and detecting carotenoids based on potential-modulated SERS, comprising the following steps:

[0008] Preparation of carbon-based microelectrodes: After cleaning and drying, carbon fibers are placed in a glass capillary tube, a small amount of silver paste is applied for fixation, epoxy resin is added and encapsulated, and carbon-based microelectrodes are obtained after fixation.

[0009] Preparation of SERS active microelectrode modified with silver nanoparticles: After ultrasonic cleaning and drying, the carbon-based microelectrode was electrochemically activated. It was then placed in an electrolyte to electrochemically anchor the linking molecules, immersing the silver nanoparticles. After modification, it was rinsed several times with pure water to obtain the SERS active microelectrode.

[0010] Constructing a carotenoid detection system: PBS was selected as the electrolyte, and the potential was set. The SERS active microelectrode was used as the working electrode to construct the monitoring system and obtain the carotenoid detection system.

[0011] Qualitative analysis of carotenoids: Adjust the potential, select a 532nm laser, set the power, and compare the Raman signals of carotenoid molecules at different potentials;

[0012] Quantitative analysis of carotenoids: At the optimal potential, a 532nm laser was selected, the power was set, and the intensity change of the Raman signal was measured.

[0013] As a preferred embodiment of the method for identifying and detecting carotenoids based on potential-controlled SERS according to the present invention, in the qualitative analysis of carotenoids, the potential is a potential of -1.0 to 1.0V, and the potential is maintained for 1 to 15 minutes to perform electro-driven adsorption and enrichment of carotenoid molecules.

[0014] As a preferred embodiment of the method for identifying and detecting carotenoids based on potential-controlled SERS according to the present invention, in the preparation of carbon-based microelectrodes, in the step of adding epoxy resin and encapsulating, the amount of epoxy resin is 20% to 70% by volume, based on A-type glue.

[0015] As a preferred embodiment of the method for identifying and detecting carotenoids based on potential-controlled SERS according to the present invention, in the preparation of carbon-based microelectrodes and silver nanoparticle-modified SERS active microelectrodes, the ultrasonic treatment is set to an ultrasonic frequency of 60 Hz.

[0016] As a preferred embodiment of the method for identifying and detecting carotenoids based on potential-controlled SERS according to the present invention, the carotenoid detection system includes a working electrode that is a SERS active electrode modified with silver nanoparticles, a platinum wire as a counter electrode, and Ag / AgCl as a reference electrode.

[0017] As a preferred embodiment of the method for identifying and detecting carotenoids based on potential-controlled SERS as described in this invention, the following steps are taken: In preparing the SERS active microelectrode modified with silver nanoparticles, 200 μL of acetone, 3 M HNO3, 1 M KOH, and 1.5 mL of pure water are added; the potential of the electrode surface is controlled appropriately to be -2.0–2.0 V and 10–40 s using a potentiostatic method; the electrode is cyclically scanned 10–30 times within a potential range of -1.0–1.0 V using a cyclic voltammetry method; the 0.1 M LiClO4 electrolyte contains 1–10 mM of 1,8-Diaminooctane, with a potential range of -0.5–2.0 V, and is scanned 3–15 times at a scan rate of 5–100 mV / s; 100 μL of silver nanoparticles concentrated 1–10 times is taken and allowed to stand for 2–10 hours to prepare the SERS active microelectrode modified with silver nanoparticles.

[0018] As a preferred embodiment of the method for identifying and detecting carotenoids based on potential-modulated SERS described in this invention, in the quantitative analysis of carotenoids, the sample is placed under a microscope, a 532nm laser is selected, and the power is selected as 1.0mW.

[0019] As a preferred embodiment of the method for identifying and detecting carotenoids based on potential-controlled SERS according to the present invention, in the qualitative analysis of carotenoids, the laser wavelength is 532nm and the laser power is 1.0mW.

[0020] As a preferred embodiment of the method for identifying and detecting carotenoids based on potential-regulated SERS according to the present invention, wherein: in the quantitative analysis of carotenoids, the potential is -0.1V for trans-β-Apo-8′-carotenal and -0.75V for β-carotene.

[0021] As a preferred embodiment of the method for identifying and detecting carotenoids based on potential-controlled SERS according to the present invention, the detection method is capable of identifying and detecting carotenoids trans-β-Apo-8′-carotenal and β-carotene in aqueous solution.

[0022] This invention proposes a method for identifying and detecting carotenoids based on potential-modulated SERS. By simply adjusting the potential, carotenoid molecules can be selectively enriched on the surface of a microelectrode, and then qualitative and quantitative detection can be performed simultaneously in two modes using electrochemical and SERS methods. This dual-channel detection method is low-cost, easy to operate, and has a short reporting time, making it widely applicable in the fields of food safety and biomedicine. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0024] Figure 1 A schematic diagram illustrating the principle of using potential-modulated SERS to identify and detect two carotenoids, provided in an embodiment of the present invention;

[0025] Figure 2 The image shows a field emission scanning electron microscope (FEM) image and elemental mapping diagram of the silver nanoparticle-modified carbon microelectrode in Example 1.

[0026] Figure 3 This is a dark-field microscope image of the silver nanoparticle-modified carbon microelectrode in Example 1.

[0027] Figure 4 The above are SERS images of the two carotenoids on the surface of a carbon microelectrode modified with silver nanoparticles in Example 1 under conditions of applied potential and no applied potential.

[0028] Figure 5 The image shows the SERS diagrams of two carotenoids on the surface of a carbon microelectrode modified with silver nanoparticles under different potential conditions in Example 1.

[0029] Figure 6 The image shows the SERS diagrams of silver nanoparticle-modified carbon microelectrodes for different concentrations of trans-β-Apo-8′-carotenal under optimal potential and enrichment time in Example 1.

[0030] Figure 7 The image shows the SERS diagrams of different concentrations of β-carotene on a carbon microelectrode modified with silver nanoparticles under optimal potential and enrichment time in Example 1. Detailed Implementation

[0031] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0032] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0033] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0034] Example 1

[0035] A method for identifying and detecting two carotenoids based on potential-modulated SERS includes the following steps:

[0036] (1) A bundle of carbon fibers approximately 1 cm long was cut, with each individual carbon fiber having a diameter of 7 μm. The bundle was then subjected to ultrasonic treatment at 60 Hz for 10 min in anhydrous ethanol and water, followed by overnight drying in a 60°C oven. A 2 cm long glass capillary with a diameter of 1 mm was cut, and a 2.5 cm long copper wire was placed inside the capillary as a carrier for the carbon fibers. The copper wire was dipped in silver paste, which was used to fix the individual carbon fibers. After 20 min, the copper wire and carbon fibers were firmly welded together, and the position of the weld point in the glass capillary was adjusted. Subsequently, a 50% volume ratio of mixed epoxy resin was added to the glass capillary. After encapsulation, the microelectrode was left to stand overnight to obtain a carbon-based microelectrode with a diameter of 7 μm.

[0037] (2) The prepared carbon-based microelectrode was subjected to ultrasonic treatment at a frequency of 60 Hz for 3 min in 200 μL acetone, 3 M HNO3, 1 M KOH, and pure water, respectively. After cleaning, the microelectrode was dried at room temperature and then electrochemically activated. Using 0.5 M H2SO4 as the electrolyte, the electrode surface was controlled at 2.0 V for 35 s using a potentiostatic method. Subsequently, cyclic voltammetry was used to scan the electrode 15 times within the potential range of 0–1.0 V. After electrode activation, an alkyl linker (1,8-Diaminooctane) was modified onto the carbon microelectrode surface through electrochemical oxidation. This was achieved using 0.1 M LiClO4 anhydrous ethanol solution as the supporting electrolyte, containing 8 mM of 1,8-Diaminooctane. Cyclic voltammetry was performed to anchor the 1,8-Diaminooctane alkyl chain on the electrode surface, with a potential range of 0–1.6 V, 10 scans, and a scan rate of 20 mV / s. The modified carbon microelectrode was rinsed several times with anhydrous ethanol and pure water, and then sonicated in pure water at a frequency of 60 Hz for 30 s to remove loosely adsorbed alkyl chains. Subsequently, the alkyl chain-functionalized carbon microelectrode was used to modify silver nanoparticles to prepare SERS active microelectrodes. Specifically, 100 μL of silver nanoparticles concentrated 5 times was dropped into a plastic petri dish, with one alkyl-functionalized carbon microelectrode immersed in each drop. After 8 hours, the SERS active microelectrode modified with silver nanoparticles was obtained.

[0038] (3) 0.1M pH 7.0 PBS was selected as the supporting electrolyte. The carotenoid stock solution (ethanol solution) was diluted 100 times with this electrolyte, so that the carotenoid concentration was increased from 10... -5 M diluted to 10 -7 M. After mixing, the mixture was transferred to a detection cell. Using a platinum wire as the counter electrode and Ag / AgCl as the reference electrode, the prepared functionalized carbon microelectrode was used as the working electrode to construct the detection system. By setting different potentials, namely trans-β-Apo-8′-carotenal at -0.1V and β-carotene at -0.75V, and maintaining the potential for 7 min, carotenoid molecules in the electrolyte were electro-driven adsorption and enrichment.

[0039] (4) The enriched carbon microelectrode was placed under a confocal microscope with a 10x lens and a 532nm laser. The laser power was adjusted to 1.0mW to record the Raman signal of carotenoid molecules on the surface of the microelectrode. The Raman signal intensity, peak position, and ratio between peaks of carotenoids at different potentials (-0.1, -0.3, -0.5, -0.7, -0.9V) were compared to qualitatively analyze the carotenoids.

[0040] (5) The carbon microelectrode that has been enriched with various carotenoid molecules at the optimal potential is placed under the 10x lens of a confocal microscope. The 532nm laser is also selected with the optimal power (1.0mW). Five Raman signals are collected at each point and the average value is taken. The concentration of carotenoid molecules is quantified by the intensity change of the Raman signal.

[0041] The steps for preparing silver nanoparticle-modified carbon microelectrodes and the potential-controlled SERS method for recognizing carotenoids in Example 1 are as follows: Figure 1 As shown in the image. Field emission scanning electron microscope (FESEM) image of the silver nanoparticle-modified carbon microelectrode. Figure 2 As shown, from Figure 2 The surface structure of the functionalized microelectrode can be clearly observed, with spherical silver nanoparticles of uniform size and evenly distributed on the carbon fiber surface. Compared to the bare carbon fiber surface, the surface modified with silver nanoparticles becomes rougher, which provides the possibility for the superior SERS activity of this electrode in detecting carotenoid molecules. Furthermore, from... Figure 2 The mapping diagrams of C and Ag also show that the silver nanoparticles are uniformly modified on the electrode surface.

[0042] Dark-field microscopy analysis of the silver nanoparticle-modified carbon microelectrode prepared in Example 1 is as follows: Figure 3 As shown, where Figure 3a shows the SPR peak of silver nanoparticles. Although this is a significant redshift compared to the SPR peak of silver nanoparticles characterized by UV-Vis, the result reveals that silver nanoparticles do indeed exist on the electrode surface. This redshift phenomenon is mainly attributed to the aggregation of nanoparticles modified on the electrode surface. Figure 3 b and Figure 3 c compares the resonant luminescence of bare carbon fiber electrodes and carbon fibers modified with silver nanoparticles. The figure clearly shows that the bare electrode exhibits no luminescence, while the luminescence profile of the silver nanoparticle-modified carbon microelectrode is visible. These results demonstrate that silver nanoparticles were successfully modified onto the surface of the carbon microelectrode.

[0043] The silver nanoparticle-modified carbon microelectrode prepared in Example 1 was used to detect trans-β-Apo-8′-carotenal and β-carotene, such as... Figure 4 As shown in the figure, the two carotenoid molecules have similar Raman signals, namely 1524 cm⁻¹. -1 C=C stretching vibration at 1157cm -1 C-C stretching vibration at 1006 cm -1 The -CH3 rocking vibration was observed at the surface. Furthermore, it was found that the Raman signal intensity was significantly weak when no potential was applied to the carbon microelectrode surface, solely through static adsorption. However, the Raman signal intensity was significantly stronger when potentials were applied (trans-β-Apo-8′-carotenal -0.1V, β-carotene -0.75V), with trans-β-Apo-8′-carotenal and β-carotene showing 12 and 21 times the Raman signal intensity respectively, indicating static adsorption. This is mainly attributed to the fact that most carotenoids are lipid-soluble molecules, making it difficult for them to approach the SERS active substrate in aqueous solution via electrostatic adsorption. However, adjusting the surface potential of the functionalized carbon microelectrode increased the possibility of charge transfer, providing a certain driving force for carotenoid molecules, leading to their enrichment and adsorption on the functionalized carbon microelectrode surface. In addition, the presence of a hydrophilic aldehyde group on the trans-β-Apo-8′-carotenal backbone, differing in functional groups from β-carotene, resulted in different responses to potential enrichment, reflected in the different enhancement factors. The above results indicate that the identification and detection of two carotenoids can be achieved by potentiometric SERS.

[0044] In Example 1, the SERS results of silver nanoparticle-modified carbon microelectrodes at different potentials for detecting trans-β-Apo-8′-carotenal and β-carotene are as follows: Figure 5As shown in the figure, the trans-β-Apo-8′-carotenal exhibits the strongest Raman signal at a potential of -0.1 V, while β-carotene shows the strongest Raman signal at -0.75 V. Conversely, the trans-β-Apo-8′-carotenal shows a very weak Raman signal at -0.75 V, and β-carotene shows a very weak Raman signal at -0.1 V. Based on this, the optimal potential for recognizing and detecting these two carotenoids can be determined.

[0045] Based on the above observation, it can be concluded that the preferred voltage settings in this invention are trans-β-Apo-8′-carotenal -0.1V and β-carotene -0.75V.

[0046] In Example 1, under optimal potential conditions (trans-β-Apo-8′-carotenal at -0.1V and β-carotene at -0.75V), the SERS results of the silver nanoparticle-modified carbon microelectrode were as follows when different concentrations of trans-β-Apo-8′-carotenal and β-carotene were present: Figure 6 and 7 As shown in the figure, as the concentration of carotenoid molecules increases, the SERS signal (1524 cm⁻¹) decreases. -1 C=C stretching vibration at 1157 cm -1 C-C stretching vibration at 1006 cm -1 The -CH3 rocking vibration at the site also gradually increases, mainly due to suitable potential conditions driving the accumulation of lipid-soluble carotenoid molecules on the surface of the SERS-active carbon microelectrode, thereby greatly improving the detection sensitivity of this method. Finally, in the range of 0–10... -5 Within the M concentration range, the Raman signal of the functionalized carbon microelectrode showed a good correlation with the concentration, which further indicates that SERS recognition and detection of two carotenoids can be successfully achieved through potential modulation.

[0047] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for identifying and detecting carotenoids based on potential-modulated SERS, characterized in that: Includes the following steps: Preparation of carbon-based microelectrodes: After cleaning and drying, carbon fibers are placed in a glass capillary tube, a small amount of silver paste is applied for fixation, epoxy resin is added and encapsulated, and carbon-based microelectrodes are obtained after fixation. Preparation of SERS active microelectrode modified with silver nanoparticles: After ultrasonic cleaning and drying, carbon-based microelectrode is subjected to electrochemical activation treatment, electrolyte is placed in it, electrically anchored linking molecules are inserted, silver nanoparticles are immersed in it, and after modification, it is rinsed several times with pure water to obtain SERS active microelectrode. Constructing a carotenoid detection system: PBS was selected as the electrolyte, and the potential was set. The SERS active microelectrode was used as the working electrode to construct the monitoring system and obtain the carotenoid detection system. Qualitative analysis of carotenoids: Adjusting the potential and comparing the Raman signals of carotenoid molecules at different potentials; Quantitative analysis of carotenoids: At the optimal potential, a 532nm laser was selected, the power was set, and the intensity change of the Raman signal was measured; In the preparation of the carbon-based microelectrode, in the step of adding epoxy resin and encapsulating it, the amount of epoxy resin is 20%~70% by volume, calculated as A-type adhesive. In the preparation of the SERS active microelectrode modified with silver nanoparticles, the ultrasonic cleaning is set to an ultrasonic frequency of 60 Hz. In the preparation of the SERS active microelectrode modified with silver nanoparticles, 200 μL of acetone, 3 M HNO3, 1 M KOH, and 1.5 mL of pure water were added. The appropriate potential on the electrode surface was controlled using a potentiostatic method at -2.0–2.0 V and 10–40 s. Cyclic voltammetry was used to scan the electrode 10–30 times within a potential range of -1.0–1.0 V. A 0.1 M LiClO4 electrolyte containing 1–10 mM of 1,8-Diaminooctane was used, with a potential range of -0.5–2.0 V, and 3–15 scans were performed at a scan rate of 5–100 mV / s. 100 μL of concentrated silver nanoparticles (1–10 times concentration) was taken and allowed to stand for 2–10 hours to prepare the SERS active microelectrode modified with silver nanoparticles. In the quantitative analysis of carotenoids, the sample was placed under a microscope, and a 532nm laser with a power of 1.0mW was selected. In the qualitative analysis of carotenoids, the laser wavelength is 532 nm and the laser power is 1.0 mW. In the quantitative analysis of carotenoids, the potentials are -0.1V for trans-β-Apo-8′-carotenal and -0.75V for β-carotene. The method can identify and detect carotenoids trans-β-Apo-8′-carotenal and β-carotene in aqueous solutions.

2. The method for identifying and detecting carotenoids based on potential-modulated SERS according to claim 1, characterized in that: In the qualitative analysis of carotenoids, the potential is -1.0~1.0V, and the potential is maintained for 1~15min to perform electro-driven adsorption and enrichment of carotenoid molecules.

3. The method for identifying and detecting carotenoids based on potential-modulated SERS according to claim 1, characterized in that: In the construction of the carotenoid detection system, the working electrode construction monitoring system includes a platinum wire as the counter electrode and Ag / AgCl as the reference electrode.

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