Preparation method and application of SERS (Surface Enhanced Raman Scattering) probe for specifically detecting polyamine

Abstract

The invention discloses a preparation method and application of an SERS (Surface Enhanced Raman Scattering) probe for specifically detecting polyamine, and belongs to the field of biosensors. The preparation method of the SERS probe comprises the following steps: (1) preparing an SERS substrate with a gold nanostructure; (2) dropwise adding the SERS substrate into the sulfydryl functionalized groove of the acupuncture needle to obtain an SERS active microneedle; (3) the SERS active microneedle is soaked in a 4-mercaptophenylboronic acid aqueous solution containing mercaptoethanol, and the hydrogen peroxide responsive SERS active microneedle is obtained; and (4) dropwise adding polyamine oxidase into a groove of the hydrogen peroxide responsive SERS active microneedle, and specifically detecting the SERS probe of polyamine. According to the probe, gaps in an SERS substrate adsorbing mercaptophenylboronic acid are filled with micromolecular mercaptoethanol, decomposition of hydrogen peroxide by a nanogold interface is reduced, the reaction rate of adsorbed mercaptophenylboronic acid and hydrogen peroxide is increased, and therefore the detection limit of the technology to hydrogen peroxide is reduced by about one order of magnitude.

Description

Technical Field

[0001] The invention relates to a preparation method and application of a SERS probe for specifically detecting polyamines, and belongs to the field of biosensors. Background Art

[0002] Pre-prepared meals refer to semi-finished or finished dishes that are made from one or more agricultural products, are standardized, large-scale pre-processed and / or pre-cooked, and are pre-packaged. They are a type of food that is rapidly developing around the world. Pre-prepared meals are convenient and fast, and are not only popular among office workers and those who are not good at cooking, but also meet the needs of hotels to reduce costs and increase efficiency. Their sales and influence in the market are increasing day by day. Pre-prepared meals originated in the United States and flourished in Japan. In recent years, the scale of my country's pre-prepared meal industry has entered a period of rapid growth and entered the fast lane. Since polyamines are more common in pre-prepared meals of aquatic products, meat products and fermented products, especially in pre-prepared meals of meat and green-skinned red meat seafood, the development of a rapid detection method for polyamines suitable for pre-prepared meals can provide technical support for the safety of pre-prepared meal processing and storage technology, and is of great significance for evaluating the degree of spoilage and safe shelf life of pre-prepared meals.

[0003] At present, the detection methods for polyamines at home and abroad mainly include immunological methods, chromatographic methods and biosensor methods. Classical immunological methods for histamine analysis include radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). The former has a slow reaction rate and produces radioactive waste; the latter needs to consider cross-reactions with similar analytes and can only perform single-target analysis. In addition, due to the low molecular weight of polyamines, it is difficult to produce specific anti-histamine antibodies with high affinity. Chromatographic separation methods for polyamine analysis mainly include capillary electrophoresis (CE), capillary electrochromatography (CEC) and high-performance liquid chromatography (HPLC). The former two have problems such as low sensitivity and low quantitative accuracy of migration time, while the latter has problems such as environmental pollution, high cost and time-consuming. Currently, common biosensors for polyamine analysis include electrochemical sensors, fluorescence sensors, colorimetric sensors, etc. In addition to the poor repeatability and low stability that affect the application of such sensing detection methods, time-consuming and complex pretreatment also limits the promotion and application of such technologies in the detection of histamine in pre-prepared dishes.

[0004] Surface-enhanced Raman scattering (SERS) technology has the advantages of convenient operation and low fluorescence background. Currently, there are still several problems in the research of SERS-based polyamine sensing analysis: (1) There are many types of polyamine molecules, and their structures are easily affected by complex matrices and substrate environments, resulting in low intrinsic Raman activity and unclear characteristic peaks; (2) The affinity between polyamine molecules and SERS substrates is low, making it difficult for them to adsorb onto SERS substrates, and the signal enhancement effect is weak; (3) Conventional SERS technology lacks selectivity and still requires complex sample pretreatment steps or target enrichment time. Therefore, it is very necessary to develop a new type of highly specific and sensitive SERS sensor for the rapid detection of histamine. Summary of the Invention

[0005] Object of the Invention: The object of the present invention is to provide a preparation method and application of a SERS probe for specifically detecting polyamines.

[0006] Technical Solution: The preparation method of the SERS probe for specifically detecting polyamines of the present invention includes the following steps:

[0007] (1) Prepare a SERS substrate with gold nanostructures;

[0008] (2) Drop the SERS substrate into the mercapto-functionalized groove of the acupuncture needle to obtain a SERS-active microneedle;

[0009] (3) Immerse the SERS-active microneedle in an aqueous solution of 4-mercaptobenzeneboronic acid containing mercaptoethanol to obtain a hydrogen peroxide-responsive SERS-active microneedle;

[0010] (4) Drop polyamine oxidase into the groove of the hydrogen peroxide-responsive SERS-active microneedle to obtain a SERS probe for specifically detecting polyamines.

[0011] Further, the SERS substrate with gold nanostructures described in step (1) includes AuNFs or GNSs.

[0012] Further, step (2) also includes a pretreatment step for the acupuncture needle, including: immersing the acupuncture needle in a polymethyl methacrylate solution until an insulating protective layer is formed on the outer layer of the acupuncture needle body, standing at room temperature until dry; etching a groove on the needle body, performing mercapto-functionalization on the groove, removing the polymer insulating protective layer on the outer layer of the acupuncture needle body, and cleaning and drying with absolute ethanol.

[0013] Further, the groove accounts for 1 / 2 - 1 / 3 of the needle body diameter.

[0014] Further, in the aqueous solution of 4-mercaptobenzeneboronic acid containing mercaptoethanol described in step (3), the molar ratio of 4-mercaptobenzeneboronic acid to mercaptoethanol is 1 - 3:1.

[0015] Further, the concentration of the polyamine oxidase described in step (4) is 0.1-10 mU / mL.

[0016] The present invention also provides a SERS probe for specifically detecting polyamines prepared by the above method.

[0017] The present invention also provides the application of the above SERS probe for specifically detecting polyamines in detecting the polyamine content of prefabricated dishes.

[0018] The present invention also provides a method for detecting the polyamine content of prefabricated dishes, comprising the following steps:

[0019] (1) Prepare hydrogels with different polyamine concentrations, pierce the SERS probe for specifically detecting polyamines described in claim 7 into the hydrogels, incubate for 5 minutes, detect the Raman spectrum, calculate the relationship between the polyamine concentration and the Raman signal ratio, and draw a polyamine concentration curve;

[0020] (2) Pierce the SERS probe for specifically detecting polyamines described in claim 7 into the prefabricated dish to be detected, incubate for 5 minutes, take it out and detect the Raman spectrum, calculate the Raman signal ratio, and substitute it into the polyamine concentration curve.

[0021] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages: 1. When the SERS probe for specifically detecting polyamines of the present invention detects the polyamine content of prefabricated dishes, it does not require steps such as meat grinding, extraction, separation, and purification of the detection sample. The probe is directly pierced into the sample to be detected, incubated for a period of time, taken out and then the Raman spectrum can be measured to obtain the detection result. This detection method saves time and labor costs, reduces reagent consumption and instrument requirements. 2. The probe of the present invention fills and adsorbs the voids on the SERS substrate of mercaptophenylboronic acid with small molecule mercaptoethanol, reduces the decomposition of hydrogen peroxide by the gold nanosurface, improves the reaction rate of the adsorbed mercaptophenylboronic acid and hydrogen peroxide, and thus improves the detection ability of the technology of the present invention for hydrogen peroxide. 3. During detection, the SERS probe for specifically detecting polyamines of the present invention directly measures the amount of polyamine molecules inside the sample to be detected, which is similar to an in-situ detection technology, and does not require separating the polyamine molecules in the detection sample. Therefore, there is no loss of polyamine molecules, and the obtained results are true and reliable. 4. The detection method of the present invention does not involve the preparation and use of any solvents and reagents, and is applicable to detection in any occasion. Description of the Drawings

[0022] Figure 1 It is a schematic diagram of the polyamine detection process of the present invention;

[0023] Figure 2 It is an electron microscope characterization diagram of the gold nanoshell of the SERS substrate of the present invention.

[0024] Figure 3SEM image of the SERS substrate in the SERS-active microneedle grooves of the present invention, where A is the SEM image of the grooves on the thiolated acupuncture needle, B is the SEM image of the grooves on the acupuncture needle after dropping gold nanoshells, and C and D are the SEM images of the gold nanoshells.

[0025] Figure 4 Raman characteristic peak intensity ratios of SERS-active microneedles adsorbed with different molar ratios of 4-mercaptobenzoic acid / thioethanol before and after reacting in 200 μmol / L hydrogen peroxide at 37 °C for 5 minutes.

[0026] Figure 5 Standard curves for hydrogen peroxide detection established by SERS-active microneedles adsorbed with 4-mercaptobenzeneboronic acid and SERS-active microneedles adsorbed with 4-mercaptobenzeneboronic acid and thioethanol (3:1).

[0027] Figure 6 SERS intensity ratios of SERS-active microneedles with polyamine responses at different concentrations of polyamine oxidase in a hydrogel (50 mg / L polyamine) concentration.

[0028] Figure 7 Graph of polyamine concentration and Raman signal ratio.

[0029] Figure 8 Polyamine concentrations measured for different meats purchased on the market during storage at room temperature (25 °C) for 24 hours. Detailed implementation mode

[0030] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0031] Example 1 Preparation of gold nanoshells (GNSs) for the SERS substrate

[0032] (a) Preparation of amino-functionalized SiO2 ethanol sol: Add 50 μL of γ-aminopropyltriethoxysilane to 100 mL of SiO2 ethanol sol (diameter about 110 nm, 0.01 mg / mL), mix well and let stand at 4 °C for 12 hours; treat at 80 °C for 120 minutes; wash three times with ethanol and concentrate to 20 mL for standby.

[0033] (b) Preparation of gold nanoshell precursor complex: Slowly add the above-mentioned amino-functionalized SiO2 ethanol sol to 750 mL of 2 - 5 nm colloidal gold solution prepared by the sodium borohydride reduction method, stir for 30 minutes, centrifuge at 3000 rpm to collect the precipitate, and redisperse it in 50 mL of pure water.

[0034] (c) Synthesis of gold nanoshells: Take 5 mL of the above gold nanoshell precursor complex and add it to 1000 mL of 0.125% chloroauric acid aqueous solution (containing 200 mg of potassium carbonate), add 5 mL of 1% hydrogen peroxide aqueous solution, stir at room temperature for 30 minutes, let it settle naturally, and collect the gold nanoshells (seeFigure 2 )。

[0035] Example 2 Integrated SERS-Active Microneedles

[0036] Take an acupuncture needle and wash it three times with absolute ethanol. After drying, a clean-surface acupuncture needle is obtained. Immerse the body of the acupuncture needle into a 30% (w / v) polymethyl methacrylate solution (PMMA). After a layer of insulating protective layer is formed on the outer layer of the acupuncture needle body, let it stand at room temperature until dry (for more than 2 hours). Use a 50X microscopic laser to etch small holes in the insulating layer, and then use electrocorrosion to etch the needle body at the rupture of the insulating layer to form a groove accounting for 1 / 2 - 1 / 3 of the needle body diameter. After functionalizing the acupuncture needle with γ-mercaptopropyltriethoxysilane, use methyl methacrylate (MMA) to dissolve and remove the polymer insulating protective layer on the outer layer of the acupuncture needle body, ensuring that only the mercapto-functionalized groove on the subsequent acupuncture needle can adsorb the SERS probe. Wash with absolute ethanol and dry to obtain a mercapto-functionalized acupuncture needle. Then, drop the SERS substrate prepared in Example 1 into the mercapto-functionalized groove, let it dry naturally, and wipe off the SERS substrate on the surface of the needle body outside the groove to obtain the SERS-active microneedle (see Figure 3 )。

[0037] Example 3 Preparation of Hydrogen Peroxide-Responsive SERS-Active Microneedles

[0038] Immerse the active microneedles prepared in Example 2 into an aqueous solution of 4-mercaptobenzeneboronic acid at 1×10 -3 mol / L (containing different concentrations of mercaptoethanol, and the molar ratios of 4-mercaptobenzeneboronic acid to mercaptoethanol are 1:1, 3:1, 9:1, 27:1, 81:1, 1:0) for more than 30 minutes to obtain hydrogen peroxide-responsive SERS-active microneedles, and measure the initial spectrum; then react with hydrogen peroxide at 200 μmol / L at 37 °C for 5 minutes and measure the spectrum after the reaction. The ratio of I 420 / I 393 is as shown in Figure 4 . The results show that co-incubation with mercaptoethanol, a molecule smaller than 4-mercaptobenzeneboronic acid, can effectively improve the reaction degree of 4-mercaptobenzeneboronic acid with hydrogen peroxide, enabling the established hydrogen peroxide-responsive SERS-active microneedles to detect lower concentrations of hydrogen peroxide. When the molar ratio of 4-mercaptobenzeneboronic acid to mercaptoethanol reaches 1:1, the ratio of I 420 / I 393 in the initial spectrum before the reaction is significantly higher, which is not conducive to accurate quantification in the detection of low-concentration hydrogen peroxide. Therefore, a molar ratio of 3:1 of 4-mercaptobenzeneboronic acid to mercaptoethanol is used for subsequent tests. Figure 5The standard curves for hydrogen peroxide detection established for SERS-active microneedles adsorbed with 4-mercaptobenzeneboronic acid and SERS-active microneedles adsorbed with 4-mercaptobenzeneboronic acid and mercaptoethanol (3:1) show that the addition of mercaptoethanol can reduce the detection limit by about one order of magnitude.

[0039] Example 4 Preparation of Polyamine-responsive SERS-active Microneedles

[0040] 0.5 μL of polyamine oxidase at different concentrations (0.001, 0.01, 0.1, 1, 10 mU / mL) was dropped into the grooves of the optimized hydrogen peroxide-responsive SERS-active microneedles in Example 3. After natural drying, polyamine-responsive SERS-active microneedles were obtained. Polyamines (the polyamines used in this patent are a mixture of cadaverine, putrescine, tyramine, and histamine, which are common in meat spoilage, in equimolar amounts) were diluted and condensed into hydrogels with 2% (w / v) agarose to prepare hydrogels with a polyamine concentration of 50 mg / L to simulate spoiled meat. The above polyamine-responsive SERS-active microneedles were inserted into the hydrogels and incubated at 37 °C for 5 minutes, and the Raman spectra were detected to optimize the concentration of polyamine oxidase used. As Figure 6 shown, when the enzyme concentration reaches 0.1 mU / mL and at 37 °C for 5 minutes, the ratio of I 420 / I 393 reaches above 1.5. Therefore, the polyamine-responsive SERS-active microneedles prepared with this concentration of polyamine oxidase were used for subsequent experiments.

[0041] Example 5 Standard Curve of Polyamine Content and Raman Characteristic Peak Ratio

[0042] Polyamines were diluted and condensed into hydrogels with 2% (w / v) agarose to prepare hydrogels with different polyamine concentrations (5, 10, 20, 50, 100, 200, 500, 1000 mg / mL) to simulate meat. The optimized polyamine-responsive SERS-active microneedles in Example 4 were inserted into the hydrogels and incubated for 5 minutes, and the Raman spectra were detected to calculate the relationship between polyamine concentration and Raman signal ratio and draw a curve as Figure 7 .

[0043] Example 6 Detection of Histamine Content in Prepared Food Samples

[0044] Fresh fish and livestock meat were purchased from the market and placed in self-sealing bags at room temperature. The polyamine content of the prepared food stored for 0, 3, 6, 12, and 24 hours was detected respectively. The prepared polyamine-responsive SERS-active microneedles were inserted into the prepared food samples and incubated for 5 minutes. After taking them out, the Raman spectra were detected to calculate the Raman signal ratio. According to Figure 7 , the polyamine content in the prepared food to be measured can be calculated ([[]] Figure 8 ).

[0045] The detection of polyamines by traditional methods, such as immunology (taking about 10 - 20 h) and chromatography methods (taking about 2 - 4 h), has a relatively slow reaction rate. The detection method described in the present invention takes less time (less than 8 min), which is shortened by 15 - 150 times, and also shortens by more than 10 times compared with the biosensor technology developed in recent years (1 - 2 h). In terms of sensitivity, the lowest detection limit (0.01 mg / kg) of the ultrasensitive detection method described in the present invention can effectively indicate the polyamine content in prefabricated dishes, timely detect spoiled prefabricated dishes, and is 1 - 2 orders of magnitude lower than most traditional methods (0.1 - 10 mg / kg). At the same time, the detection tool described in the present invention has a low production cost, and also greatly reduces the reagent cost and labor cost of sample pretreatment.

Claims

1. A method for preparing a SERS probe for specific detection of polyamines, characterized in that: The following steps are involved: (1) Preparation of gold nanostructured SERS substrate; (2) dropping the SERS substrate into the thiol-functionalized groove of the acupuncture needle to obtain a SERS-active microneedle; (3) immersing the SERS active microneedles in a 4-mercaptophenylboric acid aqueous solution containing mercaptoethanol to obtain hydrogen peroxide responsive SERS active microneedles; (4) Polyamine oxidase is added dropwise into the groove of the hydrogen peroxide-responsive SERS-active microneedle to specifically detect the polyamine SERS probe.

2. The SERS probe for specific detection of polyamines according to claim 1, characterized in that: The gold nanostructured SERS substrate described in step (1) includes AuNFs or GNSs.

3. The SERS probe for specific detection of polyamines according to claim 1, characterized in that: Step (2) also includes a pretreatment step for the acupuncture needle, including: immersing the acupuncture needle in a polymethyl methacrylate solution until an insulating protective layer is formed on the outer layer of the acupuncture needle body, and then standing at room temperature until it dries; etching a groove on the needle body, functionalizing the groove with thiol, and then removing the polymer insulating protective layer on the outer layer of the acupuncture needle body, and washing with anhydrous ethanol and drying.

4. The SERS probe for specific detection of polyamines according to claim 3, characterized in that: The groove occupies 1 / 2-1 / 3 of the diameter of the needle body.

5. The SERS probe for specific detection of polyamines according to claim 1, characterized in that: In the aqueous solution of 4-mercaptophenylboric acid containing mercaptoethanol described in step (3), the molar ratio of 4-mercaptophenylboric acid to mercaptoethanol is 1 to 3:

1.

6. The SERS probe for specific detection of polyamines according to claim 1, characterized in that: The concentration of polyamine oxidase in step (4) is 0.1-10 mU / mL. 7 . The SERS probe for specific detection of polyamines prepared by the method for preparing the SERS probe for specific detection of polyamines according to any one of claims 1 to 6 .

8. Use of the SERS probe for specific detection of polyamines as claimed in claim 7 in detecting the polyamine content in pre-prepared dishes.

9. A method for detecting the polyamine content of pre-prepared dishes, characterized in that: The following steps are involved: (1) preparing hydrogels containing different polyamine concentrations, piercing the hydrogels with the SERS probe for specific detection of polyamines as claimed in claim 7, incubating for 5 minutes, detecting Raman spectra, calculating the relationship between polyamine concentration and Raman signal ratio, and drawing a polyamine concentration curve; (2) The SERS probe for specific detection of polyamines as claimed in claim 7 is inserted into the prepared dish to be detected, and the dish is incubated for 5 minutes. After the dish is taken out, the Raman spectrum is detected, and the Raman signal ratio is calculated and then introduced into the polyamine concentration curve.

Images