Method for rapidly detecting serum trace substances based on nano-silver sol surface enhanced Raman

Abstract

The invention provides a method for rapidly detecting serum based on surface enhanced Raman of novel nano-silver sol, and belongs to the field of trace substance detection. A nano-silver sol prepared by a microwave method is used as an enhanced substrate, serum is used as a research object, and the surface enhanced Raman spectrum is used for rapidly detecting trace substances in the serum. The method is high in detection precision, short in detection time and simple to operate, and a rapid, cheap, simple, convenient and sensitive analysis and detection method is provided for detection of trace substances in serum.

Description

Technical Field

[0001] The present invention relates to the field of trace substance detection, and particularly to a method for rapidly detecting trace substances in serum based on surface-enhanced Raman of silver nano-sol Background Art

[0002] In the rapid development of current medicine and life sciences, trace substance detection has gradually become a field of great concern in research and clinical practice. As an important part of the internal circulation in the human body, blood contains rich biochemical information. Although the content of trace substances in it is tiny, they may have a profound impact on health and diseases. These trace substances cover many categories, from metabolites, hormones, cytokines to micronutrients and environmental pollutants, and the changes in their levels are often closely related to the physiological state and pathological process of the human body.

[0003] With the continuous progress of analytical techniques, the currently used detection methods include gas chromatography, high-performance liquid chromatography, capillary electrophoresis, chemical chromogenic method, colloidal gold method, etc. However, gas chromatography and liquid chromatography have deficiencies such as complex operation of detection means, slow detection speed, and strong professional limitations. The colloidal gold method is prone to false positives, and the detection limit of the chemical method is relatively high. In this context, it is extremely urgent to develop a rapid detection method for trace substances.

[0004] Raman spectroscopy is a kind of scattering spectroscopy, which can reflect the characteristic structure of molecules. However, the Raman scattering effect is a very weak process, so Raman signals are very weak. Surface-enhanced Raman spectroscopy (SERS) is to utilize a certain enhancement effect. When molecules are adsorbed on the surface of some nanoscale rough metals (such as gold, silver, copper), the Raman signals of the adsorbed substances increase geometrically, which is suitable for screening trace substances.

[0005] The success of SERS application depends on the development and preparation of the substrate. At present, the domestic and foreign research on surface-enhanced Raman scattering technology mainly focuses on the research of three kinds of substrates: nanoparticle thin films, metal nanostructure arrays, and nanoparticle sols. Nanoparticle thin films are a widely used SERS substrate. However, for monodisperse spherical particles, they need to be aggregated to improve the SERS activity. Metal nanostructure arrays are highly ordered SERS substrates, which can be prepared by nanolithography technology. This method can prepare large-scale SERS substrates in a short time, but the problem is that it is difficult to store. Nanoparticle sols are prepared by chemical methods or physical methods. Compared with traditional chemical methods, an important advantage of this method is that the colloid does not contain organic substances or ions. Therefore, under carefully controlled conditions, the chemical and physical effects of ions or other adsorbents can be studied.

[0006] The rapid detection method for trace substances in serum of the present invention is characterized by simple and fast operation, requiring few reagents and consumables, not needing professional personnel to operate, and cooperating with a surface-enhanced Raman substrate with good stability and high sensitivity, enabling non-professional personnel to complete the entire detection process in a short time, with low cost and high speed. Summary of the Invention

[0007] The object of the present invention is to provide a method for rapidly detecting trace substances in serum based on surface-enhanced Raman of silver nanosol, providing a rapid, inexpensive, simple and accurate analytical detection method for trace substances in serum.

[0008] The present invention provides a method for rapidly detecting trace substances in serum based on surface-enhanced Raman of silver nanosol, comprising the following steps:

[0009] S1: Synthesis and post-treatment of silver nanosol;

[0010] S2: Optimizing the conditions of surface-enhanced Raman spectroscopy for silver nanosol;

[0011] S3: Treating the test sample of trace substances in the serum to be detected and collecting and analyzing Raman spectra;

[0012] As a preferred embodiment of the present invention, step S1 includes the following steps:

[0013] 1) Heating the silver nitrate solution and slowly dropping it into the pre-prepared trisodium citrate solution while stirring. It is necessary to ensure an appropriate dropping speed during the process to fully mix and react; the concentration ratio of the silver nitrate solution to the trisodium citrate solution is 1-5:1.

[0014] 2) Placing the mixed solution in a microwave oven, continuing to heat without stirring and then taking it out, cooling to room temperature to obtain the silver nanosol mother liquor;

[0015] 3) Centrifuging the silver nanosol mother liquor in batches, removing the supernatant to obtain a precipitate; dispersing the precipitate in ultrapure water or absolute ethanol to obtain a uniform silver nanosol dispersion.

[0016] Furthermore, in the final mixed solution of step 1), the concentration of silver nitrate is 0.01-0.05%; the concentration of trisodium citrate is 0.005-0.01%; in step 2), the preset heating device is 300-700W and the reaction time is 1-10 minutes; in step 3), in the silver nanosol dispersion, the concentration of the precipitate is 0.01-2mg / mL.

[0017] As a preferred embodiment of the present invention, in step 1), the heating treatment time of the silver nitrate solution is 1-10 minutes.

[0018] As a preferred embodiment of the present invention, in step S2, the specific steps are as follows: Take a rhodamine standard, place the nano - silver sol treated in step 3) of step S1 into sample bottles in different ratios, mix well and then collect Raman spectra to obtain Raman spectra; wherein, the volume ratio of the rhodamine standard solution to the nano - silver sol is 1 - 0.25, the concentration of the nano - silver sol is 0.1 - 1 mg / mL; the concentration of the rhodamine standard is 0.1 - 2 mM, and the solvent is anhydrous methanol or ultrapure water.

[0019] As a preferred embodiment of the present invention, the specific steps of step S3 are as follows: Add the serum sample to be tested into a sample bottle, add the treated nano - silver sol, mix well and then collect Raman spectra;

[0020] As a preferred embodiment of the present invention, in step S3, the concentration of the nano - silver sol is 0.5 mg / mL and the dosage is 1 mL.

[0021] As a preferred embodiment of the present invention, the serum trace substances include L - serine, glutathione, phenylalanine, amide III, tryptophan, lipids, fatty acids and amide I.

[0022] As a preferred embodiment of the present invention, in step S3, the Raman spectrum collection parameters are set as follows: excitation wavelength 785 nm, energy 30 mW, scanning range 200 - 2600 cm -1 , integration time 10 s, integrated three times.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] The present invention does not use a nucleating agent and prepares silver nanomaterials by the microwave method. The preparation method is simple, with fewer reagents, fewer steps, high efficiency, low cost, and high - quality nano - silver materials can be prepared within a fixed time. The nano - silver materials prepared by the present invention are quasi - spherical nanoparticles with uniform particle size, few impurities, good repeatability and good stability.

[0025] The present invention constructs a rapid detection method for trace substances. Applying surface - enhanced Raman spectroscopy to the detection of serum trace substances has high detection accuracy, short detection time and simple operation. Description of the Drawings

[0026] Figure 1 It is a 20KV TEM image of the nano - silver sol prepared by the microwave method;

[0027] Figure 2 It is a Raman spectrum of the nano - silver sol;

[0028] Figure 3SERS spectrum of 1 mM rhodamine 6G standard solution in Example 1. The peaks marked as 612, 774, 1090, 1128, 1182, 1312, 1362, 1511, 1575, 1598, 1651 cm -1 SERS characteristic peaks of rhodamine 6G standard.

[0029] Figure 4 SERS spectra of 1 mM rhodamine 6G standard and silver nanosol with different volume ratios. The peaks marked as 612, 774, 1090, 1128, 1182, 1312, 1362, 1511, 1575, 1598, 1651 cm -1 SERS characteristic peaks of rhodamine 6G standard.

[0030] Figure 5 SERS spectrum of serum sample after substrate enhancement. The peak marked as 810 cm -1 indicating that the sample contains l-serine and glutathione; 889, 1016, 1202 cm -1 indicating that the sample contains phenylalanine; 1255 cm -1 indicating that the sample contains amide III; 1363, 1557 cm -1 indicating that the sample contains tryptophan; 1646 cm -1 indicating that the sample contains lipids and fatty acids; 1679 cm -1 indicating that the sample contains amide I. Detailed implementation mode

[0031] The present invention provides a method for rapid surface-enhanced Raman detection of trace substances in human serum based on a novel silver nanosol. The following is a detailed description of the embodiments of the present invention. The embodiments give detailed implementation methods and specific operation procedures, but the protection scope of the present invention is not limited to the following embodiments.

[0032] Example 1:

[0033] Weigh 0.0441 g of trisodium citrate and dissolve it in 5 mL of ultrapure water for standby. Take 150 mL of 0.02% silver nitrate solution and heat it in a microwave oven for 3 min. Slowly drip the heated solution into 5 mL of the pre-prepared trisodium citrate solution while stirring. Put the mixed solution back into the microwave oven and continue heating; then keep stirring until the solution temperature drops to room temperature. Then centrifuge the solution at 8000 rpm for 5 min. Remove the supernatant to obtain the precipitate. Repeat the centrifugation separation process 3 - 5 times, and disperse the precipitate in ultrapure water or absolute ethanol to obtain silver nanosol with a concentration of 0.1 g / mL; the TEM characterization diagram of the material is Figure 1As shown, the prepared silver nanoparticles have a complete morphology and an average particle size.

[0034] Example 2:

[0035] The Raman substrate material was the silver nanoparticle sol synthesized and processed in Example 1. After taking 1 mL of the processed silver nanoparticle sol, Raman spectrum collection was carried out. The Raman spectrum collection parameters were set as follows: excitation wavelength 785 nm, energy 30 mW, scanning range 200 - 2600 cm -1 , integration time 10 s, integrated three times.

[0036] Figure 2 Those marked at 805, 928, 951, 1030, 1173, 1299, 1399, 1606, 1707 cm -1 were used as the SERS characteristic peaks of the silver nanoparticle sol.

[0037] Take 1 mL of an aqueous solution of 1 mM rhodamine 6G standard and add it to a sample bottle. Then add 1 mL of the processed silver nanoparticle sol and carry out Raman spectrum collection (1 mM rhodamine 6G standard is used to evaluate the performance of the substrate material). The Raman spectrum collection parameters were set as follows: excitation wavelength 785 nm, energy 30 mW, scanning range 200 - 2600 cm -1 , integration time 10 s, integrated three times.

[0038] Figure 3 Those marked at 612, 774, 1090, 1128, 1182, 1312, 1362, 1511, 1575, 1598, 1651 cm -1 were used as the SERS characteristic peaks of the rhodamine 6G standard.

[0039] Example 3:

[0040] The Raman substrate material was the silver nanoparticle sol synthesized and processed in Example 1. Take 1 mM rhodamine 6G standard, add it to the processed silver nanoparticle sol with different volume ratios and mix them, then carry out Raman spectrum collection. The Raman spectrum collection parameters were set as follows: excitation wavelength 785 nm, energy 30 mW, scanning range 200 - 2600 cm -1 , integration time 10 s, integrated three times.

[0041] Figure 4 Those marked at 612, 774, 1090, 1128, 1182, 1312, 1362, 1511, 1575, 1598, 1651 cm -1 were used as the SERS characteristic peaks of the rhodamine 6G standard.

[0042] Example 4:

[0043] Combined with Examples 1-3; the Raman spectroscopy acquisition parameters were set as follows: excitation wavelength 785 nm, energy 30 mW, scanning range 200-2600 cm -1 , integration time 10 s, integrated three times.

[0044] In this example, trace substances in serum specimens were detected. The Raman substrate material was the nano-silver sol synthesized and processed in Example 1.

[0045] Prepare 2 mL of serum specimen, take 1 mL of the solution to be tested and add the processed nano-silver sol, and perform Raman spectroscopy acquisition after mixing. As Figure 5 shown, the SERS spectrogram was measured. The 810 cm -1 marked in the figure indicates that the specimen contains l-serine and glutathione; 889, 1016, 1202 cm -1 indicates that the specimen contains phenylalanine; 1255 cm -1 indicates that the specimen contains amide III; 1363, 1557 cm -1 indicates that the specimen contains tryptophan; 1646 cm -1 indicates that the specimen contains lipids and fatty acids; 1679 cm -1 indicates that the specimen contains amide I. (Due to the influence of factors such as instrument stability, the shift of a few wavenumbers is considered reasonable).

[0046] The above-described embodiments only represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.

Claims

1. A method for rapid detection of trace substances in serum based on surface-enhanced Raman of silver nano-sol, characterized in that, The method includes the following steps: S1: Synthesis and post-treatment of silver nanosol; S2: Optimization of surface-enhanced Raman spectroscopy conditions for silver nanosol; S3: Treatment and Raman spectroscopy acquisition and analysis of the trace substances in the serum sample to be tested.

2. The method for rapid detection of trace substances in serum based on surface-enhanced Raman of silver nanosol according to claim 1, wherein: Step S1 includes the following steps: 1) Heat the silver nitrate solution and slowly dropwise add the pre-prepared trisodium citrate solution while stirring; the concentration ratio of the silver nitrate solution to the trisodium citrate solution is 1-5:1; 2) Place the mixed solution in a microwave oven, continue heating without stirring and then take it out, cool it to room temperature to obtain the silver nanosol mother liquor; 3) Centrifuge the silver nanosol mother liquor in batches, remove the supernatant to obtain a precipitate; disperse the precipitate in ultrapure water or absolute ethanol to obtain a uniform silver nanosol dispersion.

3. The method for rapid detection of trace substances in serum based on surface-enhanced Raman of silver nanosol according to claim 2, wherein: In the final mixed solution of step 1), the concentration of silver nitrate is 0.01-0.05%; the concentration of trisodium citrate is 0.005-0.01%; in step 2), the heating power is 300-700W and the reaction time is 1-10 minutes; in step 3), in the silver nanosol dispersion, the concentration of the precipitate is 0.01-2mg / mL.

4. The method for rapid detection of trace substances in serum based on surface-enhanced Raman of silver nano-sol according to claim 2, wherein: In step 1), the heating treatment time of the silver nitrate solution is 1-10 minutes.

5. The method for rapid detection of trace substances in serum based on surface-enhanced Raman of silver nanoparticles sol according to claim 2, wherein: The specific steps of S2 are: Take 1 volume of rhodamine standard, put the silver nanosol treated in step 3) of step S1 into the sample bottle in different proportions, perform Raman spectroscopy acquisition to obtain Raman spectra; among them, the volume ratio of the rhodamine standard solution to the silver nanosol is 1-0.25, the concentration of the silver nanosol is 0.1-1mg / mL; the concentration of the rhodamine standard is 0.1-2mM, and the solvent is absolute methanol or ultrapure water.

6. The method for rapid detection of trace substances in serum based on surface-enhanced Raman of silver nanosol according to claim 1, wherein: The specific steps of S3 are: Add the serum sample to be tested into the sample bottle, and then add the treated silver nanosol for Raman spectroscopy acquisition.

7. The method for rapid surface-enhanced Raman detection of trace substances in serum based on the novel silver nano-sol according to claim 6, characterized in that: In step S3, the concentration of the silver nanosol is 0.5mg / mL and the dosage is 1mL.

8. The method for rapid detection of trace substances in serum based on surface-enhanced Raman of silver nanosol according to claim 1, wherein: In step S3, the serum trace substances include L-serine, glutathione, phenylalanine, amide III, tryptophan, lipids, fatty acids and amide I.

9. The method for rapid detection of trace substances in serum based on surface-enhanced Raman of silver nanoparticles sol according to claim 1, wherein: In step S3, the Raman spectroscopy acquisition parameters are set as follows: excitation wavelength 785 nm, energy 30 mW, scanning range 200 - 2600 cm -1 , integration time 10 s, integrated three times.

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