Preparation method of 3D nano-silver tree / urchin-like gold and application of 3D nano-silver tree / urchin-like gold in uric acid SERS detection

Abstract

The invention relates to a preparation method of 3D nano-silver tree / urchin-shaped gold and application of the 3D nano-silver tree / urchin-shaped gold in uric acid SERS (Surface Enhanced Raman Scattering) detection, based on the characteristics of gold and silver nanoparticles and the advantage that a 3D SERS substrate is easy to form more hot spots, the 3D nano-silver tree / urchin-shaped gold is prepared by applying a simple and easy-to-control replacement reduction method to serve as an SERS substrate, and the SERS substrate is used for detecting uric acid. The SERS substrate has a remarkable Raman enhancement effect on uric acid, the concentration ranges from 5 * 10 <-4 > mol / L to 1 * 10 <-7 > mol / L, and the SERS intensity (ISERS) at the position of 1400 cm <-1 > and the concentration logarithm value of the uric acid are in a good linear relation. The SERS substrate has the advantages of being easy to prepare, high in sensitivity, good in stability and uniformity and the like, and has good application value in uric acid detection.

Description

Technical Field

[0001] The present invention relates to the field of surface enhanced Raman detection technology, and in particular to a preparation method of a 3D nano silver tree / sea urchin-shaped gold and an application thereof in uric acid SERS detection. Background Art

[0002] Uric acid is a product of purine decomposition in the human body. It is the best biochemical marker for diagnosing gout caused by purine metabolism disorders. It is also an important marker for cardiovascular and cerebrovascular diseases, diabetes, hypertension, renal failure and other diseases. In addition, it can also be used as an important indicator of preeclampsia. Under normal circumstances, the concentration of uric acid in urine is 160-1000 mg / L, and the concentration of uric acid in blood is 35-70 mg / L. The saturation value of uric acid in the blood is 70 mg / L. Once it reaches or exceeds this level, uric acid precipitates in the form of crystals and deposits in the kidneys, subcutaneous tissue, tendons, cartilage, joint capsules and synovium, leading to a series of complications, affecting the normal function of human cells, and is diagnosed as hyperuricemia. When the uric acid concentration is greater than 0.4 mmol / L, it indicates severe preeclampsia. In addition, with the continuous improvement of people's living standards, the incidence of diseases such as gout caused by hyperuricemia has gradually increased, and the incidence population is getting younger and younger. Therefore, the detection of uric acid content is of great significance in clinical diagnosis. At present, the main methods for detecting uric acid include phosphotungstic acid reduction method, enzyme detection method, liquid chromatography, electrochemical method, etc., but these methods have some shortcomings, such as being mostly suitable for late preeclampsia testing, or cumbersome and time-consuming operation, or low sensitivity, etc. Therefore, it is necessary to develop a fast, simple, sensitive and accurate method for detecting uric acid.

[0003] Chinese invention patent 201110129164.0 discloses a quantitative detection method of uric acid based on surface enhanced Raman spectroscopy technology, wherein NaOH and hydroxylamine hydrochloride are added to AgNO3 solution to obtain silver colloid; the silver colloid is centrifuged to obtain high-concentration silver colloid; different concentrations of uric acid solution are mixed with high-concentration silver colloid and K2SO4 solution is added for Raman spectroscopy test to obtain uric acid SERS spectrum; high-concentration silver colloid is taken for test to obtain silver colloid background SERS spectrum; the silver colloid background Raman signal is used as internal standard for intensity normalization to establish a uric acid SERS spectrum line relative intensity-concentration standard working curve; the SERS spectrum of unknown concentration uric acid solution is compared with the uric acid SERS spectrum relative intensity-concentration standard working curve to infer the uric acid concentration. This technical solution uses common silver colloid aggregates as SERS substrates, the sensitivity of detecting uric acid is not high, and the preparation process requires the use of electrolyte K2SO4 to induce silver colloid aggregation, and the preparation process is complicated. Summary of the invention

[0004] The technical problem to be solved by the present invention is: to provide a preparation method of a 3D nano silver tree / sea urchin-shaped gold and its application in uric acid SERS detection. The 3D nano silver tree / sea urchin-shaped gold is prepared by chemical reduction method. It has a 3D nanostructure and has the advantages of simple preparation, high sensitivity, good stability and uniformity as a SERS substrate. It has good application value in uric acid detection.

[0005] The technical solution to solve the above technical problem is: a method for preparing a 3D nano silver tree / sea urchin-shaped gold, comprising the following steps: A. Pretreatment of copper sheet: The copper sheet was cut into (1-1.5) × (1-1.5) cm specifications, polished with sandpaper, and then ultrasonically treated in anhydrous ethanol and ultrapure water; B. Preparation of 3D silver nanotrees: Place the pretreated copper sheet in a silver nitrate solution and soak it for 5-10 minutes at room temperature, wait for the formation of the silver nanotrees, and finally wash it with ultrapure water and blow dry it to obtain a 3D silver nanotree copper sheet for use; C. Preparation of sea urchin-shaped gold nanoparticles: 10-20 mL of ultrapure water, 250-350 μL of 12-17 mmol / L chloroauric acid solution and 350-450 μL of 58-62 mmol / L hydroxylamine hydrochloride solution were added to a beaker, magnetically stirred to uniformly mix, and then 1000-1100 μL of 38-42 mmol / L sodium hydroxide solution was added, and magnetically stirred at room temperature for 15-25 min to obtain sea urchin-shaped gold nanoparticles; D. Assembly of silver nanotrees / sea urchin-shaped gold nanoparticles: The sea urchin-shaped gold nanoparticles were assembled onto the 3D silver nanotrees by physical deposition, i.e., the 3D silver nanotree copper sheet was placed in a crucible, just covering the bottom of the crucible, and 10-15 mL of the sea urchin-shaped gold nanoparticle solution prepared in step C was added, and the mixture was allowed to stand at room temperature for 45-55 min. After drying at room temperature, the mixture was washed alternately with up water and ethanol to obtain the 3D nanosilver tree / sea urchin-shaped gold.

[0006] Furthermore, in step B, the concentration of the silver nitrate solution is 0.006-0.008 mol / L, and the amount used is 65-75 mL.

[0007] Furthermore, in step A, the surface is polished with sandpaper to remove copper oxide and stains until it turns rose red, and then placed in anhydrous ethanol and ultrapure water for ultrasonic treatment for 4-6 minutes respectively.

[0008] Another technical solution of the present invention is: application of the 3D nano silver tree / sea urchin-shaped gold prepared by the above method in uric acid SERS detection, comprising the following steps: (1) Drawing a standard curve: Using 3D nanosilver trees / sea urchin-shaped gold as SERS substrates, immerse multiple SERS substrates in 1.5 mL of uric acid solutions of different concentrations, take them out, dry them naturally, detect their Raman signals, and draw a uric acid standard curve; (2) The 3D nanosilver tree / sea urchin-shaped gold SERS substrate was immersed in the human serum to be tested, taken out, and naturally dried. The Raman signal was detected and the uric acid content in the human serum was calculated using the uric acid standard curve.

[0009] Furthermore, the uric acid standard curve in step (1) is: I SERS = ̶890(log C )+8066, R 2 =0.983, where I SERS represents the Raman intensity of uric acid on the substrate material, log C It represents the logarithm of uric acid concentration.

[0010] Furthermore, in step (1), the concentration of the series of uric acid solutions is 5×10 -4 ~1×10 -7 mol / L.

[0011] Furthermore, in step (1), when drawing the uric acid standard curve, the peak used as the internal standard is the Raman spectrum of the 3D nanosilver tree / sea urchin-shaped gold SERS substrate at 1400 cm -1 Spectrum Peak.

[0012] Surface enhanced Raman spectroscopy (SERS) has the advantages of high sensitivity, non-destructive detection, resistance to water interference, fingerprint recognition, etc., and is widely used in the qualitative and quantitative analysis of chemical and biological molecules. Most chemical and biological molecules have Raman activity and can be detected label-free by SERS. The key to SERS detection technology is to prepare a substrate with high activity and good uniformity. Precious metals (such as copper, silver, and gold) are common SERS substrates. Relevant studies have shown that a stronger local electromagnetic field can be generated at the sharp corners, tips, and gaps between nanoparticles of the substrate, which can easily form hot spots ("hot spots") and have a strong Raman enhancement effect, with an enhancement factor of up to 10. Compared with 1D and 2D SERS substrates, 3D SERS substrates are easy to achieve multidimensional surface plasmon coupling and are more likely to form high-density hot spots. Relevant studies have shown that compared with nano-gold, nano-silver has stronger SERS activity, but nano-gold has good biocompatibility and high stability.

[0013] Based on the characteristics of gold and silver nanoparticles and the advantage of 3D SERS substrates that they can easily form more "hot spots", the present invention uses a simple and easy-to-control replacement reduction method to prepare 3D nano silver trees / sea urchin-shaped gold as SERS substrates and uses them for the detection of uric acid. The principle is as follows Figure 1 The SERS substrate has the advantages of simple preparation, high sensitivity, good stability and uniformity, and has good application value in uric acid detection.

[0014] The present invention investigates the SERS performance of uric acid on the substrate, and the results show that the SERS substrate has a significant Raman enhancement effect on uric acid. -4 mol / L~1×10 -7 mol / L concentration range, 1400 cm -1 SERS intensity ( I SERS ) has a good linear relationship with the logarithm of uric acid concentration, and the linear fitting equation is: I SERS =890.56(log C )+8066.99 (R 2 =0.987), detection limit 2.5×10 -9 mol / L. The recoveries of the spiked samples in human serum were between 82.86% and 125.13%, with RSD (n=3) <3.5%.

[0015] The present invention uses a chemical reduction method to prepare a 3D nano silver tree / sea urchin-shaped gold, which has a 3D nanostructure and can form a high-density "hot spot" to further improve the Raman spectrum enhancement effect and detection sensitivity. Compared with China's invention patent 201110129164.0, the present invention is a solid SERS substrate, while the above patent uses common silver colloid aggregates as the SERS substrate; the morphology structure of the solid SERS substrate of the present invention is 3D nano silver tree / sea urchin-shaped gold, while the above patent is a SERS substrate of silver colloid aggregates, and the electrolyte K2SO4 is required to trigger the aggregation of silver colloids; the sensitivity of the method of the present invention in detecting uric acid is significantly higher than that of the above patent.

[0016] Below, the technical features of the preparation method of a 3D nano silver tree / sea urchin-shaped gold and its application in uric acid SERS detection of the present invention are further explained in conjunction with the accompanying drawings and examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : Schematic diagram of the preparation of the SERS substrate of the 3D nanosilver tree / sea urchin-shaped gold and the detection principle of uric acid in the present invention.

[0018] Figure 2: The results of 3D silver nanotree / sea urchin-shaped gold SERS substrate detecting RB solutions of different concentrations. Among them, a is the SERS spectra of RB solutions of different concentrations, and b is the linear relationship between the logarithm of the RB standard solution concentration and the SERS intensity.

[0019] Figure 3 : SERS spectra of uric acid on different substrates.

[0020] Figure 4 : The results of 3D silver nanotree / sea urchin-shaped gold SERS substrate detecting different concentrations of uric acid standard solutions. Among them, a is the SERS spectra of different concentrations of uric acid standard solutions, and b is the linear relationship between the logarithm of uric acid solution concentration and SERS intensity.

[0021] Figure 5 : Reproducibility test result diagram. In the figure, a is the Raman signal diagram of uric acid standard solution measured 5 times in parallel, and b is the summary diagram of Raman spectrum.

[0022] Figure 6 : Uniformity test result diagram.

[0023] Figure 7 : Stability test results. DETAILED DESCRIPTION

[0024] The reagents used in the present invention include copper sheet (99.9%, Tianjin Guangfu Fine Chemical Research Institute), silver nitrate (AR, Sinopharm Group Chemical Reagent Co., Ltd.), chloroauric acid (AR, Aladdin Industries, USA), rhodamine B (RB) (AR, Shanghai McLean Biochemical Technology Co., Ltd.), hydroxylamine hydrochloride (AR, Guangdong Guanghua Technology Co., Ltd.), uric acid (AR, West Asia Reagent), and human serum purchased from Shanghai Jinghang Biology. Example 1

[0025] A method for preparing a 3D nano silver tree / sea urchin-shaped gold comprises the following steps: A. Pretreatment of copper sheet: The copper sheet was cut into 1.3×1.3 cm size, polished with sandpaper, and the copper oxide and stains on the surface were removed until it turned rose red. It was then placed in anhydrous ethanol and ultrapure water for 5 minutes each.

[0026] B. Preparation of 3D silver nanotrees: Place the pretreated copper sheet in a silver nitrate solution (0.007 mol / L, 70 mL) and soak for 7 minutes at room temperature to allow the silver nanotrees to form. Finally, wash with ultrapure water for 3 times and blow dry with N2 to obtain the 3D silver nanotree copper sheet for later use.

[0027] C. Preparation of sea urchin-shaped gold nanoparticles: Add 15 mL of ultrapure water, 300 μL of 15 mmol / L chloroauric acid solution, and 400 μL of 60 mmol / L hydroxylamine hydrochloride solution into a beaker, stir magnetically to mix them evenly, then add 1050 μL of 40 mmol / L sodium hydroxide solution, and stir magnetically at room temperature for 20 min to obtain sea urchin-shaped gold nanoparticles.

[0028] D. Assembly of 3D silver nanotree / sea urchin-shaped gold nanoparticles: The sea urchin-shaped gold nanoparticles were assembled onto the 3D silver nanotree by physical deposition. The 3D silver nanotree copper sheet was placed in a 20 mL crucible, just covering the bottom of the crucible, and then 12 mL of the above-prepared sea urchin-shaped gold nanoparticle solution was added, and the mixture was allowed to stand at room temperature for 50 min. After drying at room temperature, the mixture was washed alternately with up water and ethanol to obtain the 3D nanosilver tree / sea urchin-shaped gold. Example 2

[0029] The application of the 3D nano silver tree / sea urchin-shaped gold prepared in Example 1 in uric acid SERS detection comprises the following steps: (1) Drawing a standard curve: Using 3D nanosilver trees / sea urchin-shaped gold as SERS substrates, multiple SERS substrates were immersed in 1.5 mL of 5×10 -4 ~1×10 -7 mol / L series of uric acid solutions, take them out, dry them naturally, detect their Raman signals, and draw a uric acid standard curve; the uric acid standard curve is: I SERS = ̶890(log C )+8066, R 2 =0.983, where I SERS represents the Raman intensity of uric acid on the substrate material, log C It represents the logarithm of uric acid concentration.

[0030] (2) The 3D nanosilver tree / sea urchin-shaped gold SERS substrate was immersed in 1.5 mL of human serum to be tested, taken out, and naturally dried. The Raman signal was then detected, and the uric acid content in human serum was calculated using the uric acid standard curve.

[0031] In this embodiment, the instrument parameters for detecting Raman signals are: 638 nm, 37.5 mW, integration time uric acid is 20 s, filter 25%. In step (1) of drawing the standard curve, when drawing the uric acid standard curve, the peak used as the internal standard is the 3D nanosilver tree / sea urchin-shaped gold SERS substrate Raman spectrum 1400 cm -1 Spectrum Peak.

[0032] The following is a description of the development process of this invention: 1. Enhancement effect of 3D silver nanotree / sea urchin-shaped gold nanoparticle SERS substrate on RB Raman spectroscopy.

[0033] Taking RB as the Raman signal molecule, the Raman enhancement effect of the substrate was investigated. The SERS spectra of a series of RB solutions on the substrate are shown in Figure 2. Figure 2 As shown in a. Figure 2 It can be seen from a that as the concentration of RB solution increases, its SERS signal gradually increases. -1 The characteristic peak enhancement effect at is significant, so the SERS signal at this location is selected as the quantitative basis for RB. Figure 2 b shows that in 1×10 -5 mol / L~1×10 -8 In the range of mol / L, the logarithm of RB solution concentration is I SERS It shows a good linear relationship, and its linear fitting equation is: I SERS = 2058 (log C )+17816 ( R 2 =0.989). The RB enhancement factor is calculated using the formula , where I SERS is the Raman signal value on the substrate, I bare is the Raman signal value on the die, C SERS for The concentration of the signal molecule detected on the substrate, C bare for The concentration of signal molecules on the die. According to the experimental results, I SERS =1420, C SERS =1.0×10 -8 mol / L、I bare =749, C bare =1.0 mol / L, substituting into the above formula, the enhancement factor EF is 1.9×10 7 , indicating that the substrate has a good enhancement effect on the Raman spectrum of RB, providing a basis for the SERS detection of uric acid.

[0034] 2. Raman enhancement effect of uric acid on 3D silver nanotree / sea urchin-shaped gold nanoparticle SERS substrate.

[0035] Take 1.5 mL of 1.0×10 -4The copper sheet, 3D silver nanotree sheet, and 3D silver nanotree / sea urchin-shaped gold were soaked in 100 mol / L uric acid solution. After 20 min, the SERS signals were measured after natural drying. The results are shown in Figure 2. Figure 3 .Depend on Figure 2 It can be seen that when the copper sheet is used as the substrate, no Raman peak is detected; when the 3D silver nanotree is used as the substrate, multiple Raman peaks appear, among which 1400 cm -1 The Raman peak at 1400 cm is the most obvious, with an intensity of 2538. When 3D silver nanotrees / gold nanoparticles are used as SERS substrates, -1 The Raman peak at is significantly enhanced, indicating that the Raman enhancement effect of the SERS substrate is significantly improved after the gold nanoparticles are assembled into the 3D silver nanotrees.

[0036] 3. Performance of 3D silver nanotree / sea urchin-shaped gold SERS substrate in detecting uric acid.

[0037] The 3D silver nanotree / sea urchin-shaped gold SERS substrate was used to detect different concentrations of uric acid standard solutions. The results are as follows Figure 4 As shown. Figure 4 As can be seen from a, the SERS signal intensity increases with the concentration of uric acid, with the 1400 cm -1 The characteristic peak of 1400 cm -1 The SERS signal value at is quantified. Figure 4 b The standard curve of uric acid shows that at 5×10 -4 ~1×10 -7 mol / L, the logarithm of uric acid concentration is I SERS It shows a good linear relationship, and the linear fitting equation is: I SERS = ̶890(log C )+8066( R 2 =0.983), and the detection limit was 2.5×10 -9 mol / L (LOD=3δ / k).

[0038] Reproducibility study. -7 The mol / L uric acid standard solution was measured 5 times in parallel, and the Raman signals of 3 different points were collected each time. The results are shown in Figure 5 As shown in a, the relative standard deviation (RSD) is 6.45%, indicating good reproducibility. -6 mol / L standard solution, collect Raman signals at the same point 15 times, summarize and compare their Raman spectra as shown in the figure below Figure 5 As shown in b, the results show that after multiple laser irradiations, the 1400 cm-1 The Raman peak intensity at the position only decreased slightly, but still showed good repeatability, with a relative standard deviation (RSD) of 4.78%.

[0039] Investigation of the uniformity of SERS substrate signals. -5 The test was conducted at 5 different locations on the same substrate immersed in 1 mol / L uric acid standard solution. Each point was tested 3 times in parallel. The results are shown in the figure. Figure 6 As shown in the figure, the relative standard deviation (RSD) is 3.01%, which shows that the SERS signal of the substrate has good uniformity.

[0040] Stability test. Take 1.5 mL 5×10 -4 The SERS substrate was immersed in mol / L standard uric acid solution, dried naturally, and then placed for different days (1, 7, 15, 22, and 31) to detect the 1400 cm -1 The Raman peak intensity at , each time parallel detection 3 times, the results are as follows Figure 7 As shown. Figure 7 It can be seen that as the placement time of the SERS substrate increases, the Raman signal intensity changes little before 22 days, and the Raman signal intensity decreases slightly on the 31st day, indicating that the SERS substrate has good stability within 22 days.

[0041] In order to verify the feasibility of this method for detecting actual samples, the SERS substrate was used to detect the uric acid content in human serum, and a spike test was performed. The results are shown in Table 1.

[0042]

[0043] As shown in Table 1, the RSDs were all ≤3.10%, and the recovery rates were between 82.43% and 125.13%. According to the determination results, the uric acid content in human serum was about 3.27 mg / dL, which was lower than the uric acid concentration range of human blood (3.5-7.0 mg / dL). The uric acid content in human serum was about 3.27 mg / dL (1.95×10 -4 mol / L), which is consistent with the 120-480 μmol / L in the literature. Therefore, it can be used to detect the uric acid content in serum samples.

Claims

1. A method for preparing 3D nano silver tree / sea urchin-shaped gold, characterized in that: The following steps are involved: A. Pretreatment of copper sheet: The copper sheet was cut into (1-1.5) × (1-1.5) cm specifications, polished with sandpaper, and then ultrasonically treated in anhydrous ethanol and ultrapure water; B. Preparation of 3D silver nanotrees: Place the pretreated copper sheet in a silver nitrate solution and soak it for 5-10 minutes at room temperature, wait for the formation of the silver nanotrees, and finally wash it with ultrapure water and blow dry it to obtain a 3D silver nanotree copper sheet for use; C. Preparation of sea urchin-shaped gold nanoparticles: 10-20 mL of ultrapure water, 250-350 μL of 12-17 mmol / L chloroauric acid solution and 350-450 μL of 58-62 mmol / L hydroxylamine hydrochloride solution were added to a beaker, magnetically stirred to uniformly mix, and then 1000-1100 μL of 38-42 mmol / L sodium hydroxide solution was added, and magnetically stirred at room temperature for 15-25 min to obtain sea urchin-shaped gold nanoparticles; D. Assembly of silver nanotrees / sea urchin-shaped gold nanoparticles: The sea urchin-shaped gold nanoparticles were assembled onto the 3D silver nanotrees by physical deposition, i.e., the 3D silver nanotree copper sheet was placed in a crucible, just covering the bottom of the crucible, and then 10-15 mL of the sea urchin-shaped gold nanoparticle solution prepared in step C was added, and the mixture was allowed to stand at room temperature for 45-55 min. After drying at room temperature, the mixture was washed alternately with up water and ethanol to obtain the 3D nanosilver tree / sea urchin-shaped gold.

2. The method for preparing the 3D nano silver tree / sea urchin-shaped gold according to claim 1, characterized in that: In step B, the concentration of the silver nitrate solution is 0.006-0.008 mol / L, and the amount used is 65-75 mL.

3. The method for preparing the 3D nano silver tree / sea urchin-shaped gold according to claim 1 or 2, characterized in that: In step A, the surface is polished with sandpaper to remove copper oxide and stains until it turns rose red, and then placed in anhydrous ethanol and ultrapure water for ultrasonic treatment for 4-6 minutes respectively.

4. Application of the 3D nano silver tree / sea urchin-shaped gold prepared according to any one of claims 1 to 3 in uric acid SERS detection, characterized in that: The following steps are involved: (1) Drawing a standard curve: Using 3D nanosilver trees / sea urchin-shaped gold as SERS substrates, immerse multiple SERS substrates in 1.5 mL of uric acid solutions of different concentrations, take them out, dry them naturally, detect their Raman signals, and draw a uric acid standard curve; (2) The 3D nanosilver tree / sea urchin-shaped gold SERS substrate was immersed in the human serum to be tested, taken out, and naturally dried. The Raman signal was detected and the uric acid content in the human serum was calculated using the uric acid standard curve.

5. The use of the 3D nano silver tree / sea urchin-shaped gold in uric acid SERS detection according to claim 4, characterized in that: The uric acid standard curve in step (1) is: I SERS = ̶890(log C )+8066, R 2 =0.983, where I SERS represents the Raman intensity of uric acid on the substrate material, log C It represents the logarithm of uric acid concentration.

6. The use of the 3D nano silver tree / sea urchin-shaped gold in uric acid SERS detection according to claim 4, characterized in that: In step (1), the concentration of the series of uric acid solutions is 5×10 -4 ~1×10 -7 mol / L.

7. The use of the 3D nano silver tree / sea urchin-shaped gold in uric acid SERS detection according to claim 4, characterized in that: In step (1), when drawing the uric acid standard curve, the peak used as the internal standard is the 3D nanosilver tree / sea urchin-shaped gold SERS substrate Raman spectrum 1400 cm -1 Spectrum Peak.

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