A method for rapid detection of orlistat based on protein-modified gold nanoparticles

Abstract

The present invention discloses a method for rapid detection of orlistat based on protein-modified gold nanoparticles. Based on the strong interaction between orlistat and protein and the affinity of protein to metal nanoparticles, gold nanoparticles are modified with protein to obtain Au@BSA composite substrate, and a SERS detection method for orlistat is established. The method solves the problem that orlistat cannot be close to the surface of metal nanoparticles due to its hydrophobicity, thus failing to obtain signal enhancement. The modification process is simple and easy to operate, and no complex instruments are required. Rapid qualitative and quantitative analysis of orlistat in traditional Chinese medicine can be achieved in a short time, and the method has great application potential in on-site detection.

Description

Technical Field

[0001] The invention relates to a method for quickly detecting orlistat based on protein-modified gold nanoparticles, and belongs to the field of traditional Chinese medicine detection. Background Art

[0002] Orlistat is a potent and specific gastrointestinal lipase inhibitor that blocks the hydrolysis and absorption of dietary fat and has the effect of controlling body weight. The main adverse reactions reported so far are constipation, abdominal pain, diarrhea, dizziness, menstrual disorders, rash, etc., and there is a risk of liver damage and breast cancer.

[0003] At present, there are few studies on the detection of orlistat, especially the determination method of illegally added drugs. In 2006, the State Food and Drug Administration approved the supplementary inspection method for the drug inspection of traditional Chinese medicines for the treatment of obesity, which is only applicable to the detection of illegal additions of sibutramine, ephedrine and other drugs in traditional Chinese medicines for weight loss, and there is no detection method for the illegal addition of orlistat. The currently reported detection methods of orlistat are mainly concentrated in liquid chromatography and liquid chromatography-mass spectrometry. However, chromatographic technology has problems such as complex sample pretreatment, large instruments, and high difficulty in operation, and is not suitable for on-site rapid detection.

[0004] Surface enhanced Raman spectroscopy (SERS) has the advantages of small equipment, fast detection speed, and simple operation, and is more suitable for rapid on-site detection. Patent CN113834714A discloses the use of acetylcholinesterase inhibition rate to detect orlistat; Patent CN114280029A discloses a method for detecting orlistat using Raman spectroscopy, using silver sol and silica aerogel to prepare an enhanced matrix, which can be effectively detected at an addition ratio of 0.05% by mass; the above method has the problems of cumbersome preparation and difficult operation. In addition, the inventors found in previous studies that due to the hydrophobicity of the orlistat molecule, it is difficult for it to directly approach the surface of the metal nanoparticles, so it is difficult to obtain the Raman signal of orlistat through direct SERS detection.

[0005] Therefore, developing a method based on gold nanoparticles that can amplify the orlistat detection signal is beneficial to improving the accuracy of gold nanoparticles in the detection of orlistat and broadening the application of gold nanoparticles in the detection of orlistat. Summary of the invention

[0006] In view of the problem that orlistat cannot be directly detected by SERS using gold colloid, the present invention provides a method for directly detecting orlistat by SERS using Au@BSA as an enhanced substrate based on the strong interaction between orlistat and protein. The composite substrate first uses a simple sodium citrate reduction method to obtain gold nanosol, which is then incubated with a protein solution to prepare an Au@BSA composite substrate, thereby obtaining the SERS signal of orlistat.

[0007] The first object of the present invention is to provide a gold nanoparticle-bovine serum albumin composite substrate, and the preparation method of the gold nanoparticle-bovine serum albumin composite substrate comprises:

[0008] (1) Preparation of gold nanosol:

[0009] The potassium chloroaurate solution is heated to boiling, and a sodium citrate solution is added to obtain a mixed solution, and the mixed solution is heated until the color of the solution gradually changes from a transparent light yellow to an opaque purple-red sol state, thereby preparing a gold nanosol;

[0010] (2) Preparation of gold nanoparticle-bovine serum albumin composite substrate:

[0011] The gold nano-sol and the bovine serum albumin solution are mixed to obtain a reaction solution, which is shaken, ultrasonicated and centrifuged; the precipitate is taken out and redissolved in water to prepare a gold nano-particle-bovine serum albumin composite substrate.

[0012] In one embodiment, the mass ratio of potassium chloroaurate to sodium citrate in the mixed solution of step (1) is 0.2-1.0:0.2-0.5; and the mass concentration of potassium chloroaurate in the mixed solution is 5-20 mg / mL.

[0013] In one embodiment, the concentration of the potassium chloroaurate solution in step (1) is 0.2-1.0 mg / mL; and the concentration of sodium citrate in the mixed solution is 0.2-0.5 mg / mL.

[0014] In one embodiment, the heating condition in step (1) is heating at 100° C. to 150° C. for 30 to 120 min.

[0015] In one embodiment, in the reaction solution of step (2), the molar ratio of gold nanosol to bovine serum albumin is 1-2:10-200; and the concentration of gold nanosol in the reaction solution is 1-2 nmol / L.

[0016] In one embodiment, the concentration of the gold nanosol in step (2) is 1-2 nmol / L, and the concentration of bovine serum albumin is 10-200 nmol / L; the concentration of the gold nanosol in the re-dissolved gold nanoparticle-bovine serum albumin composite substrate is 2.1-4.3 nmol / L, and the concentration of bovine serum albumin is 1.4-28.6 nmol / L.

[0017] In one embodiment, the ultrasonic condition in step (2) is 100-150W for 10-20min.

[0018] In one embodiment, the centrifugation in step (1) and step (2) is performed at 10,000 to 15,000 r / min for 20 min.

[0019] A second object of the present invention is to provide a method for detecting orlistat, using any of the above-mentioned gold nanoparticle-bovine serum albumin composite substrates to detect orlistat, comprising the steps of:

[0020] The gold nanoparticle-bovine serum albumin composite substrate and the sample to be tested are mixed and reacted, and the Raman intensity of the characteristic peak at 620cm-1 is detected using surface enhanced Raman spectroscopy. The Raman intensity is brought into the calibration curve to calculate the content of orlistat in the sample.

[0021] In one embodiment, the standard curve is:

[0022] y=9.68x+243.72; wherein y represents the Raman intensity (au); and x represents the concentration of orlistat (mg / L).

[0023] In one embodiment, the volume ratio of the gold nanoparticle-bovine serum albumin composite substrate to the sample to be tested is 1 to 5:1; and the reaction is carried out for 10 to 30 minutes.

[0024] In one embodiment, the sample to be tested is a Chinese patent medicine.

[0025] In one embodiment, the sample sampling amount is 1 g.

[0026] In one embodiment, the sample extraction solvent is methanol, and the amount used is 10 mL.

[0027] In one embodiment, the ultrasonic time during sample extraction is 5 min.

[0028] In one embodiment, the centrifuge speed during the sample extraction process is 8000 r / min and the time is 10 min.

[0029] In one embodiment, the volume multiple of the supernatant diluted with ultrapure water during the sample extraction process is 20 to 200 times.

[0030] In one embodiment, a portable Raman spectrometer is used for detection, and the wavelength of the laser light source is 785, 633, and 532 nm; the power of the portable Raman spectrometer is 10 to 300 mW, the integration time is 1 to 10 s, and the number of integrations is 1 to 10 times.

[0031] The third object of the present invention is to provide an application of bovine serum albumin in enhancing the detection of orlistat by gold nanoparticles, wherein bovine serum albumin and gold nanoparticles are used to prepare a gold nanoparticle-bovine serum albumin composite substrate, and the gold nanoparticle-bovine serum albumin composite substrate is used to detect orlistat in a sample to be tested.

[0032] The fourth object of the present invention is to provide a product for detecting orlistat, wherein the product comprises any one of the above-mentioned gold nanoparticle-bovine serum albumin composite substrates.

[0033] Beneficial effects of the present invention:

[0034] The present invention is based on the molecular docking results between orlistat and protein, which show that there is a strong interaction between the two, and the protein has a strong affinity for metal nanoparticles. The gold nanoparticles are modified by protein to synthesize the Au@BSA composite substrate, providing a new method for detecting orlistat in Chinese patent medicines for weight loss, which is simple, fast, low-cost and accurate.

[0035] Specific:

[0036] (1) The detection limit of orlistat by the method of the present application is 0.5 mg / L, and the linear range is 0.5-40 mg / L, which is much higher than the conventional detection limit of 0.5 mg / g (CN114280029A) in the prior art;

[0037] (2) The gold nanoparticle-bovine serum albumin composite substrate is simple to synthesize and the detection process is convenient to operate, which has great application prospects in on-site detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the common Raman spectrum of the orlistat solid powder in Example 1;

[0039] Figure 2 The SERS spectra of Au@BSA blank substrate and the SERS spectra of orlistat enhanced by Au@BSA in Example 1;

[0040] Figure 3 The SERS spectra of orlistat with different concentrations using Au@BSA as the enhancement substrate in Example 1;

[0041] Figure 4The standard curve of orlistat established in Example 1 using Au@BSA as the enhanced substrate;

[0042] Figure 5 This is the SERS spectrum of Comparative Example 1 in which unmodified gold nanoparticles are used as the enhanced substrate to detect orlistat with a concentration of 100 mg / L.

[0043] Figure 6 The SERS spectra of the Au@LYS blank substrate in Comparative Example 2 and the SERS spectra of orlistat with Au@LYS and Au@BSA as enhanced substrates, respectively;

[0044] Figure 7 The SERS spectra of the Au@ALB blank substrate in Comparative Example 3 and the SERS spectra of orlistat with Au@ALB and Au@BSA as enhanced substrates, respectively;

[0045] Figure 8 The SERS spectra of orlistat prepared with Au@BSA as the enhanced substrate at different BSA protein concentrations in Comparative Example 4;

[0046] Fig. 9 The SERS spectra of the blank Ag@BSA substrate in Comparative Example 5 and the SERS spectra of orlistat with Ag@BSA and Au@BSA as enhanced substrates, respectively;

[0047] Fig.10 This is the SERS spectra of the blank matrix and the sample extract spiked with orlistat of the Chinese patent medicine detected using Au@BSA as the enhanced substrate in Example 1. DETAILED DESCRIPTION

[0048] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0049] The instruments involved in the present invention are:

[0050] VORTEX-5 vortex oscillator (Qilin Bell Co., Ltd.), PS-10 ultrasonic cleaner (Guangdong Dongguan Jiekang Ultrasonic Equipment Co., Ltd.), refrigerated centrifuge (Shanghai Luxiang Centrifuge Instrument Co., Ltd.), FI-FO785E60X-W portable Raman spectrometer (Beijing Zhuoli Hanguang Instrument Co., Ltd.).

[0051] Source of raw materials

[0052] Potassium chloroaurate powder was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., CAS number: 13682-61-6;

[0053] Bovine serum albumin was purchased from Shanghai Yuanye Biotechnology Co., Ltd., CAS number: 9048-46-8;

[0054] Lysozyme was purchased from Beijing Bailingwei Technology Co., Ltd., CAS No.: 12650-88-3;

[0055] Chicken egg albumin was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., CAS number: 9006-59-1.

[0056] Example 1: Preparation of Au@BSA composite substrate and Raman spectroscopy detection

[0057] Preparation of gold nanoparticle-bovine serum albumin (Au@BSA) composite substrate

[0058] 1. Solution preparation: Weigh 0.1000 g of potassium chloroaurate powder and dissolve it in 10 mL of ultrapure water. Ultrasonicate for 1 min to completely dissolve it to obtain a potassium chloroaurate solution with a concentration of 10 mg / mL.

[0059] Weigh 0.1 g of trisodium citrate and dissolve it in 10 mL of ultrapure water. Ultrasonicate for 1 min to completely dissolve it to obtain a sodium citrate solution with a mass fraction of 1%.

[0060] Weigh 0.0660 g of bovine serum albumin (BSA) and dissolve it in 10 mL of ultrapure water. Ultrasonicate for 1 min to completely dissolve it to obtain a BSA solution with a concentration of 100 μmol / L, and then dilute it with ultrapure water to obtain a BSA solution with a concentration of 150 nmol / L.

[0061] 2. Synthesis of gold nanoparticles:

[0062] Take a 250mL round-bottom flask, add 47mL of ultrapure water, then add 3mL of 10mg / mL potassium chloroaurate solution, heat to boiling in an oil bath at 120°C and a rotation speed of 565r / min, quickly add 2mL of 1% (w / v) sodium citrate solution to the flask, and continue heating for 15min; it is observed that the color of the solution gradually changes from transparent light yellow to an opaque purple-red sol state, gold nanoparticles are generated, and a gold nanosol (gold nanoparticle concentration of 1.41nmol / L) is prepared. After heating, cool to room temperature (20-30°C) for use.

[0063] 3. Preparation of Au@BSA composite substrate:

[0064] Take a 2mL centrifuge tube and add 1.5mL of cooled gold nanosol (prepared in step 2), then add 100μL of 0.15μmol / L BSA solution, vortex oscillate for 30s, and ultrasonicate for 15min; then centrifuge at 12000r / min for 20min; use a pipette to remove 1.4mL of supernatant and add 500μL of ultrapure water for re-dissolution, and ultrasonicate for 30s to obtain the Au@BSA composite substrate.

[0065] 4. SERS detection: Wrap a layer of tin foil on a clean glass slide, add 20 μL of Au@BSA, then add 10 μL of orlistat solutions of different concentrations (0.5 mg / L, 1 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, 50 mg / L), mix well, wait for the reaction for 30 minutes, collect SERS spectra, set the instrument power to 300 mW, the integration time to 3 s, and the number of integrations to 1. For orlistat solid powder, directly perform Raman spectroscopy scanning to obtain the normal Raman spectrum of orlistat.

[0066] The Raman spectrum results of orlistat solid powder are as follows Figure 1 As shown in the figure; the SERS spectra of Au@BSA blank substrate and the SERS spectra of orlistat detected by Au@BSA composite substrate are shown in the figure Figure 2 shown.

[0067] The results showed that compared with the blank substrate without orlistat, the SERS spectrum of the substrate containing orlistat increased at 1280 cm -1 A characteristic peak appeared at , which has a good correspondence with the characteristic peak of the solid powder, indicating that it is the characteristic peak of orlistat. This peak is not interfered by the background characteristic peak of the substrate and can be used as a characteristic peak for qualitative and quantitative identification of orlistat.

[0068] The results of the detection of orlistat at different concentrations using Au@BSA composite substrate are shown in Figure 2. Figure 3 As shown, the results show that 1280cm -1 The characteristic peak intensity at 1280cm has a significant concentration dependence, and the peak intensity increases with the increase of orlistat concentration. When the orlistat concentration is as low as 0.5mg / L, the peak intensity at 1280cm -1 The characteristic peaks at the 400 μg / mL can still be identified, so the detection limit of this method is 0.5 mg / L, which can meet the actual detection requirements. The standard curve of Raman intensity and orlistat concentration is shown in Figure 2. Figure 4 The results showed that in the concentration range of orlistat 0.5-40 mg / L, 1280 cm -1 There is a good linear relationship between the peak intensity at and the logarithm of the concentration. 2 Greater than 0.96. The calibration curve is: y=9.68x+243.72; wherein y represents the Raman intensity (au); and x represents the concentration of orlistat (mg / L).

[0069] Comparative Example 1: SERS detection of orlistat based on unmodified gold nanoparticles

[0070] SERS detection: Wrap a layer of tin foil on a clean glass slide, add 20 μL of unmodified gold sol (gold nanoparticles prepared in step 2 of Example 1), and then add 10 μL of orlistat solution. After mixing evenly, collect the SERS spectrum. The instrument power is set to 300 mW, the integration time is set to 3 s, and the number of integrations is set to 1.

[0071] The results are as follows Figure 5 The results show that the SERS signal of orlistat cannot be directly obtained by using unmodified gold nanoparticles as the enhanced substrate, but when using BSA-modified gold nanoparticles as the substrate, the SERS signal of orlistat can still be obtained at 1280 cm at a concentration of 0.5 mg / L. -1 The presence of orlistat can be judged by the characteristic peak at , which indicates that the surface enhanced Raman method based on molecular docking provided by the present invention for detecting orlistat illegally added in weight loss Chinese patent medicines has good sensitivity and detection accuracy.

[0072] Comparative Example 2: Preparation of composite substrate using lysozyme

[0073] On the basis of Example 1, bovine serum albumin was replaced by lysozyme, and the remaining steps were consistent with Example 1 to prepare the Au@LYS composite substrate.

[0074] Comparative Example 3: Preparation of composite substrate using egg white protein

[0075] On the basis of Example 1, bovine serum albumin was replaced with chicken egg white albumin, and the remaining steps were consistent with Example 1 to prepare the Au@ALB composite substrate.

[0076] Comparative Example 4: Changing the ratio of bovine serum albumin and gold nanoparticles

[0077] On the basis of Example 1, the concentration / dosage of bovine serum albumin and gold nanoparticles in step 3 was changed (as shown in Table 1), and the remaining steps were consistent with Example 1 to prepare an Au@BSA composite substrate, and its Raman spectrum intensity was detected.

[0078] Table 1 Different ratios of bovine serum albumin and gold nanoparticles

[0079] Bovine serum albumin (nmol / L) Gold nanoparticles (nmol / L) Combination 1 1500 1.41 Combination 2 750 1.41 Combination 3 150 1.41 Combination 4 75 1.41 Combination 5 15 1.41

[0080] The composite matrix prepared using lysozyme and egg albumin Figure 6 and Figure 7 As shown in the figure, after replacing BSA with lysozyme and egg albumin, orlistat -1The characteristic peak at the position of BSA did not appear or was very weak. The results showed that the interaction between BSA and orlistat was stronger than that between BSA and other proteins, and the rapid detection of orlistat could be achieved. The detection results of orlistat by the composite substrate prepared under different BSA concentration ratios are shown in Figure 2. Figure 8 As shown in the figure, with the increase of protein concentration, the 1280cm -1 The peak intensity showed a trend of first increasing and then decreasing. When the concentration of bovine serum albumin was 150nmol / L, the detection effect of the prepared composite substrate was the best.

[0081] Comparative Example 5: Preparation of composite substrate using silver nanoparticles

[0082] On the basis of Example 1, the gold nanoparticles were replaced with silver nanoparticles (preparation method: under heating conditions, 200 mL of a silver nitrate aqueous solution with a molar concentration of 0.001 M was slightly boiled, and then 4 mL of a sodium citrate aqueous solution with a mass ratio of 1% was quickly added for reduction. After reacting for 1 hour, the heating was stopped and the mixture was cooled at room temperature to prepare silver nanoparticles), and the remaining steps were consistent with Example 1 to prepare an Au@BSA composite substrate, and its Raman spectrum intensity was detected.

[0083] The detection results of orlistat on the prepared Ag@BSA composite substrate are shown in Fig. 9 As shown in the figure, the results show that the Ag@BSA composite substrate has no significant enhancement effect on orlistat. -1 The characteristic peak at , indicating that the Ag@BSA composite substrate is not suitable for SERS detection of orlistat.

[0084] Example 2 SERS detection of orlistat in Chinese patent medicine

[0085] 1. Sample pretreatment:

[0086] Take an appropriate amount of Qingshen Xiaopang Pills (Tongrentang) and grind it in a small grinder, then weigh 1.000g of powdered sample and add it to a 10mL centrifuge tube, weigh 0.0100g of orlistat solid powder and add it to the sample, and mix well. Then add 5mL of methanol and shake it thoroughly, ultrasonicate it for 5min, centrifuge it at 8000r / min for 10min, remove the precipitate and retain the supernatant, use nitrogen to purge and concentrate, concentrate the solution to 1mL, add 4mL of ultrapure water to re-dissolve, and obtain the original sample solution, and continue to dilute it 80 times with ultrapure water to obtain the solution to be tested.

[0087] 2. SERS detection: Wrap a layer of tin foil on a clean glass slide, add 20 μL of Au@BSA (prepared in Example 1), then 10 μL of sample solution, mix well and wait for the reaction for 30 minutes, collect SERS spectra, set the instrument power to 300 mW, the integration time to 3 s, and the number of integrations to 1.

[0088] 3. Result analysis: According to the 1280cm -1 The intensity of the characteristic peak at was quantitatively analyzed using a standard curve.

[0089] Test results such as Fig.10 As shown in the figure, the results show that when the concentration of orlistat in the actual sample solution is 10 mg / L, the -1 The qualitative and quantitative judgment of the characteristic peak at is reliable, and the characteristic peak will not be interfered by the sample matrix, which further proves the feasibility of this method in practical applications.

[0090] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for detecting orlistat, characterized in that: The method for detecting orlistat using a gold nanoparticle-bovine serum albumin composite substrate comprises the following steps: The gold nanoparticle-bovine serum albumin composite substrate and the sample to be tested were mixed in a volume ratio of 1 to 5:1 and reacted for 10 to 30 minutes. The surface enhanced Raman spectroscopy was used to detect the 1280 cm -1 The Raman intensity of the characteristic peak at is taken into account, and the Raman intensity is brought into a standard curve related to the Raman intensity and the content of orlistat to calculate the content of orlistat in the sample to be tested; The preparation method of the gold nanoparticle-bovine serum albumin composite substrate comprises: (1) Preparation of gold nanosol: The potassium chloroaurate solution is heated to boiling, and a sodium citrate solution is added to obtain a mixed solution, and the mixed solution is heated until the color of the solution gradually changes from a transparent light yellow to an opaque purple-red sol state, thereby preparing a gold nanosol; (2) Preparation of gold nanoparticle-bovine serum albumin composite substrate: The gold nano-sol and the bovine serum albumin solution are mixed to obtain a reaction solution, which is shaken, ultrasonicated, and centrifuged; the precipitate is taken out and redissolved in water to prepare a gold nano-particle-bovine serum albumin composite substrate; In the reaction solution, the molar ratio of the gold nanosol to the bovine serum albumin is 1-2:10-200; and in the reaction solution, the concentration of the gold nanosol is 1-2 nmol / L.

2. The method according to claim 1, characterized in that: The mass ratio of potassium chloroaurate to sodium citrate in the mixed solution of step (1) is 0.2-1.0:0.2-0.5; and the concentration of potassium chloroaurate in the mixed solution is 5-20 mg / mL.

3. The method according to claim 1, characterized in that: The heating condition in step (1) is 100° C. to 150° C. for 30 to 120 min.

4. The method according to claim 1, characterized in that The ultrasonic condition in step (2) is 100-150W for 10-20min.

5. The method according to claim 1, characterized in that The standard curve is: y=9.68x+243.72; wherein y represents the Raman intensity, au; and x represents the concentration of orlistat, mg / L.

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