Method for rapidly detecting phenobarbital based on protein modified gold nanoparticles

Abstract

The invention discloses a method for rapidly detecting phenobarbital based on protein modified gold nanoparticles, which is characterized in that based on the strong interaction between phenobarbital and protein and the affinity of the protein to the metal nanoparticles, the protein is used for modifying the gold nanoparticles to obtain an Au-coated LA composite substrate, and an SERS (Surface Enhanced Raman Scattering) detection method of phenobarbital is established. The method solves the problem of poor detection effect caused by weak SERS detection signal of phenobarbital directly using gold sol, the modification process is simple and easy to operate, no complex instrument is needed, rapid qualitative and quantitative analysis of phenobarbital in the Chinese patent medicine can be realized in a short time, and the application potential in field detection is great.

Description

Technical Field

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

[0002] Barbiturates are the most commonly used sedatives and hypnotic drugs, and phenobarbital is a long-acting drug. The adverse reactions of this type of drug include rash, exfoliative dermatitis and Stevens-Johnson syndrome. Long-term and high-dose use of barbiturates may lead to drug dependence, which is manifested as a strong demand for continued use or a request for an increase in dosage, or the occurrence of psychogenic dependence, withdrawal syndrome, etc. Since Chinese patent medicines have the characteristics of few adverse reactions and mild pharmacological effects, they are easily the first choice of people. With the continuous expansion of the sedative and hypnotic drug market, some lawless elements illegally add trace amounts of chemical ingredients to them, which can improve the efficacy of the drug in a relatively short period of time. Consumers who take them for a long time or in excess without knowing it will cause serious adverse reactions and endanger their health.

[0003] At present, the research on detecting Chinese patent medicines and illegally added sedatives and hypnotic drugs mainly includes high performance liquid chromatography-tandem mass spectrometry and high performance liquid chromatography-diode array method. Chromatographic methods have the characteristics of high sensitivity and good accuracy, but due to the high detection cost, large instruments, complex operation process, long detection time and other problems, they are more suitable for use in laboratories, but not in field detection scenarios.

[0004] Surface enhanced Raman spectroscopy (SERS) has the advantages of simple operation, high detection efficiency, and no interference from moisture, and meets the needs of rapid on-site detection. Patent CN104614361B discloses the use of gold nanomaterials combined with Raman spectroscopy to detect phenobarbital in urine samples. However, in the early experiments, the inventors found that the gold nanoparticles prepared in this scheme had poor adsorption of phenobarbital, resulting in weak Raman signals obtained by direct SERS detection, high detection limits, and difficulty in accurately determining phenobarbital in samples.

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

[0006] Aiming at the problem that the SERS detection signal of phenobarbital using gold sol is weak, the detection limit is high, and it does not meet the actual detection needs, the present invention provides a method for directly detecting phenobarbital using Au@LA as an enhanced substrate based on the strong binding effect between phenobarbital and α-lactalbumin, which greatly reduces the detection limit and improves the sensitivity. The composite substrate first uses a simple sodium citrate reduction method to obtain gold nanosol, and then incubates it with a protein solution to prepare an Au@LA composite substrate, thereby obtaining the SERS signal of phenobarbital.

[0007] The first object of the present invention is to provide a gold nanoparticle-α-lactalbumin composite substrate, wherein the preparation method of the gold nanoparticle-α-lactalbumin 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-α-lactalbumin composite substrate:

[0011] The gold nano sol and α-lactalbumin 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 nanoparticle-α-lactalbumin composite substrate.

[0012] In one embodiment, the mass ratio of potassium chloroaurate to sodium citrate in the mixed solution of step (1) is 0.6-3:0.4-1, and the mass concentration of potassium chloroaurate is 5-20 mg / mL.

[0013] In one embodiment, the heating condition in step (1) is heating at 100° C. to 150° C. for 30 to 120 min.

[0014] In one embodiment, in the reaction solution of step (2), the molar ratio of gold nanosol to α-lactalbumin is 1-2:50-2000; and the concentration of gold nanosol in the reaction solution is 1-2 nmol / L.

[0015] In one embodiment, the concentration of the gold nanosol in step (2) is 1-2 nmol / L, and the concentration of α-lactalbumin is 100-1000 nmol / L; the concentration of the gold nanosol in the re-dissolved gold nanoparticle-α-lactalbumin composite substrate is 2.1-4.3 nmol / L, and the concentration of α-lactalbumin is 14.3-285.8 nmol / L.

[0016] In one embodiment, the ultrasonic condition in step (2) is 100-150W for 10-20min.

[0017] In one embodiment, the centrifugation in step (1) and step (2) is performed at 10,000 to 15,000 r / min for 20 min.

[0018] A second object of the present invention is to provide a method for detecting phenobarbital, using any of the above-mentioned gold nanoparticle-α-lactalbumin composite substrates to detect phenobarbital, comprising the steps of:

[0019] The gold nanoparticle-α-lactalbumin 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 phenobarbital in the sample.

[0020] In one embodiment, the standard curve is:

[0021] y=704.25x+247.51; wherein y represents the Raman intensity (au); and x represents the lg value of phenobarbital concentration (mg / L).

[0022] In one embodiment, the volume ratio of the gold nanoparticle-α-lactalbumin composite substrate to the sample to be tested is 1 to 5:1; and the reaction is carried out for 20 to 60 minutes.

[0023] In one embodiment, the sample to be tested is a Chinese patent medicine.

[0024] In one embodiment, the sample sampling amount is 1 g.

[0025] In one embodiment, the sample extraction solvent is methanol, and the amount used is 10 mL.

[0026] In one embodiment, the ultrasonic time during sample extraction is 5 min.

[0027] In one embodiment, the centrifuge speed during the sample extraction process is 10000 r / min and the time is 15 min.

[0028] In one embodiment, the volume multiple of the supernatant diluted with ultrapure water during the sample extraction process is 20 to 200 times.

[0029] In one embodiment, a portable Raman spectrometer is used for detection, and the wavelength of the laser light source is 785, 633 or 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.

[0030] The third object of the present invention is to provide an application of α-lactalbumin in enhancing the detection of phenobarbital by gold nanoparticles, wherein α-lactalbumin and gold nanoparticles are used to prepare a gold nanoparticle-α-lactalbumin composite substrate, and the gold nanoparticle-α-lactalbumin composite substrate is used to detect phenobarbital in a sample to be tested.

[0031] The fourth object of the present invention is to provide a product for detecting phenobarbital, wherein the product comprises any one of the above-mentioned gold nanoparticle-α-lactalbumin composite substrates.

[0032] Beneficial effects of the present invention:

[0033] Based on the strong binding effect between phenobarbital and α-lactalbumin, the present invention provides a method for directly detecting phenobarbital by using Au@LA as an enhanced substrate, which greatly reduces the detection limit and improves the sensitivity.

[0034] Specific:

[0035] (1) The detection limit of the method of the present application for phenobarbital is 1 mg / L, and the linear range is 1 to 100 mg / L, which is much higher than the conventional detection limit of 100 mg / L in the prior art;

[0036] (2) The gold nanoparticle-α-lactalbumin composite substrate of the present application is simple to synthesize and the detection process is convenient to operate, and has great application prospects in on-site detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is the common Raman spectrum of the phenobarbital solid powder of Example 1;

[0038] Figure 2 The SERS spectra of Au@LA blank substrate and the SERS spectra of phenobarbital with Au@LA as the enhanced substrate in Example 1 are shown in FIG.

[0039] Figure 3 The SERS spectra of phenobarbital at different concentrations using Au@LA as the enhanced substrate in Example 1;

[0040] Figure 4 The standard curve of phenobarbital established in Example 1 with Au@LA as the enhanced substrate;

[0041] Figure 5 This is the SERS spectrum of comparative example 1 when unmodified gold nanoparticles are used as the enhanced substrate to detect phenobarbital with a concentration of 100 mg / L.

[0042] Figure 6 The SERS spectra of Au@LYS blank substrate and the SERS spectra of phenobarbital with Au@LYS and Au@LA as enhanced substrates, respectively, are shown in Comparative Example 2.

[0043] Figure 7 The SERS spectra of Au@ALB blank substrate and the SERS spectra of phenobarbital with Au@ALB and Au@LA as enhanced substrates, respectively, are shown in Comparative Example 3;

[0044] Figure 8 This is the SERS spectrum of phenobarbital prepared in comparative example 4 with Au@LA as the enhanced substrate at different α-lactalbumin concentrations;

[0045] Fig. 9 The SERS spectra of the blank Ag@LA substrate in comparative example 5 and the SERS spectra of phenobarbital with Ag@LA and Au@LA as enhanced substrates, respectively;

[0046] Fig.10 This is the SERS spectra of the blank matrix and the sample extract spiked with phenobarbital of the Chinese patent medicine detected using Au@LA as the enhanced substrate in Example 1. DETAILED DESCRIPTION

[0047] 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.

[0048] The instruments involved in the present invention are:

[0049] 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.).

[0050] Source of raw materials

[0051] Potassium chloroaurate powder was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., CAS number: 13682-61-6;

[0052] α-Lactalbumin was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., CAS number: 9051-29-0;

[0053] Lysozyme was purchased from Beijing Bailingwei Technology Co., Ltd., CAS No.: 12650-88-3;

[0054] Chicken egg albumin was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., CAS number: 9006-59-1.

[0055] Example 1: Preparation of Au@LA composite substrate and Raman spectroscopy detection

[0056] Preparation of gold nanoparticle-α-lactalbumin (Au@LA) composite substrate

[0057] 1. Solution preparation:

[0058] 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 0.0140 g of α-lactalbumin (LA) drug and dissolve it in 10 mL of ultrapure water. Ultrasonicate for 1 min to completely dissolve it to obtain a LA solution with a concentration of 100 μmol / L, and then dilute it with ultrapure water to obtain a LA solution with a concentration of 1000 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@LA composite substrate:

[0064] Take a 2mL centrifuge tube and add 1.5mL of cooled gold nanosol (prepared in step 2), then add 100μL of LA solution with a concentration of 1μmol / L, vortex oscillate for 30s, and ultrasonicate for 15min (120W, 40kHz); then centrifuge at 12000r / min for 20min; use a pipette to remove 1.4mL of supernatant and add 500μL of ultrapure water to re-dissolve, and ultrasonicate for 3min to obtain the Au@LA composite substrate.

[0065] 4. SERS detection:

[0066] A layer of tin foil was covered on a clean glass slide, 20 μL of Au@LA was added, and then 10 μL of phenobarbital solution of different concentrations (0.5 mg / L, 1 mg / L, 2 mg / L, 5 mg / L, 10 mg / L, 25 mg / L, 50 mg / L, 100 mg / L) was added. After mixing evenly, the reaction was allowed to proceed for 40 min, and the SERS spectrum was collected. The instrument power was set to 300 mW, the integration time was set to 3 s, and the number of integrations was set to 1. The phenobarbital solid powder was directly scanned by Raman spectroscopy to obtain the ordinary Raman spectrum of phenobarbital.

[0067] The Raman spectrum results of phenobarbital solid powder are as follows Figure 1 As shown; the SERS spectra of Au@LA blank substrate and the SERS spectra of phenobarbital detected by Au@LA composite substrate are shown in Figure 2 shown.

[0068] The results show that compared with the blank substrate without phenobarbital, the SERS spectrum of the substrate containing phenobarbital at 620 cm -1 There is an obvious characteristic peak at the bottom, which has a good correspondence with the characteristic peak of the solid powder, indicating that it is the characteristic peak of phenobarbital. This peak represents the breathing vibration of the benzene ring and the pyrimidine ring. Since this peak is not affected by the background characteristic peak of the substrate, it can be used as a characteristic peak for qualitative and quantitative identification of phenobarbital.

[0069] The results of different concentrations of phenobarbital are as follows Figure 3 As shown, the results show that 620cm -1 The characteristic peak intensity at 620cm has a clear concentration dependence, and the peak is not disturbed. The peak intensity increases with the increase of phenobarbital concentration. When the phenobarbital concentration is as low as 1 mg / L, the peak intensity at 620cm -1 The characteristic peaks at can still be identified, so the detection limit of this method is 1 mg / L, which can meet the actual detection needs.

[0070] The standard curve of Raman intensity and phenobarbital concentration is as follows Figure 4 The results show that in the concentration range of phenobarbital 1-100 mg / L, 620 cm -1 There is a good linear relationship between the peak intensity at and the logarithm of the concentration. 2 Greater than 0.98. The calibration curve is: y=704.25x+247.51; wherein y represents the Raman intensity (au); and x represents the lg value of phenobarbital concentration (mg / L).

[0071] Comparative Example 1: SERS detection of phenobarbital without using LA-modified gold nanoparticles as substrate

[0072] SERS detection: A layer of tin foil was coated on a clean glass slide, 20 μL of unmodified gold sol (gold nanoparticles prepared in step 2 of Example 1) was added, and then 10 μL of 100 mg / L phenobarbital solution was added. After mixing evenly, the SERS spectrum was collected. The instrument power was set to 300 mW, the integration time was set to 3 s, and the number of integrations was set to 1.

[0073] The results are as follows Figure 5 The results show that the SERS signal of phenobarbital directly detected by using unmodified gold nanoparticles as an enhanced substrate is weak and the detection limit is extremely high (the detection limit is as high as 100 mg / L). However, when using LA-modified gold nanoparticles as a substrate, the SERS signal can still be detected by 620 cm at a concentration of 1 mg / L. -1 The presence of phenobarbital can be judged by the characteristic peak at , which indicates that the surface enhanced Raman method for detecting illegally added phenobarbital in Chinese patent medicines based on modified gold nanoparticles provided by the present invention has good sensitivity and detection accuracy.

[0074] Comparative Example 2: Preparation of composite substrate using lysozyme

[0075] On the basis of Example 1, α-lactalbumin was replaced by lysozyme (Lysozyme, molecular weight is 14-15 kDa, close to the molecular weight of α-lactalbumin), and the remaining steps were consistent with Example 1 to prepare the Au@LYS composite substrate.

[0076] Comparative Example 3: Preparation of composite substrate using egg white protein

[0077] On the basis of Example 1, α-lactalbumin was replaced with Albumin from chicken eggwhite, and the remaining steps were consistent with Example 1 to prepare the Au@ALB composite substrate.

[0078] Comparative Example 4: Changing the ratio of α-lactalbumin and gold nanoparticles

[0079] On the basis of Example 1, the concentrations of α-lactalbumin and gold nanoparticles in step 3 were changed (as shown in Table 1), and the remaining steps were consistent with Example 1 to prepare the Au@LA composite substrate, and its Raman spectrum intensity was detected.

[0080] Table 1 Different ratios of α-lactalbumin and gold nanoparticles

[0081] α-Lactalbumin (nmol / L) Gold nanoparticles (nmol / L) Combination 1 100 1.41 Combination 2 500 1.41 Combination 3 1000 1.41 Combination 4 2000 1.41 Combination 5 5000 1.41

[0082] The composite matrix prepared using lysozyme and egg albumin Figure 6 and Figure 7As shown in the figure, after replacing α-lactalbumin with lysozyme and egg albumin, phenobarbital -1 The characteristic peaks at the site did not appear, indicating that the interaction between α-lactalbumin and phenobarbital was stronger than that between other proteins, and rapid detection of phenobarbital could be achieved.

[0083] The test results of the composite substrate prepared under different concentration ratios of α-lactalbumin for phenobarbital are shown in Figure 8 As shown in Figure 2, with the increase of protein concentration, the 620 cm -1 The peak intensity showed a trend of first increasing and then decreasing. When the concentration of α-lactalbumin was 1000nmol / L (i.e., 1μmol / L), the detection effect of the prepared composite substrate was the best.

[0084] Comparative Example 5: Preparation of composite substrate using silver nanoparticles

[0085] On the basis of Example 1, the gold nanoparticles were replaced with silver nanoparticles (the preparation method is as follows: under heating conditions, after a 200 mL silver nitrate aqueous solution with a molar concentration of 0.001 M is slightly boiled, 4 mL of a 1% by mass sodium citrate aqueous solution is quickly added for reduction, and the heating is stopped after the reaction for 1 hour, and the silver nanoparticles are cooled at room temperature to prepare the silver nanoparticles), and the remaining steps are consistent with Example 1 to prepare the Ag@LA composite substrate, and its Raman spectrum intensity is detected.

[0086] The test results of the prepared Ag@LA composite substrate on phenobarbital are shown in Fig. 9 As shown in the figure, the results show that the Ag@LA composite substrate has a certain enhancement effect on phenobarbital, but this enhancement effect is only about 40% of the Au@LA composite substrate. If the Ag@LA composite substrate is used for SERS detection of phenobarbital, the detection limit is much higher than that of the Au@LA composite substrate, which is not conducive to its application in the actual detection process.

[0087] Example 2: SERS detection of phenobarbital in Chinese patent medicine

[0088] 1. Sample pretreatment:

[0089] Take Zaoren Anshen Capsules (Tongjitang) and remove the capsule shell. Weigh 1.000g of powdered sample and add it to a 15mL centrifuge tube. Weigh 0.0100g of phenobarbital solid powder and add it to the sample and mix well. Then add 10mL of methanol and shake it thoroughly. After ultrasonication for 5min, centrifuge at 10000r / min for 15min. After removing the precipitate, obtain the original sample solution, and continue to dilute it 200 times with ultrapure water to obtain the test solution.

[0090] 2. SERS detection:

[0091] Wrap a layer of tin foil on a clean glass slide, add 20 μL of Au@LA (prepared in Example 1), then 10 μL of sample solution, mix well and wait for the reaction for 40 minutes, collect SERS spectra, set the instrument power to 300 mW, the integration time to 3 s, and the number of integrations to 1.

[0092] 3. Result analysis:

[0093] According to the SERS spectrum, 620 cm -1 The intensity of the characteristic peak at was quantitatively analyzed using a standard curve.

[0094] The results are as follows Fig.10 As shown in the figure, the results show that when the concentration of phenobarbital in the actual sample solution is 5 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.

[0095] 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 gold nanoparticle-α-lactalbumin composite substrate, characterized in that: The preparation method of the gold nanoparticle-α-lactalbumin 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 changes from transparent light yellow to an opaque purple-red sol state, thereby preparing a gold nanosol; (2) Preparation of gold nanoparticle-α-lactalbumin composite substrate: The gold nano sol and α-lactalbumin 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 nanoparticle-α-lactalbumin composite substrate.

2. The gold nanoparticle-α-lactalbumin composite substrate according to claim 1, characterized in that: The mass ratio of potassium chloroaurate to sodium citrate in the mixed solution of step (1) is 0.6-3:0.4-1; and the mass concentration of potassium chloroaurate in the mixed solution is 5-20 mg / mL.

3. The gold nanoparticle-α-lactalbumin composite substrate 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 gold nanoparticle-α-lactalbumin composite substrate according to claim 1, characterized in that: In the reaction solution of step (2), the molar ratio of gold nanosol to α-lactalbumin is 1-2:100-2000; and the concentration of gold nanosol in the reaction solution is 1-2 nmol / L.

5. The gold nanoparticle-α-lactalbumin composite substrate according to claim 1, characterized in that: The ultrasonic condition in step (2) is 100-150W for 10-20min.

6. A method for detecting phenobarbital, characterized in that, Using the gold nanoparticle-α-lactalbumin composite substrate according to any one of claims 1 to 4 to detect phenobarbital comprises the steps of: The gold nanoparticle-α-lactalbumin composite substrate and the sample to be tested were mixed and reacted, and surface enhanced Raman spectroscopy was used to detect 620 cm -1 The Raman intensity of the characteristic peak at is taken into account, and the Raman intensity is brought into the calibration curve to calculate the content of phenobarbital in the sample.

7. The method according to claim 6, characterized in that The standard curve is: y=704.25x+247.51; wherein y represents the Raman intensity (au); and x represents the lg value of phenobarbital concentration (mg / L).

8. The method according to claim 6, characterized in that The volume ratio of the gold nanoparticle-α-lactalbumin composite substrate to the sample to be tested is 1 to 5:1; the reaction is carried out for 20 to 60 minutes.

9. Application of α-lactalbumin in enhancing the detection of phenobarbital by gold nanoparticles, characterized in that: A gold nanoparticle-α-lactalbumin composite substrate is prepared by using α-lactalbumin and gold nanoparticles, and the gold nanoparticle-α-lactalbumin composite substrate is used to detect phenobarbital in a sample to be tested.

10. A product for detecting phenobarbital, characterized in that: The product comprises the gold nanoparticle-α-lactalbumin composite substrate as described in any one of claims 1 to 5.

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