Surface enhanced Raman spectroscopy test strip for detecting fentanyl and preparation method thereof

Abstract

The invention discloses a detection method and a preparation method of a fentanyl surface enhanced Raman spectroscopy test strip which are rapid, sensitive and simple and convenient to operate. The test strip comprises a sample pad, a combination pad, an NC membrane, a water absorption pad and a PVC bottom plate. A sample pad, a combination pad, an NC (nitrocellulose) membrane and a water absorption pad are sequentially and continuously adhered to the PVC bottom plate. The combination pad is coated with a Raman immune probe (Au-DTNB-Ag-FYL ab) marked with a fentanyl antibody, and the NC membrane is coated with a fentanyl-bovine serum albumin (BSA) conjugate forming a detection line and goat anti-mouse IgG forming a quality control line. The preparation method of the fentanyl surface enhanced Raman spectrum test strip comprises the following steps: preparing nanogold, modifying the surface of the nanogold with Raman signal molecules DTNB, preparing a silver-coated gold composite core shell (Au-DTNB (at) Ag), synthesizing a Raman immune probe, preparing a sample pad, preparing a combination pad, treating the sample pad, and assembling the test strip. The test strip disclosed by the invention has the advantages of high sensitivity, strong specificity, short detection time, on-site operation, low detection cost, simplicity and convenience in operation and the like.

Description

Technical Field

[0001] The present invention relates to an immunochromatographic rapid detection technology for fentanyl, in particular to a surface-enhanced Raman spectroscopy test strip for rapid detection of fentanyl and a preparation method thereof. Background Art

[0002] Fentanyl is a synthetic opioid drug with powerful analgesic effects and is the main anesthetic drug in clinical practice. Due to its similar or stronger excitatory and hallucinogenic effects to drugs, in recent years, fentanyl and its analogs have been mixed into heroin or other illegal drugs and sold, causing a large number of abuse-related deaths in North America and other countries. According to the report of the US Centers for Disease Control and Prevention, among the 67,367 drug overdose death cases in the United States in 2018, 46,802 cases involved opioid drugs. Among them, the number of deaths involving synthetic opioid drugs increased by 10% compared with 2017, mainly due to the illegal supply of fentanyl and its analogs. Fentanyl-related new psychoactive substances have strong pharmacological effects, high lethality, and serious abuse. Therefore, various countries have formulated corresponding laws and regulations for control. China issued the "Announcement on Listing Fentanyl-related Substances in the Supplementary Catalogue of Controlled Non-medical Narcotic and Psychotropic Substances" on May 1, 2016, to implement comprehensive control over fentanyl-related substances. Due to the characteristics of fentanyl-related new psychoactive substances, such as strong toxicity, rapid changes, diverse varieties, and difficult detection, it poses a huge challenge to customs supervision and anti-drug work.

[0003] Common drug detection methods include chromatography, capillary electrophoresis, chemical colorimetry, and colloidal gold method, etc. Chromatography has many advantages such as high efficiency, high selectivity, and less sample consumption, and is favored by the drug detection industry. However, the pretreatment processes of these methods are complex and cumbersome, the detection time is long, professional personnel are required for operation, and there are significant detection limitations. For example, HPLC-MS can only analyze and detect polar, non-volatile, and thermally unstable substances. For non-polar substances, derivatization treatment is still needed, which increases the complexity of drug detection. The chemical method mainly uses specific chemical reagents to react with drug samples to produce color reactions, precipitation and other chemical reactions to identify the types of drugs. Its characteristic is that it can quickly conduct qualitative detection, but its detection limit is high, the sensitivity is low, the specificity is poor, and it cannot detect trace amounts and drugs with similar chemical structures. Compared with conventional instrumental analysis methods, immunochromatography has the characteristics of convenience, rapidity, and easy operation. This type of method has a short detection time and low cost, and is suitable for on-site monitoring and large-scale sample screening, but there are deficiencies in sensitivity, broad-spectrum specificity, or detection range, and the detection sensitivity urgently needs to be improved. Surface-enhanced Raman spectroscopy (SERS) has been widely used for detecting trace targets due to its excellent selectivity, ability to perform fingerprint recognition, and detection sensitivity that can reach the single-molecule level. The technology that combines immunochromatography and surface-enhanced Raman spectroscopy to establish SERS immunochromatography detection technology not only has the high sensitivity and spectral distinguishability of SERS, but also has the high specificity and convenience of immunoassay. In this study, a nano-precious metal with a gold-silver composite core-shell structure was prepared. By connecting fentanyl antibodies and Raman signal molecules, combined with a confocal Raman spectrometer, a rapid, sensitive, and easy-to-operate surface-enhanced Raman spectroscopy test strip detection method for fentanyl was developed, aiming to solve the bottleneck technical problem of low detection sensitivity of current fentanyl drugs and provide better technical support for customs supervision and drug seizure work. Summary of the Invention

[0004] The preparation method of the surface-enhanced Raman spectroscopy test strip for fentanyl of the present invention includes the following steps: 1. Preparation of gold nanoparticles: The gold nanoparticles (AuNPs) are prepared by the modified sodium citrate reduction method. Add 1 mL of 1% chloroauric acid solution to 99 mL of ultrapure water, heat and stir in a microwave extraction synthesizer. When the temperature rises to 98 °C and stabilizes for 5 min, add 1% sodium citrate, continue heating and stirring for reaction. After the color turns wine red, take it out and stir to cool to room temperature to obtain the gold nanoparticle solution (AuNPs), and store it at 4 °C for standby; 2. Preparation of DTNB (Raman signal molecule)-modified gold nanoparticles: Take 10 mL of the gold nanoparticle solution prepared in step (1), add 20 μL of 0.01 mol / L DTNB solution, stir and react at room temperature for 2 h, centrifuge at 12000 rpm / min for 10 min, remove the supernatant, and resuspend the precipitate with 2 mL of ultrapure water to obtain the AuNPs-DTNB solution; 3. Preparation of silver-coated AuNPs-DTNB core-shell: Take 2 mL of the AuNPs-DTNB solution prepared in step (2), add it to 1 mL of 1% PVP, stir at room temperature for 5 min, then add 2 mL of 0.01 mol / L ascorbic acid, stir and react for 15 min, and then gradually add 0.6 mL of 0.01 mol / L silver nitrate dropwise. The color of the colloid changes from wine red to orange-yellow, then stir and react for 30 min, wash twice with ultrapure water, centrifuge at 8000 rpm / min for 10 min, and resuspend the precipitate with an equal volume of ultrapure water and disperse it by ultrasound to obtain the silver-coated AuNPs-DTNB core-shell complex (Au-DTNB@Ag) solution; 4. Preparation of Raman immunoprobe: Take 1 mL of the Au-DTNB@Ag solution prepared in step (3), add 30 μL of 0.05 mol / L potassium carbonate solution, and then add 4 μg of fentanyl antibody diluted with 0.02 mol / L borate buffer (pH 7.4) to the solution. After stirring and reacting for 30 min, add 10% BSA solution to a final concentration of 1%, stir and react for 30 min, centrifuge at 4 °C and 10000 rpm / min for 10 min, and resuspend the precipitate with the preservation solution (0.02 mol / L borate buffer (pH 7.4), containing 2% BSA, 0.25% Proclin-300) to prepare the Raman immunoprobe (Au-DTNB@Ag-FYL Ab); 5. Preparation of conjugate pad: Dilute the Au-DTNB@Ag-FYL Ab prepared in step (4) 20-fold with 0.01 mol / L phosphate buffer (pH 7.4), evenly coat it on a 1 cm-wide conjugate pad, dry it at 37 °C, and seal it for later use; 6. Treatment of sample pad: Immerse the sample pad in 0.01 mol / L phosphate buffer (pH 7.4) containing 0.5% Tween-20 and 1% BSA for 30 min, dry it at 37 °C, and seal it for later use; 7. NC membrane coating: Dilute FYL-BSA conjugate and goat anti-mouse IgG antibody with 0.01 mol / L phosphate buffer (pH 7.4) to a final concentration of 0.1 mg / mL and 0.25 mg / mL, set the membrane liquid volume of the membrane sprayer to 30 µL / 25-30cm, and spray them on the NC membrane in parallel as the test line and quality control line, with a spacing of 5-8 mm between the test line and the quality control line. Dry at 37°C and package for later use; 8. Assembly of test strips: The sample pad, binding pad, NC membrane and absorbent pad are adhered to the PVC base plate in sequence, cut into 4 mm wide test strips with a strip cutter, vacuum packed, and stored at 4-8°C.

[0005] The present invention also provides a method for detecting fentanyl in milk using the surface enhanced Raman spectroscopy test strip of fentanyl, comprising the following steps: 1. Sample pretreatment: Mix milk and 3% trichloroacetic acid at a ratio of 1:1 (v:v), centrifuge at 4°C, 10,000 rpm / min for 15 min to remove protein and fat, transfer the middle layer of supernatant to a clean centrifuge tube, dilute 5 times with 0.01 mol / L phosphate buffer (PH7.4), and take 100 µL of the sample for detection.

[0006] 2. Surface enhanced Raman spectroscopy test strip detection: directly add the above-treated sample to be tested on the test strip, react for 10 minutes, and measure the characteristic peak intensity of the Raman signal molecule DTNB using a confocal Raman spectrometer.

[0007] 3. Analysis of test results: Draw the fentanyl standard curve with the relative Raman signal molecule characteristic peak intensity as the ordinate and the standard concentration as the abscissa. Substitute the measured sample Raman signal molecule DTNB characteristic peak value into the fentanyl standard curve equation to obtain the concentration of the measured sample.

[0008] The detection principle of the present invention is: The fentanyl in the test sample competes with the FYL-BSA conjugate coated on the T line for binding to the Raman immunoprobe Au-DTNB@Ag-FYL Ab. When the test sample contains fentanyl, the antibody connected to the Raman immunoprobe binds to fentanyl and does not bind to the FYL-BSA coated on the T line, so no band appears on the T line, and the result is positive. When there is no fentanyl in the test sample, the Raman immunoprobe binds to the FYL-BSA coated on the T line, a band appears on the T line, and the result is negative. The present invention determines the fentanyl content by measuring the intensity of the characteristic peak of the T-line Raman signal molecule DTNB by a confocal Raman spectrometer.

[0009] Compared with the prior art, the present invention has the following advantages: 1. The test strip of the present invention has the advantages of strong specificity, high sensitivity, and short detection time (10 minutes).

[0010] 2. The test strip of the present invention can be operated on-site with low detection cost.

[0011] 3. The test strip of the present invention is easy to operate and does not require professional personnel to operate.

[0012] 4. The test strip of the present invention is convenient to store and has low temperature requirements. Its effective storage period can reach one year at 4 - 8 °C; it can be stored for six months at room temperature. Description of the Drawings

[0013] Figure 1 The drawing is a fentanyl standard curve Embodiment

[0014] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0015] The embodiment of the present invention discloses a surface-enhanced Raman spectroscopy test strip for detecting fentanyl and its preparation method.

[0016] Raw materials and reagents not mentioned in the embodiments are obtained through conventional commercial channels, and the required equipment are all conventional test equipment. For example, fentanyl antigen and antibody are purchased from Beijing Zhongke Kainuo Technology Co., Ltd.; goat anti-mouse IgG is purchased from Wuhan Boster Biological Technology Co., Ltd., which will not be elaborated one by one here. Embodiment

[0017] A preparation method of a surface-enhanced Raman spectroscopy test strip for detecting fentanyl (1) Preparation of gold nanoparticles: The gold nanoparticles (AuNPs) are prepared by the modified sodium citrate reduction method. Add 1 mL of 1% chloroauric acid solution to 99 mL of ultrapure water, heat and stir in a microwave extraction synthesizer. When the temperature rises to 98 °C and stabilizes for 5 min, add 1% sodium citrate, continue heating and stirring for reaction. After the color turns wine red, take it out and stir to cool to room temperature to obtain the gold nanoparticle solution (AuNPs), and store it at 4 °C for later use; (2)Preparation of DTNB (Raman signal molecule)-modified gold nanoparticles: Take 10 mL of the gold nanoparticle solution prepared in step (1), add 20 μL of 0.01 mol / L DTNB solution, stir and react at room temperature for 2 h, centrifuge at 12000 rpm / min for 10 min, remove the supernatant, and resuspend the precipitate with 2 mL of ultrapure water to obtain the AuNPs-DTNB solution; (3)Preparation of silver-coated AuNPs-DTNB core-shell: Take 2 mL of the AuNPs-DTNB solution prepared in step (2), add it to 1 mL of 1% PVP, stir at room temperature for 5 min, then add 2 mL of 0.01 mol / L ascorbic acid, stir and react for 15 min, and then dropwise add 0.6 mL of 0.01 mol / L silver nitrate. The color of the colloid changes from wine red to orange-yellow, then stir and react for 30 min, wash twice with ultrapure water, centrifuge at 8000 rpm / min for 10 min, and resuspend the precipitate with an equal volume of ultrapure water and disperse it by ultrasound to obtain the silver-coated AuNPs-DTNB core-shell complex (Au-DTNB@Ag) solution; (4)Preparation of Raman immunoprobe: Take 1 mL of the Au-DTNB@Ag solution prepared in step (3), add 30 μL of 0.05 mol / L potassium carbonate solution, and then add 4 μg of fentanyl antibody diluted with 0.02 mol / L borate buffer (pH 7.4) to the solution. After stirring and reacting for 30 min, add 10% BSA solution to a final concentration of 1%, stir and react for 30 min, centrifuge at 4°C and 10000 rpm / min for 10 min, and resuspend the precipitate with the preservation solution (0.02 mol / L borate buffer (pH 7.4), containing 2% BSA, 0.25% Proclin-300) to prepare the Raman immunoprobe (Au-DTNB@Ag-FYL Ab); (5)Preparation of conjugate pad: Dilute the Au-DTNB@Ag-FYL Ab prepared in step (4) 20-fold with 0.01 mol / L phosphate buffer (pH 7.4), evenly coat it on a 1 cm-wide conjugate pad, dry it at 37°C, and seal it for standby; (6)Treatment of sample pad: Immerse the sample pad in 0.01 mol / L phosphate buffer (pH 7.4) containing 0.5% Tween-20 and 1% BSA for 30 min, dry it at 37°C, and seal it for standby; Coating of NC membrane: Dilute the fentanyl-BSA conjugate and goat anti-mouse IgG antibody with 0.01 mol / L phosphate buffer (pH 7.4) respectively, and the final concentrations are 0.1 mg / mL and 0.25 mg / mL. Set the membrane liquid volume of the membrane spraying instrument to 30 µL / 25 - 30 cm, and spray them on the NC membrane in parallel as the test line and the quality control line. The distance between the test line and the quality control line is 5 - 8 mm, dry at 37 °C, and seal for standby; Assembly of the test strip: Attach a sample pad, a conjugate pad, an NC membrane and an absorbent pad to the PVC bottom plate in sequence, cut it into test strips with a width of 4 mm with a strip cutting machine, vacuum package, and store at 4 - 8 °C. Example

[0018] Establishment of the standard curve of the test strip of the present invention Prepare fentanyl standard products with different concentrations: Dilute the high-concentration 10 mg / mL fentanyl standard product to final concentrations of 0 ng / mL, 0.01 ng / mL, 0.1 ng / mL, 1 ng / mL, 2.5 ng / mL, and 5 ng / mL respectively, and detect with the SERS immunochromatographic test strip. The fentanyl standard product solution and the Au-DTNB@Ag-FYL Ab complex on the conjugate pad chromatograph on the surface of the NC membrane due to capillary action, and the T line and C line on the NC membrane will show color. Detect the characteristic peak of the Raman signal molecule DTNB of the T line with a Raman spectrometer. Draw the standard curve of fentanyl with the relative Raman light intensity as the ordinate and the mass concentration of different standard products as the abscissa. Example

[0019] Determination of the sensitivity of the test strip of the present invention Take the average value (X) and standard deviation (SD) of the light intensity at the zero concentration point of 20 standard curves, and find the corresponding concentration from the standard curve for the count obtained by adding 3 times the SD to the light intensity of X. This concentration is the detection limit.

[0020] The results show that the lowest detection limit of fentanyl of the test strip developed by the present invention is 0.01 ng / mL, and the linear range is 0.01 - 5 ng / mL. Example

[0021] Specific detection of the test strip of the present invention Prepare standard products of fentanyl, methamphetamine, morphine, and ketamine respectively, detect with the SERS immunochromatographic test strip, determine the characteristic peak of the DTNB signal molecule with a Raman spectrometer, and calculate according to the formula: Cross-reactivity (CR) = concentration of fentanyl at 50% inhibition rate / concentration of analog at 50% inhibition rate × 100%.

[0022] Table 1 Specificity of the test strip (cross-reactivity) Name CR(%) Fentanyl 100 Methamphetamine <0.1 Morphine <0.1 Ketamine <0.1 The results in Table 1 show that the test strip prepared by the present invention has high specificity for detecting fentanyl. Example

[0023] Detection of milk samples with the test strip of the present invention Mix milk and 3% trichloroacetic acid at a ratio of 1:1 (v:v), centrifuge at 4°C, 10,000 rpm for 15 min to remove proteins and fats. Transfer the middle layer supernatant to a clean centrifuge tube, dilute it 5-fold with 0.01 mol / L PBS (pH 7.4). Take 100 μL of the sample to be tested for surface-enhanced Raman spectroscopy test strip detection, and then use a confocal Raman spectrometer to measure the characteristic peak of the DTNB signal molecule, substitute it into the fentanyl standard curve equation to obtain the concentration of the tested sample. Example

[0024] Shelf life test of the test strip of the present invention For the test strip prepared in Example 1, after being stored at 2 - 8°C for 6 months, measurements were carried out. The measurement results show that the maximum Raman light intensity (zero standard), 50% inhibition concentration, and the actual measured value of added fentanyl of the test strip are all within the normal range.

[0025] Considering that abnormal storage conditions may occur during transportation and use, the test strip was placed at 37°C for 4 days for an accelerated aging experiment. The results show that all indicators of the test strip fully meet the requirements. From the above results, it can be concluded that the test strip can be stored at 2 - 8°C for at least 6 months.

[0026] Note: Term explanations in this specification: 5,5’-dithiobis-2-nitrobenzoic acid (DTNB).

Claims

1. A surface-enhanced Raman spectroscopy test strip for detecting fentanyl and its preparation method, characterized in that: a sample pad, a conjugate pad, a nitrocellulose membrane (NC membrane) and an absorbent pad are sequentially adhered to a PVC bottom plate, the conjugate pad is coated with a Raman immuno-probe Au-DTNB@Ag-fentanyl antibody solution, and the NC membrane is coated with a fentanyl-bovine serum albumin (BSA) conjugate constituting a test line (T line) and a goat anti-mouse IgG constituting a quality control line (C line).

2. The preparation method of the surface-enhanced Raman spectroscopy test strip according to claim 1, characterized in that, comprising the following steps: (1) Preparation of gold nanoparticles: Gold nanoparticles (AuNPs) are prepared by an improved sodium citrate reduction method. 1 mL of 1% chloroauric acid solution is added to 99 mL of ultrapure water, heated and stirred in a microwave extraction synthesizer. When the temperature rises to 98 °C and stabilizes for 5 min, 1% sodium citrate is added, and the reaction continues with heating and stirring. After the color turns wine red, it is taken out and stirred to cool to room temperature to obtain a gold nanoparticle solution (AuNPs), which is stored at 4 °C for later use.

3. (2) Preparation of gold nanoparticles surface-modified with Raman signal molecules (DTNB): Take 10 mL of the gold nanoparticle solution prepared in step (1), add 20 μL of 0.01 mol / L DTNB solution, stir and react at room temperature for 2 h, centrifuge at 12000 rpm / min for 10 min, remove the supernatant, and resuspend the precipitate with 2 mL of ultrapure water to obtain an AuNPs-DTNB solution; (3) Preparation of a silver-coated AuNPs-DTNB core-shell: Take 2 mL of the AuNPs-DTNB solution prepared in step (2), add it to 1 mL of 1% PVP, stir at room temperature for 5 min, then add 2 mL of 0.01 mol / L ascorbic acid, stir and react for 15 min, and then gradually add 0.6 mL of 0.01 mol / L silver nitrate dropwise. The color of the colloid changes from wine red to orange yellow, and then stir and react for 30 min. Wash it twice with ultrapure water, centrifuge at 8000 rpm / min for 10 min, and resuspend the precipitate with an equal volume of ultrapure water and ultrasonicate to obtain a silver-coated AuNPs-DTNB core-shell complex (Au-DTNB@Ag) solution; (4) Preparation of Raman immunoprobe: Take 1 mL of the Au-DTNB@Ag solution prepared in step (3), add 30 µL of 0.05 mol / L potassium carbonate solution, and then add 4 µg of fentanyl antibody (FYLAb) diluted with 0.02 mol / L sodium borate buffer (pH 7.4) to the solution. After stirring for 30 min, add 10% BSA solution to a final concentration of 1%. After stirring for 30 min, centrifuge at 4°C and 10,000 rpm / min for 10 min, and suspend the precipitate with storage solution (0.02 mol / L sodium borate buffer (pH 7.4), containing 2% BSA, 0.25% Proclin-300) to prepare the Raman immunoprobe (Au-DTNB@Ag-FYLAb). (5) Preparation of binding pad: The Au-DTNB@Ag-FYL Ab prepared in step (4) was diluted 20 times with 0.01 mol / L phosphate buffer (pH 7.4), evenly coated on a 1 cm wide binding pad, dried at 37°C, and packaged for later use; (6) Sample pad treatment: Soak the sample pad in 0.01 mol / L pH 7.4 phosphate buffer (containing 0.5% Tween-20 and 1% BSA) for 30 min, dry at 37°C, and seal for later use; (7) NC membrane coating: Dilute fentanyl-BSA conjugate and goat anti-mouse IgG antibody with 0.01 mol / L phosphate buffer (pH 7.4) to a final concentration of 0.1 mg / mL and 0.25 mg / mL, respectively. Set the membrane liquid volume of the membrane sprayer to 30 µL / 25-30 cm, and spray them on the NC membrane in parallel as the test line and quality control line. The test line and quality control line are separated by 5-8 mm. Dry at 37°C and package for later use. (8) Assembly of test strips: The sample pad, binding pad, NC membrane and absorbent pad are adhered to the PVC base in sequence, cut into 4 mm wide test strips using a strip cutter, vacuum packed and stored at 4°C.

4. The detection method of the surface enhanced Raman spectroscopy test strip according to claim 1, It is characterized in that The following steps are involved: (1) Sample pretreatment: Mix milk and 3% trichloroacetic acid at a ratio of 1:1 (v:v), centrifuge at 4°C, 10,000 rpm / min for 15 min to remove protein and fat, transfer the middle layer of the supernatant to a clean centrifuge tube, dilute it 5 times with 0.01 mol / L phosphate buffer (pH 7.4), and take 100 µL of the sample for detection.

5. (2) Surface enhanced Raman spectroscopy test strip detection: directly add the above-treated sample to be tested onto the test strip, react for 10 min, and measure the characteristic peak intensity of the Raman signal molecule DTNB using a confocal Raman spectrometer.

6. (3) Analysis of test results: Draw a fentanyl standard curve with the relative Raman signal molecular characteristic peak intensity as the ordinate and the standard concentration as the abscissa. Substitute the sample Raman signal molecular characteristic peak value into the fentanyl standard curve equation to obtain the concentration of the measured sample.

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