Detection method and detection platform for psychoactive substances in sewage

Abstract

The invention provides a detection method and a detection platform for mental active substances in sewage, the preparation method is based on the specificity of aptamers and a fluorescence quenching mechanism, cocaine, methamphetamine or codeine aptamers are used as recognition elements, CdSe / ZnS core-shell quantum dots are used as fluorescent agents for marking the aptamers, and the fluorescent agents are used for detecting the mental active substances in the sewage. Gold nanoparticles are introduced as a fluorescence quencher and are used for reducing background signals of detection, so that high-sensitivity detection of a target substance is realized. By adopting the technical scheme provided by the invention, on-site detection of psychoactive substances in sewage can be realized, the detection time is short, the operation is simple, and the sensitivity and the specificity are high. Furthermore, a plurality of aptamers can be adopted, so that synchronous detection of a plurality of mental active substances can be realized.

Description

Technical Field

[0001] The present invention relates to the technical field of sewage detection, and in particular to a detection method and a detection platform for psychoactive substances in sewage. Background Art

[0002] At present, there are four mainstream detection methods for detecting psychoactive substances, namely chromatography, enzyme-linked immunosorbent assay, colloidal gold method, and fluorescence method. In the traditional detection of psychoactive substances, chromatography is the main detection method. However, it often requires expensive detection instruments, cannot be detected on site, has a cumbersome and complicated sample pretreatment process, and requires professional operators to perform experimental operations, which greatly limits the application and development of traditional detection methods in the detection of psychoactive substances. Compared with chromatography, enzyme-linked immunosorbent assay has lower sensitivity. Although the colloidal gold method has a short detection time and can realize on-site detection, it has low sensitivity and specificity and large errors. Only the fluorescence method can not only realize on-site detection, but also has a short detection time, simple operation, high sensitivity and specificity. Compared with antibodies and aptamers, the reaction conditions of microorganisms and enzymes used as recognition elements are more stringent, and the cost is high and difficult to preserve. There are few studies on the detection of cocaine. Antibodies are less stable and have strong immunogenicity to antigens. Summary of the invention

[0003] In response to the above technical problems, the present invention discloses a detection method and detection platform for psychoactive substances in sewage, which solves the problems that the existing methods require expensive instruments and equipment, time-consuming and complicated pre-treatment procedures, long detection time, low specificity and sensitivity, and single detection of psychoactive substances.

[0004] To this end, the technical solution adopted by the present invention is:

[0005] A method for detecting psychoactive substances in sewage, comprising the following steps:

[0006] Step S1, adding a sodium phosphate buffer containing Tween 20 to an aqueous solution of gold nanoparticles, mixing and incubating, centrifuging and washing, removing excess Tween 20, then adding a sodium phosphate buffer, and ultrasonically treating to obtain an AuNP solution; adding at least one of a cocaine aptamer, a methamphetamine aptamer, and a codeine aptamer to the AuNP solution to obtain an aptamer-AuNP solution, incubating the solution by shaking, and then freezing it at -20° for 2 minutes to obtain an aptamer-AuNP conjugate solution; dialyzing the aptamer-AuNP conjugate solution to obtain an aptamer-AuNP conjugate colloid;

[0007] The sequence of the cocaine aptamer is SEQ ID NO.1, the sequence of the methamphetamine aptamer is SEQ ID NO.2, and the sequence of the codeine aptamer is SEQ ID NO.3;

[0008] Cocaine aptamer sequence: 5′NH2-C6-AGACAAGGAAAATCCTTCAATGAAGTGGGTCG-C6-SH3′;

[0009] Methamphetamine: 5′-SH-ACGGTTGCAAGTGGGACTCTGGTAGGCTGGGTTAATTTGG-3′;

[0010] Codeine: 5'-SH-ACAGGCACGTTGGTTAGGTCAGGTTTGGGTTTCGTGCTTG-3';

[0011] Step S2, preparing a poly-lysine-coated porous plate;

[0012] Step S3, modifying the polylysine-coated porous plate with water-soluble quantum dots and activating the carboxyl groups;

[0013] Step S4, extracting the dialyzed aptamer-AuNP conjugate colloid, adding it to the multi-well plate treated in step S3, allowing it to react with the surface of the activated carboxyl quantum dots, shaking and incubating for 2 hours, and then washing with sodium phosphate buffer to obtain a quantum dot-aptamer-gold nanoparticle fluorescent probe solution;

[0014] Step S5, adding the sample to be tested to the quantum dot-aptamer-gold nanoparticle fluorescent probe solution of step S4, exciting it within the range of 400nm-500nm, and detecting the fluorescence intensity using a fluorescence spectrophotometer. If there is fluorescence at 450nm, there is a response of the psychoactive substance of cocaine.

[0015] This technical solution introduces aptamers as recognition elements to identify cocaine to improve the selection specificity of the method. Aptamers are obtained by in vitro screening using the systematic evolution of ligands by exponential enrichment (SELEX) technique and are a short RNA or DNA single-stranded oligonucleotide sequence that can specifically and tightly bind to the target substance. Aptamers have extremely strong binding affinity and specificity to the target substance, so they are often used as molecular recognition elements in biosensors. The established aptamer biosensors have high sensitivity and specificity, can be label-free and cost-effective, and are often used in electrochemical, fluorescent, colorimetric, chemiluminescent and other sensors.

[0016] The aptamer using the technical solution of the present invention has strong specificity. After the target substance is identified, it does not have the ability to convert the identification information into a detectable signal. The fluorescent substance can be used as a marker to combine with the aptamer to convert the information into a detectable signal. Traditional fluorescent dyes show shortcomings such as easy quenching and transient in the detection process, and quantum dots have unique advantages. It makes up for the shortcomings of traditional fluorescent dyes, improves the sensitivity of analytical detection, and realizes the simultaneous detection of multiple components. Therefore, the optical specificity of quantum dots is combined with the specific recognition technology of aptamers to provide an efficient, simple, fast, sensitive, and high-throughput analytical detection platform.

[0017] In addition, gold nanoparticles are introduced as fluorescence quenchers to reduce the background signal of the detection and achieve high-sensitivity detection of cocaine. Gold nanoparticles are generally called colloidal gold in biology. The particle size is generally between 1-100nm. It presents different colors with the change of particle size and morphology, which is conducive to naked eye observation. It has strong color development ability and is widely used in optical and biosensors due to its advantages such as simple preparation, uniform particle size, stable chemical properties, strong affinity, good biocompatibility, and easy fixation and modification of biomolecules. It has the advantages of easy-to-control particle size, good biocompatibility and excellent fluorescence quenching ability.

[0018] As a further improvement of the present invention, in step S1, the concentration of Tween 20 in the sodium phosphate buffer containing Tween 20 is 1.80 mg / mL; further, the size of the gold nanoparticles is 38-42 nm, and the concentration of the aqueous solution of the gold nanoparticles is 10 nM; further, the volume ratio of the aqueous solution of the gold nanoparticles to the sodium phosphate buffer containing Tween 20 is 1:1.

[0019] As a further improvement of the present invention, in step S1, the incubation time is 35 to 45 minutes.

[0020] As a further improvement of the present invention, the centrifugal speed is 10000 rpm, the time is 5 min, and the gold nanoparticles are washed with sodium phosphate buffer after centrifugation. The centrifugal washing steps are repeated three times, and the gun tip is aimed at the gold nanoparticles for washing.

[0021] As a further improvement of the present invention, in step S1, the concentration of the sodium phosphate buffer is 10 mM, and the pH value is 7.4; the ultrasonic treatment time is 20 min.

[0022] Furthermore, the stock solution concentration of the cocaine aptamer, methamphetamine aptamer or codeine aptamer is 10 μM.

[0023] Furthermore, the shaking incubation was performed at a speed of 1000 rpm, a temperature of 25° C., and a time of 3 h;

[0024] As a further improvement of the present invention, in step S1, the dialysate used for dialysis is 5 mM PBS buffer, the dialysis time is 12 h, and the dialysis bag specification is MWCO: 12-14.

[0025] As a further improvement of the present invention, step S2 includes: preparing 100 μg / ml polylysine working solution, filtering and sterilizing it through a 5 μm filter, adding it to the porous plate so that the bottom of the porous plate can be completely covered, placing it in a 37-degree incubator for 1 hour, then absorbing the liquid and allowing the surface to dry.

[0026] As a further improvement of the present invention, in step S3, PBS buffer, QD, EDC, and NHS are added to the poly-lysine-coated multi-well plate, and incubated for 3 hours at room temperature and in the dark using a micro-oscillator adjusted to 1000 rpm.

[0027] As a further improvement of the present invention, the concentration of the PBS buffer is 25 mM, the pH value is 6.0, the concentration of EDC is 0.5 M, the concentration of NHS is 0.125 M, and the volume ratio of the PBS buffer, QD, EDC, and NHS is 10:10:4:4.

[0028] Furthermore, in step S3, a light shielding material is used to cover the micro-oscillator, and the light absorption rate of the light shielding material reaches 98-99%.

[0029] As a further improvement of the present invention, in step S4, the rotation speed of the shaking incubation is 1000 rpm and the time is 2 h.

[0030] As a further improvement of the present invention, step S5 also includes obtaining the fluorescence linear equation of quantum dot-aptamer-gold nanoparticle composite detection through experiments, and calculating the concentration of the target substance according to the detected fluorescence intensity.

[0031] As a further improvement of the present invention, the fluorescence linear equation of quantum dot-aptamer-gold nanoparticle composite detection obtained by experiment includes:

[0032] A series of standard solutions of cocaine, methamphetamine or codeine with concentrations of 4 nmo / L, 6 nmo / L, 7 nmo / L, 9 nmo / L and 15 nmo / L were prepared, and added to the quantum dot-aptamer-gold nanoparticle fluorescent probe solution obtained in step S4 for 30 min, and then rinsed with a sodium phosphate buffer (10 mM, pH 7.4) solution; excited it in the range of 400 nm-500 nm, detected the fluorescence intensity by a fluorescence spectrophotometer, and calculated the relationship between the change of the fluorescence intensity of the standard solution of each concentration and the concentration of the target substance, and fitted to obtain the corresponding fluorescence linear equation.

[0033] The present invention also discloses a detection platform for psychoactive substances in sewage, which is detected by using any of the above detection methods for psychoactive substances in sewage.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] The technical solution of the present invention can realize on-site detection of psychoactive substances in sewage, and the detection time is short, the operation is simple, the sensitivity and specificity are high, and the cost is low. Furthermore, multiple aptamers can be used to realize the simultaneous detection of multiple psychoactive substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is a flow chart of a method for detecting psychoactive substances in sewage according to an embodiment of the present invention.

[0037] Figure 2 It is a reaction mechanism diagram of a method for detecting psychoactive substances in sewage according to an embodiment of the present invention.

[0038] Figure 3 It is a schematic diagram of the material reactions of the experimental steps of the method for detecting psychoactive substances in sewage according to an embodiment of the present invention.

[0039] Figure 4 1 is a graph showing changes in fluorescence intensity of standard solutions of different cocaine concentrations according to an embodiment of the present invention.

[0040] Figure 5 4 is a graph showing the relationship between fluorescence intensity and cocaine concentration according to an embodiment of the present invention.

[0041] Figure 6 1 is a fitting curve diagram of fluorescence intensity and cocaine concentration in an embodiment of the present invention.

[0042] Figure 7 3 is an analysis diagram of the effect of interferences on the detection of cocaine according to an embodiment of the present invention. DETAILED DESCRIPTION

[0043] The preferred embodiments of the present invention are described in further detail below.

[0044] Example 1

[0045] A method for detecting psychoactive substances in sewage. This embodiment is mainly exemplified for cocaine. The method is the same for methamphetamine and codeine, except that the sequence of the aptamer is different. The flow chart of this embodiment is as follows Figure 1 shown.

[0046] The detection mechanism of this embodiment is as follows Figure 2 As shown, specifically:

[0047] In the absence of cocaine, AuNPs are relatively far from QDs, resulting in a high-intensity fluorescence signal. Then, by adding cocaine, the aptamer forms three helical stems at the three-way junction. The Y-shaped structure of the cocaine-binding aptamer brings AuNPs close to QDs, resulting in a decrease in fluorescence intensity due to the fluorescence quenching effect of AuNPs.

[0048] The experimental steps of this example are as follows Figure 3 Specifically, the detection method comprises the following steps:

[0049] (1) Coupling of aptamers and AuNPs:

[0050] 100 μL of gold nanoparticles (AuNPs) were mixed with 100 μL of sodium phosphate buffer (10 mM pH 7.4) containing 1.80 mg / mL Tween 20. The size of the gold nanoparticles was 39 nm, the concentration was 10 nM, and the mixture was incubated for 40 min. After incubation, the mixture was centrifuged to remove excess Tween 20. Centrifuge at 10,000 rpm / 5 min, and then the gold nanoparticles were rinsed with sodium phosphate buffer (PBS). In order to remove tween 20 from the surface of the gold nanoparticles, a needle was used to extract PBS buffer to wash the gold nanoparticle stack. The Tween was washed off by centrifugation three times. Furthermore, after washing, PBS solution was added to 200 ul, and the centrifugation and washing steps were repeated three times. After activation and centrifugation, gold nanoparticles will aggregate, which will affect the ability of the aptamer to bind to its gold nanoparticles. In order to evenly disperse the gold nanoparticles in the PBS buffer, they are first vortexed for 5 minutes and then ultrasonicated for 20 minutes to evenly disperse the gold nanoparticles in the solution.

[0051] Sodium phosphate buffer (10mM pH 7.4) was used to make the volume to 1.0mL, and then ultrasonicated for 20min, 25μL of cocaine aptamer (10μM stock solution) was added to the AuNP solution, and the Apt-AuNP solution was incubated for 3h under the oscillation conditions of 25℃ and 1000rpm / S, and then frozen for 2min under -20℃ freezing state to generate Au-SH linkage between the cocaine aptamer and the AuNP. In order to improve the binding ability and stability of gold nanoparticles and their aptamers: the binding ability of gold nanoparticles and their aptamers in the traditional salt-old method is relatively low, and the present embodiment first uses a micro-oscillator to oscillate during the reaction, and the oscillation speed is adjusted to 1000rmp / s, and incubated for 3 hours, which is conducive to the binding of gold nanoparticles and their aptamers. In addition, the freezing method is used to freeze the uniformly mixed gold nanoparticles and aptamers at -20℃ for 20 minutes. This method increases the number of gold nanoparticles binding aptamers by 20% on the basis of the salt-old method.

[0052] The final Apt-AuNP conjugate solution was dialyzed for 12 hours using a dialysis bag (dialysis solution was 5mM PBS buffer), and the dialysis bag specification was MWCO: 12-14. The aptamer was combined with gold nanoparticles, and the solution contained a small amount of salt, and other small molecules remained in the solution. In order to eliminate their influence, a dialysis bag was used to intercept them. The dialysis bag material was selected as cellulose, and the dialysis bag was cut to a length of 7cm, leaving a certain space for the free diffusion of the sample solution. At the same time, in order to prevent the interference of bubbles on the dialysis efficiency, it was necessary to empty it. The dialysis time was 12 hours, and the dialysis solution was replaced every 3 hours to preserve the concentration gradient of the dialysis. The dialysis solution was consistent with the experiment. PBS was selected as the dialysis buffer, and the magnetic rotor was adjusted to 300rmp / s for rotation to increase its dialysis effect. The dialysis bag specification selected a molecular weight cutoff of 12KD-14KD. After the experiment, it needs to be thoroughly cleaned and properly stored in an aqueous solution containing a small amount of ethanol.

[0053] In this example, the cocaine aptamer sequence is:

[0054] 5′NH2-C6-AGACAAGGAAAATCCTTCAATGAAGTGGGTCG-C6-SH3′.

[0055] If targeting methamphetamine and codeine, the methamphetamine aptamer sequence is:

[0056] 5,-SH-ACGGTTGCAAGTGGGACTCTGGTAGGCTGGGTTAATTTGG-3'

[0057] Codeine: 5'-SH-ACAGGCACGTTGGTTAGGTCAGGTTTGGGTTTCGTGCTTG-3'

[0058] The methamphetamine, codeine and cocaine aptamers were coupled to AuNPs in the same manner.

[0059] (2) Polylysine coated 96-well plate:

[0060] Prepare 100 μg / ml poly-lysine working solution, filter and sterilize through a 5 μm filter, and store at -20°C. Add to a 96-well plate to completely cover the bottom of the well plate, place in a 37°C incubator for 1 hour, then aspirate the liquid and let the surface dry.

[0061] In this step, the polylysine solution is stored at -20 ° C and needs to be prepared before use to ensure its activity. Prepare 100 μg / ml polylysine working solution. In order to remove insoluble polylysine particles, filter through a 5 μm filter and sterilize for use. In order to prevent errors caused by different amounts of polylysine and quantum dots, use a pipette to take 50 μl of polylysine solution from each well and add it evenly to each well. Incubate at room temperature for 1 hour to allow polylysine to be evenly coated on the surface of the well plate. After incubation, use a pipette to extract 100 μl PBS to gently rinse each well to remove unabsorbed polylysine and avoid leaving droplets in the well. Rinse 3 times. Too few rinses will result in the inability to completely remove unabsorbed polylysine, and too many rinses will damage the polylysine layer. In order to remove the residual PBS liquid in the well, turn the 96-well plate upside down on a sterile paper towel and gently tap to remove the residual liquid. Under sterile conditions, allow the plate to dry at room temperature, usually for 30 minutes, until the wells are completely dry.

[0062] (3) Activating the carboxyl groups modified on the water-soluble quantum dots:

[0063] Three portions of 100ul PBS buffer (25mM pH 6.0), 100ul QD, 40ul LEDC (0.5M), and 40ul NHS (0.125M) were added to a poly-lysine-coated 96-well plate, and incubated for 3 hours at room temperature in the dark using a micro-oscillator set to 1000 rpm.

[0064] Furthermore, due to the easy hydrolysis of EDC and NHS, the efficacy of the drug will be reduced after they are prepared and stored, which will affect the activation of the quantum dot surface and the coupling of gold nanoparticles. Therefore, they need to be prepared before each experiment to achieve the purpose of not affecting the efficacy. Because EDC is too active, exposure to white light will affect its properties and lead to poor reaction effects. Therefore, it is necessary to perform light shielding treatment and make a light shielding layer during the reaction. A plastic light shielding layer with a length, width and height of 9 / 6 / 6.5cm can be made. It is covered on the micro-oscillator during the reaction. The light shielding layer is sprayed with Black 3.0 acrylic polyethylene paint. The surface is gently polished with fine sandpaper to increase the adhesion of the paint. Then, Rust-Oleum Specialty Plastic Primer primer is sprayed. After multiple sprayings, Black 3.0 acrylic polyethylene is sprayed. The visible light absorption rate reaches 98-99%.

[0065] (4) The activated carboxyl quantum dots are connected to the amino-modified psychoactive substance nucleic acid aptamer-gold nanoparticle conjugate to form a fluorescent probe detection device:

[0066] The aptamer carries the -NH2 group. 60 μL (cocaine) Apt-AuNP colloid extracted from the dialysis bag was added to the wells of a 96-well plate to react with the surface of the carboxyl quantum dots activated by EDC. The micro-oscillator was adjusted to 1000 rpm and incubated for 2 h. After incubation, each well was rinsed with sodium phosphate buffer (pH 7.4, 10 mM) to form an aptamer-gold nanoparticle-quantum dot reagent.

[0067] (5) Combination of the target with the quantum dot-aptamer-gold nanoparticle-96-well plate fluorescent probe detection device:

[0068] The sample to be tested is added to the constructed quantum dot-aptamer-gold nanoparticle fluorescent probe solution, and it is excited in the range of 400nm-500nm. The fluorescence intensity is detected by a fluorescence spectrophotometer. If there is fluorescence at 450nm, there is cocaine. The fluorescence intensity at 450nm is recorded.

[0069] In this step, the fluorescence linear equation of quantum dot-aptamer-gold nanoparticle composite detection can be obtained through experiments, and the concentration of the target substance can be calculated based on the detected fluorescence intensity. Specifically, it includes:

[0070] Prepare a series of standard solutions of cocaine with concentrations of 4nmo / L, 6nmo / L, 7nmo / L, 9nmo / L, and 15nmo / L (the same applies to methamphetamine or codeine). Take 100μL of sample respectively and add it to the obtained quantum dot-aptamer-gold nanoparticle fluorescent probe solution for 30 minutes, and then rinse with sodium phosphate buffer (10mM, pH 7.4) solution. All measurements were performed on a poly-L-Lys-coated 96-well plate at 25°C. Detect and calculate the relationship between the change in fluorescence intensity of each concentration solution and the target cocaine. The fluorescence intensity change diagram of the standard solution of different cocaine concentrations obtained in this embodiment is shown in the figure below. Figure 4 The relationship between fluorescence intensity and cocaine concentration is shown in Figure 5 As shown, the corresponding fluorescence linear equation was established, and the fitting curve of fluorescence intensity and cocaine concentration was shown in Figure 6 As shown, the linear range of the cocaine standard curve is 10 nmol / L, the linear equation is Y=51.24X+1855, R2=0.9938, and the detection limit is calculated by the ratio of 3 times the standard deviation (s) to the slope (k) of the linear regression equation (3s / k). The detection limit of cocaine is calculated to be 0.138 nmol / L; wherein Y is the fluorescence intensity, and X is the concentration of cocaine, in nmol / L.

[0071] This embodiment cites CdSe / ZnS core-shell quantum dots as fluorescent agents for labeling nucleic acid aptamers, and achieves highly sensitive detection of cocaine through changes in fluorescence intensity. Quantum dots are nanocrystals composed of II-VI elements or III-V elements, and the three dimensions are all less than 100nm. Because its diameter is less than or close to the exciton Bohr radius, it exhibits unique physical properties, such as quantum size effect, surface effect, macroscopic quantum tunneling effect, etc. Quantum dots have attracted much attention for their advantages such as rich colors, stable photochemical properties, and strong anti-bleaching ability. Traditional organic fluorescent dyes have insurmountable defects, such as narrow excitation spectrum: the emission spectrum of fluorescent dyes is very wide, and the fluorescence spectrum peaks sometimes have very long tails, resulting in overlap between spectrum peaks, limiting the number of fluorescent probes that can be used simultaneously; organic dyes are prone to photobleaching and photolysis; in addition, each organic fluorescent dye requires a specific method for connection with the detection substance. Compared with organic fluorescent dyes, quantum dots have a wide excitation spectrum and can effectively excite and collect emitted fluorescence. Quantum dots have narrow, symmetrical fluorescence peaks, adjustable emission spectra, and emission wavelengths that can be single or multiple. The fluorescence intensity can be adjusted by changing the number of core substances or by changing the amount of a certain component of the core substance. Compared with organic fluorescent dyes, quantum dots have strong resistance to degradation by chemicals and physiological metabolism, and their photobleaching threshold is also high. Quantum dots also have the advantages of strong stability, strong labeling durability, and a single and easy method of connecting to biomolecules. They can overcome the various defects of organic fluorescent dyes and serve as a suitable substitute for organic fluorescent dyes.

[0072] Example 2

[0073] On the basis of Example 1, in order to study the specificity of the experiment, an interference experiment was set up. The specific steps include:

[0074] 100 nmol / L cocaine, methamphetamine, codeine, acetamide (1.0%) and 3-acetaminophen were prepared respectively, and 100 ul was added to the quantum dot-aptamer-gold nanoparticle solution reagent prepared in Example 1, and the fluorescence intensity was detected by a fluorescence spectrophotometer. Figure 7 As shown, it can be seen that the added interfering substances have little effect on the detection process of cocaine by quantum dots-aptamer-gold nanoparticles.

[0075] Comparative Example 1

[0076] Based on Example 1, the difference of this comparative example is that: (3) the modified carboxyl groups on the water-soluble quantum dots are activated, and MES (pH=5.5) buffer is added to the polylysine-coated 96-well plate. It is found that the fluorescence intensity of the quantum dot-gold nanoparticle-aptamer samples of the same batch obtained at last is quite different, which affects the accuracy of its detection. It is later found that no matter how evenly the MES buffer itself is mixed, the error of its own fluorescence intensity is always large.

[0077] In Example 1, 0.1 mol / L phosphoric acid was used to adjust the pH to (5.5-5.8), and the mixture was mixed evenly to reach a suitable pH range. The fluorescence intensity error of the sample obtained by the PBS buffer solution was small, and the pH of the PBS buffer solution was in the range of (7.2-7.4). Furthermore, the pH span adjustment required to adjust to the optimal pH range (5.5-5.8) of EDC and NHS is smaller. Therefore, the use of PBS solution as the buffer solution for the reaction of EDC and NHS has a better effect.

[0078] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the protection scope of the present invention.

Claims

1. A method for detecting psychoactive substances in sewage, Features: The steps include: Step S1, adding a sodium phosphate buffer containing Tween 20 to an aqueous solution of gold nanoparticles, mixing and incubating, centrifuging and washing, removing excess Tween 20, then adding a sodium phosphate buffer, and ultrasonically treating to obtain an AuNP solution; adding at least one of a cocaine aptamer, a methamphetamine aptamer, and a codeine aptamer to the AuNP solution to obtain an aptamer-AuNP solution, incubating the solution by shaking, and then freezing it at -20° for 2 minutes to obtain an aptamer-AuNP conjugate solution; dialyzing the aptamer-AuNP conjugate solution to obtain an aptamer-AuNP conjugate colloid; The sequence of the cocaine aptamer is SEQ ID NO.1, the sequence of the methamphetamine aptamer is SEQ ID NO.2, and the sequence of the codeine aptamer is SEQ ID NO.3; Step S2, preparing a poly-lysine-coated porous plate; Step S3, modifying the polylysine-coated porous plate with water-soluble quantum dots and activating the carboxyl groups; Step S4, extracting the dialyzed aptamer-AuNP conjugate colloid, adding it to the multi-well plate treated in step S3, allowing it to react with the surface of the activated carboxyl quantum dots, shaking and incubating for 2 hours, and then washing with sodium phosphate buffer to obtain a quantum dot-aptamer-gold nanoparticle fluorescent probe solution; Step S5, adding the sample to be tested to the quantum dot-aptamer-gold nanoparticle fluorescent probe solution of step S4, exciting it within the range of 400nm-500nm, and detecting the fluorescence intensity using a fluorescence spectrophotometer. If there is fluorescence at 450nm, there is a response of psychoactive substances.

2. The method for detecting psychoactive substances in sewage according to claim 1, Features: In step S1, the concentration of Tween 20 in the sodium phosphate buffer containing Tween 20 is 1.80 mg / mL; The size of the gold nanoparticles is 38-42 nm, the concentration of the aqueous solution of the gold nanoparticles is 10 nM, and the volume ratio of the aqueous solution of the gold nanoparticles to the sodium phosphate buffer containing Tween 20 is 1:

1.

3. The method for detecting psychoactive substances in sewage according to claim 2, Features: In step S1, the incubation time is 35 to 45 minutes; the centrifugal speed is 10000 rpm, the time is 5 minutes, and the gold nanoparticles are washed with sodium phosphate buffer after centrifugation. The centrifugal washing step is repeated three times, and the needle is aligned with the gold nanoparticles for washing; the concentration of the washing sodium phosphate buffer is 10 mM, and the pH value is 7.4; the ultrasonic treatment time is 20 minutes; The stock solution concentration of the cocaine aptamer, methamphetamine aptamer or codeine aptamer is 10 μM; The shaking incubation was performed at a speed of 1000 rpm / S, a temperature of 25°C, and a time of 3 h; In step S1, the dialysate used for dialysis is 5 mM PBS buffer, the dialysis time is 12 h, and the dialysis bag specification is MWCO: 12-14.

4. The method for detecting psychoactive substances in sewage according to claim 1, Features: Step S2 includes: preparing 100 μg / ml poly-lysine working solution, filtering and sterilizing it through a 5 μm filter, adding it to the multi-well plate so that the bottom of the multi-well plate can be completely covered, placing it in a 37-degree incubator for 1 hour, then absorbing the liquid and allowing the surface to dry.

5. The method for detecting psychoactive substances in sewage according to claim 1, Features: In step S3, PBS buffer, QD, EDC, and NHS were added to the poly-lysine-coated multi-well plate, and incubated for 3 h at room temperature and in the dark using a micro-oscillator set to 1000 rpm.

6. The method for detecting psychoactive substances in sewage according to claim 5, Features: The concentration of the PBS buffer is 25 mM, the pH value is 6.0, the concentration of EDC is 0.5 M, the concentration of NHS is 0.125 M, and the volume ratio of the PBS buffer, QD, EDC, and NHS is 10:10:4:4; In step S3, a light shielding object is used to cover the micro-oscillator, and the light absorption rate of the light shielding object reaches 98-99%.

7. The method for detecting psychoactive substances in sewage according to claim 6, Features: In step S4, the shaking incubation speed is 1000 rpm and the time is 2 h.

8. The method for detecting psychoactive substances in sewage according to any one of claims 1 to 7, Features: Step S5 also includes obtaining a fluorescence linear equation for quantum dot-aptamer-gold nanoparticle composite detection through experiments, and calculating the concentration of the target substance according to the detected fluorescence intensity.

9. The method for detecting psychoactive substances in sewage according to claim 8, Features: The fluorescence linear equation for quantum dot-aptamer-gold nanoparticle composite detection obtained by experiment includes: A series of standard solutions of cocaine, methamphetamine or codeine with concentrations of 4 nmo / L, 6 nmo / L, 7 nmo / L, 9 nmo / L and 15 nmo / L are prepared, and the solutions are added to the quantum dot-aptamer-gold nanoparticle fluorescent probe solution obtained in step S4 for 30 minutes, and then rinsed with a sodium phosphate buffer solution; the solution is excited within a range of 400 nm to 500 nm, the fluorescence intensity is detected by a fluorescence spectrophotometer, and the relationship between the change in the fluorescence intensity of the standard solution of each concentration and the concentration of the target substance is calculated, and the corresponding fluorescence linear equation is obtained by fitting.

10. Detection platform for psychoactive substances in sewage, Features: The detection is carried out using the method for detecting psychoactive substances in sewage as described in any one of claims 1 to 9.

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