Multi-aptamer sensor for detecting novel psychoactive substances and application of multi-aptamer sensor

Abstract

The invention discloses a multi-aptamer sensor for detecting novel psychoactive substances and application of the multi-aptamer sensor. Belongs to the technical field of poison detection. The invention provides a simple and universal chemically-coupled multi-aptamer sensor design prototype, a double-chain aptamer is coupled to the surface of a two-dimensional nano-carrier through a chemical bond by virtue of a liquid-phase amide reaction, and the chemical coupling efficiency of the double-chain aptamer and the carrier is synergistically improved by virtue of a surface passivation post-treatment technology, so that the chemical coupling efficiency of the double-chain aptamer and the carrier is improved. Compared with other rapid detection technologies, the multiple aptamer sensor for detecting the novel psychoactive substances is successfully established, and compared with other rapid detection technologies, the multiple aptamer sensor can rapidly detect multiple NPS at the same time through specific fluorescence signal enhancement, realizes no cross reaction, high sensitivity and strong specificity, and develops a new thought for rapid detection system design and performance enhancement of NPS.

Description

Technical Field

[0001] The present invention relates to the technical field of poison detection, and more particularly to a multi-aptamer sensor for detecting novel psychoactive substances and application thereof. Background Art

[0002] Drug-related crimes have become a common public hazard worldwide, with serious impacts on developed and developing countries. With the increase in law enforcement against traditional drugs and the advancement of detection technology, new drug crimes have begun to surpass traditional drug crimes. In particular, the rapid spread and dissemination of new psychoactive substances (NPS) around the world has triggered new changes in the global drug landscape and brought new challenges to the formulation of drug control policies and the development of detection technology. NPS include synthetic cannabinoids, cathinones, phenethylamines, ketamine (KET), methadone (MET) and fentanyl (FET), which have stronger hallucinogenic effects than traditional drugs. Long-term use or excessive use can cause irreversible damage, and in severe cases, even pose a threat to social security and public health. Therefore, effectively identifying NPS and analyzing its content has become a key focus for law enforcement agencies.

[0003] At present, a variety of NPS detection technologies have been established, including liquid chromatography-tandem mass spectrometry (LC-MS-MS) and gas chromatography-mass spectrometry (GC-MS). These technologies have high sensitivity, selectivity and accuracy, but they also have certain limitations, such as the inability to perform on-site analysis and the need for cumbersome sample preparation. In addition, immunoassays have become an important tool for forensic identification and clinical diagnosis in recent years as a powerful on-site tool due to their high sensitivity, simplicity and rapidity. However, cross-reactions in immunoassays lead to reduced specificity and delayed detection, limiting their practical applications. For example, KET assays often cross-react with methadone and dextromethorphan.

[0004] Multiplexing of multiple NPSs is another major challenge, as drugs currently available on the market often come in the form of mixtures containing trace amounts of multiple NPSs in order to evade detection. This trend highlights the urgent need to develop novel detection technologies that are not only highly sensitive and specific, but also minimize the possibility of cross-reactivity.

[0005] Therefore, developing a sensitive, specific and rapid method for simultaneously detecting multiple NPS is a technical problem that needs to be solved urgently by those skilled in the art. Summary of the invention

[0006] In view of this, the present invention aims at the problems existing in the prior art and provides a multi-aptamer sensor for detecting new psychoactive substances and its application.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A multiple aptamer sensor for detecting novel psychoactive substances, the multiple aptamer sensor comprising a two-dimensional nanocarrier and a plurality of double-stranded aptamers grafted onto the two-dimensional nanocarrier by a liquid phase amide reaction;

[0009] The two-dimensional nanocarrier is carboxylated;

[0010] The double-stranded aptamer is composed of a probe sequence for a specific novel psychoactive substance labeled with a fluorescent dye and a complementary sequence with a terminal modified amino group through base pairing, and the fluorescent dyes used in multiple double-stranded aptamers are different.

[0011] Preferably, after the multiple double-stranded aptamers are grafted onto the two-dimensional nanocarrier, the surface of the two-dimensional nanocarrier may be passivated using a passivating agent.

[0012] The beneficial effects of the above operation are as follows: the multi-aptamer sensor of the present invention is prepared by reacting a plurality of aptamers with a carboxylated two-dimensional nanocarrier through a liquid phase amide reaction. The surface of the carboxylated two-dimensional nanocarrier has a high density of carboxyl groups, so it can be used as a good nanocarrier to efficiently chemically connect multiple aptamers. At the same time, the ends of the designed double-stranded aptamers all contain amino groups, so that a plurality of aptamers can be fixed to the surface of the two-dimensional nanocarrier in the form of chemical bonds by condensing carboxyl groups and amino groups to form amide bonds. Moreover, compared with single-stranded aptamers (only probe sequences), since most of the hydrophobic bases of the double-stranded aptamers are arranged in a double-stranded structure, the strong physical adsorption on the surface of the two-dimensional nanocarrier can be significantly reduced, thereby increasing the efficiency of chemical coupling during the amide reaction. In addition, after the surface of the two-dimensional nanocarrier is passivated by a passivating agent, the physically adsorbed aptamers can be further eliminated, thereby enhancing the efficiency of chemical coupling. In short, the combination of the chemical grafting method and the passivated surface technology can minimize the nonspecific signals caused by the physically adsorbed aptamers to the greatest extent.

[0013] Preferably, the plurality of double-stranded aptamers target different types of novel psychoactive substances, including ketamine, fentanyl, methadone, synthetic cannabinoids, methamphetamine, phenethylamine, cathinone, piperazine, fluamine, psilocin and dimethyltryptamine;

[0014] Preferably, the two-dimensional nanocarrier is carboxylated graphene oxide CGO, graphene oxide, MoS2, or MnO2 nanosheets;

[0015] Preferably, the fluorescent dye may be Cy3, Cy5, FAM, rhodamine, fluorescein, coumarin, NBD-amines, naphthylimides, BODIPYs, cyanine dyes, thiazines or oxazines.

[0016] Preferably, when the novel psychoactive substances detected are ketamine KET, fentanyl FET and methadone MET, the two-dimensional nanocarrier is carboxylated graphene oxide CGO; the passivator is salmon sperm DNA; and the double-stranded aptamers are dsCy5-KA, dsCy3-FA and dsFAM-MA, respectively, and the specific sequences are as follows:

[0017] KA probe sequence: 5'-GGGGGGACGGGGCGGGACGTGGTGTGTGGTT CGTGTCCCC-3', SEQ ID NO.1; the 5' end of the sequence is labeled with Cy5;

[0018] cKA complementary sequence: 5'-ACACACCACGTCCCGCCCCGTCCCCTCAAAAAA-3', SEQ ID NO.2; the 3' end of the sequence is labeled with NH2;

[0019] FA probe sequence: 5'-CTTACGACGAGCGCGTGTGGCCGGCGTGAGGGA GGTGAGTCG-3', SEQ ID NO. 3; the 5' end of the sequence is labeled with Cy3;

[0020] cFA complementary sequence: 5'-GCCACACGCGCTCGTCGTATGAAAAAA-3', SEQ ID NO.4; the 3' end of the sequence is labeled with NH2;

[0021] MA probe sequence: 5'-ACCTTAAGTGGGGTTCGGGTGGAGTTTATGG GGT-3', SEQ ID NO.5; the 5' end of the sequence is labeled with FAM;

[0022] cMA complementary sequence: 5'-CCGAACCCCACTTAAGGTAAAAAA-3', SEQ ID NO.6; the 3' end of the sequence is labeled with NH2.

[0023] The beneficial effect of the above operation is: when detecting ketamine KET, fentanyl FET and methadone MET, the multi-aptamer sensor includes three aptamers, dsCy5-KA, dsCy3-FA and dsFAM-MA, which have specific binding abilities to the three NPSs of KET, FET and MET respectively. In the absence of NPS, the dye-labeled aptamer is fixed on the CGO surface by chemical bonds, and the fluorescence of the dye is effectively quenched due to the fluorescence resonance energy transfer (FRET) effect. However, when a specific NPS target appears, the probe sequence of the aptamer hybridizes with the NPS target to form a more stable complex, causing the dye marker to dissociate from the CGO surface following the probe sequence, thereby causing the fluorescence signal of the dye to be enhanced. Therefore, the rapid non-cross detection of the three NPSs can be realized by the fluorescence signal changes of the respective dyes (the maximum fluorescence intensity peaks of the three dyes, Cy5, Cy3 and FAM, appear at 665nm, 565nm and 520nm wavelengths, respectively).

[0024] Another object of the present invention is to provide: a method for preparing a multi-aptamer sensor for detecting novel psychoactive substances, comprising the following steps:

[0025] (1) Carboxylation of graphene oxide: adding graphene oxide CGO into MES buffer containing EDC and NHS to activate the carboxyl group;

[0026] (2) Liquid phase amide reaction: Add three double-stranded aptamers, dsCy5-KA, dsCy3-FA and dsFAM-MA, to the carboxylated graphene oxide solution of step (1) at the same time, incubate with shaking at room temperature, centrifuge and filter to obtain the precipitate after the incubation, and wash the precipitate with buffer to obtain the initial product of the multiple aptamer sensor;

[0027] (3) Passivation: The initial product of the multiple aptamer sensor in step (2) is placed in a buffer solution containing SSD for incubation. After the incubation, the precipitate is collected by centrifugation and washed with a buffer solution containing SSD to prepare a multiple aptamer sensor for detecting novel psychoactive substances.

[0028] Preferably, in the MES buffer of step (1), the final concentrations of EDC and NHS are both 1-100 mM, the final concentration of MES is 25 mM, and the final concentration of graphene oxide is 1-300 μg / mL; the pH of the MES buffer is 3-9; and the time for carboxyl activation is 5-60 min.

[0029] Further preferably, in step (1), the final concentrations of EDC and NHS in the MES buffer are both 10 mM, and the final concentration of graphene oxide is 50 μg / mL; the time for the carboxyl activation is 15 min, which greatly enhances the efficiency of the amide reaction.

[0030] Preferably, in step (2), the final concentration of the three double-stranded aptamers dsCy5-KA, dsCy3-FA and dsFAM-MA in the carboxylated graphene oxide solution is 0.01-10 μM; the incubation time is 10-600 min; the buffer composition is as follows: 1-200 mM HEPES, 1-300 mM Na + and 0.01-100 mM Mg 2+ , pH is 5-10; the number of washing times of the precipitate is ≥2.

[0031] Further preferably, in step (2), the final concentration of the three double-stranded aptamers dsCy5-KA, dsCy3-FA and dsFAM-MA in the carboxylated graphene oxide solution is 0.1 μM; the incubation time is 180 min. This concentration can not only reduce the steric hindrance between the aptamers, but also reduce the electrostatic repulsion between graphene oxide and the aptamers, thereby increasing the chemical grafting efficiency of the three aptamers on the graphene oxide surface.

[0032] Preferably, the composition of the buffer in step (3) is as follows: containing SSD, 1-200mM HEPES, 1-300mM Na + and 0.01-100 mM Mg 2+ The final concentration of SSD in the buffer is 0.1-100 mg / mL; the pH is 5-10; the incubation time is 1-120 min; and the number of washing times of the precipitate is ≥ 2. The SSD passivation post-treatment can reduce the physical adsorption of the unhybridized aptamer on the CGO surface and further enhance the efficiency of chemical grafting.

[0033] Further preferably, in step (3), the final concentration of SSD in the buffer solution is 1 mg / mL, and the mass fraction is 0.2%; and the incubation time is 30 min.

[0034] Another object of the present invention is to provide: application of the above-mentioned multiple aptamer sensor in detecting new psychoactive substances.

[0035] Another object of the present invention is to provide: application of the multi-aptamer sensor prepared by the above method in the simultaneous detection of ketamine KET, fentanyl FET and methadone MET.

[0036] It can be seen from the above technical solution that compared with the prior art, the present invention has the following beneficial effects:

[0037] The present invention provides a simple and universal chemically coupled multiple aptamer sensor design prototype, which synergistically improves the chemical coupling efficiency by utilizing liquid phase amide reaction and surface passivation post-treatment technology of double-stranded aptamers and two-dimensional nanocarriers, and a multiple aptamer sensor is successfully established. Compared with other rapid detection technologies, the present invention synergistically utilizes multiple grafted aptamers to simultaneously and rapidly detect multiple NPSs through specific fluorescence signal enhancement, and achieves no cross-reaction, high sensitivity, and strong specificity, thus developing new ideas for the design of rapid detection systems for NPSs and performance enhancement.

[0038] In addition, compared with single-stranded aptamers (only probe sequences), since most of the hydrophobic bases of double-stranded aptamers are arranged in a double-stranded structure, the strong physical adsorption on the surface of the two-dimensional nanocarrier can be significantly reduced, thereby increasing the efficiency of chemical coupling during the amide reaction. In addition, after the surface of the two-dimensional nanocarrier is passivated by a passivating agent, the physically adsorbed aptamers can be further eliminated, and the interference of DNA and protein molecules in the biological matrix can be reduced, thereby enhancing the efficiency of chemical coupling. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0040] Figure 1 : Characterization of the structure and physical and chemical properties of CGO before and after chemical grafting, where a and b are the atomic force microscope images of CGO before and after chemical grafting, respectively; c and d are the diameters of CGO before and after chemical grafting, respectively;

[0041] Figure 2 The following are the characteristic UV-visible spectra of CGO samples before and after chemical grafting.

[0042] Figure 3 The Zeta potential diagram of CGO samples before and after chemical grafting.

[0043] Figure 4 : Fourier transform infrared spectra of CGO samples before and after chemical grafting;

[0044] Figure 5 The present invention provides: a prototype of the CGO-KFM multi-aptamer sensor design prepared in Example 1 of the present invention and the principle of rapid detection of new psychoactive substances (NPS) such as ketamine (KET), fentanyl (FET) and methadone (MET);

[0045] Figure 6Figure 1: Sensitivity detection of single-modified CGO-aptamer sensors, where a, c and e are the fluorescence intensity changes of CGO-KA, CGO-FA and CGO-MA aptamer sensors with the increase of their respective target NPS (KET, FET and MET) concentrations; b, d and f are the correlation diagrams between the fluorescence change ratio (F-F0) / F0 of CGO-KA, CGO-FA and CGO-MA aptamer sensors and the corresponding NPS concentrations;

[0046] Figure 7 The coupling efficiency of three aptamers dsCy5-KA, dsCy3-FA and dsFAM-MA in CGO-KFM multi-aptamer sensor;

[0047] Figure 8 Figure 1 is the cross-reaction evaluation of the CGO-KFM aptamer sensor of Example 1 and the PCGO-KFM aptamer sensor of Control Group 1, wherein a is the fluorescence emission spectrum response of CGO-KFM to KET (dotted line), FET (dashed line) and MET (solid line); b is the absence of cross-reaction of CGO-KFM; c is the presence of cross-reaction of PCGO-KFM, indicating the presence (+) or absence (-) of a specific NPS, and light fonts show any false negative / positive results. DETAILED DESCRIPTION

[0048] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0049] Example 1

[0050] A multi-aptamer sensor CGO-KFM for detecting new psychoactive substances such as ketamine KET, fentanyl FET and methadone MET, the multi-aptamer sensor comprising carboxylated graphene oxide CGO and double-stranded aptamers dsCy5-KA, dsCy3-FA and dsFAM-MA grafted onto CGO by liquid phase amide reaction; after the carboxylated graphene oxide CGO is grafted with double-stranded aptamers, salmon sperm DNA (purchased from Sigma Life Science in St. Louis, USA) is used to passivate the CGO surface to obtain the obtained product;

[0051] The double-stranded aptamers dsCy5-KA, dsCy3-FA and dsFAM-MA have the following specific sequences:

[0052] KA probe sequence: 5'-GGGGGGACGGGGCGGGACGTGGTGTGTGGTT CGTGTCCCC-3', SEQ ID NO.1; the 5' end of the sequence is labeled with Cy5;

[0053] cKA complementary sequence: 5'-ACACACCACGTCCCGCCCCGTCCCCTCAAAAAA-3', SEQ ID NO.2; the 3' end of the sequence is labeled with NH2;

[0054] FA probe sequence: 5'-CTTACGACGAGCGCGTGTGGCCGGCGTGAGGGA GGTGAGTCG-3', SEQ ID NO. 3; the 5' end of the sequence is labeled with Cy3;

[0055] cFA complementary sequence: 5'-GCCACACGCGCTCGTCGTATGAAAAAA-3', SEQ ID NO.4; the 3' end of the sequence is labeled with NH2;

[0056] MA probe sequence: 5'-ACCTTAAGTGGGGTTCGGGTGGAGTTTATGG GGT-3', SEQ ID NO.5; the 5' end of the sequence is labeled with FAM;

[0057] cMA complementary sequence: 5'-CCGAACCCCACTTAAGGTAAAAAA-3', SEQ ID NO.6; the 3' end of the sequence is labeled with NH2.

[0058] The preparation method of the above-mentioned multi-aptamer sensor for detecting the new psychoactive substances of ketamine KET, fentanyl FET and methadone MET is as follows:

[0059] (1) Carboxylation of graphene oxide: Graphene oxide CGO was added to MES buffer containing EDC and NHS, the final concentrations of EDC and NHS in the MES buffer were both 10 mM, the final concentration of MES was 25 mM, and the final concentration of graphene oxide was 50 μg / mL; the pH of the MES buffer was 6.5, and the carboxyl group was activated for 15 min;

[0060] (2) Liquid phase amide reaction: To the carboxylated graphene oxide solution of step (1), three double-stranded aptamers, dsCy5-KA, dsCy3-FA and dsFAM-MA, were added simultaneously, and the final concentration of the three double-stranded aptamers in the carboxylated graphene oxide solution was 0.1 μM; the mixture was incubated at room temperature with shaking for 180 min. After the incubation, the precipitate was collected by centrifugation and filtered, and the mixture was washed with a buffer solution (25 mM HEPES, 50 mM Na + and 1.0 mM Mg2+ , pH 8.0) washing the precipitate twice to purify the product, thereby obtaining a preliminary product of a multiplex aptamer sensor;

[0061] (3) Passivation: The initial product of the multi-aptamer sensor in step (2) was placed in a buffer containing SSD (containing SSD, 25 mM HEPES, 50 mM Na + and 1.0 mM Mg 2+ ), the final concentration of SSD in the buffer solution was 1 mg / mL, the pH was 8.0, and the incubation was carried out for 30 minutes. After the incubation, the precipitate was collected by centrifugation, and the precipitate was washed twice with a buffer solution containing SSD. Finally, the precipitate was redispersed in a buffer solution containing SSD and stored at -20°C to prepare a multiple aptamer sensor for detecting new psychoactive substances.

[0062] Example 2

[0063] A multi-aptamer sensor CGO-KFM for detecting new psychoactive substances such as ketamine KET, fentanyl FET and methadone MET is the same as Example 1, except that the preparation method is as follows:

[0064] The preparation method of the above-mentioned multi-aptamer sensor for detecting the new psychoactive substances of ketamine KET, fentanyl FET and methadone MET is as follows:

[0065] (1) Carboxylation of graphene oxide: Graphene oxide CGO was added to MES buffer containing EDC and NHS, the final concentrations of EDC and NHS in the MES buffer were both 1 mM, the final concentration of MES was 25 mM, and the final concentration of graphene oxide was 10 μg / mL; the pH of the MES buffer was 5, and the carboxyl group was activated for 5 min;

[0066] (2) Liquid phase amide reaction: To the carboxylated graphene oxide solution of step (1), three double-stranded aptamers, dsCy5-KA, dsCy3-FA and dsFAM-MA, were added simultaneously, and the final concentration of the three double-stranded aptamers in the carboxylated graphene oxide solution was 0.01 μM; the mixture was incubated at room temperature with shaking for 10 min. After the incubation, the precipitate was collected by centrifugation and filtered, and the mixture was washed with a buffer solution (25 mM HEPES, 50 mM Na + and 1.0 mM Mg 2+ , pH 8.0) washing the precipitate twice to purify the product, thereby obtaining a preliminary product of a multiplex aptamer sensor;

[0067] (3) Passivation: The initial product of the multi-aptamer sensor in step (2) was placed in a buffer containing SSD (containing SSD, 25 mM HEPES, 50 mM Na + and 1.0 mM Mg 2+), the final concentration of SSD in the buffer solution was 0.1 mg / mL, the mass fraction was 0.01%, the pH was 8.0, and the incubation was 1 min. After the incubation, the precipitate was centrifuged and washed twice with a buffer solution containing SSD. Finally, the precipitate was redispersed in a buffer solution containing SSD and stored at -20°C to prepare a multiple aptamer sensor for detecting new psychoactive substances.

[0068] Example 3

[0069] A multi-aptamer sensor CGO-KFM for detecting new psychoactive substances such as ketamine KET, fentanyl FET and methadone MET is the same as Example 1, except that the preparation method is as follows:

[0070] The preparation method of the above-mentioned multi-aptamer sensor for detecting the new psychoactive substances of ketamine KET, fentanyl FET and methadone MET is as follows:

[0071] (1) Carboxylation of graphene oxide: Graphene oxide CGO was added to MES buffer containing EDC and NHS, the final concentrations of EDC and NHS in the MES buffer were both 100 mM, the final concentration of MES was 25 mM, and the final concentration of graphene oxide was 300 μg / mL; the pH of the MES buffer was 10, and the carboxyl group was activated for 60 min;

[0072] (2) Liquid phase amide reaction: To the carboxylated graphene oxide solution of step (1), three double-stranded aptamers, dsCy5-KA, dsCy3-FA and dsFAM-MA, were added simultaneously, and the final concentration of the three double-stranded aptamers in the carboxylated graphene oxide solution was 10 μM; the mixture was incubated at room temperature with shaking for 600 min. After the incubation, the precipitate was collected by centrifugation and filtered, and the mixture was washed with a buffer solution (25 mM HEPES, 50 mM Na + and 1.0 mM Mg 2+ , pH 8.0) washing the precipitate twice to purify the product, thereby obtaining a preliminary product of a multiplex aptamer sensor;

[0073] (3) Passivation: The initial product of the multi-aptamer sensor in step (2) was placed in a buffer containing SSD (containing SSD, 25 mM HEPES, 50 mM Na + and 1.0 mM Mg 2+ )The final concentration of SSD in the buffer solution is 100 mg / mL, the mass fraction is 0.5%, the pH is 8.0, and the incubation is carried out for 120 minutes. After the incubation, the precipitate is obtained by centrifugation, and the precipitate is washed twice with a buffer solution containing SSD. Finally, the precipitate is redispersed in a buffer solution containing SSD and stored at -20°C to prepare a multiple aptamer sensor for detecting new psychoactive substances.

[0074] Comparative Example 1

[0075] Preparation of fluorescent aptamer sensor (PCGO-KFM) based on physical adsorption strategy

[0076] First, 50 μg / mL CGO nanosheets were dispersed in a buffer solution (25 mM HEPES, 50 mM Na + and 1.0 mM Mg 2+ , pH 8.0), and then the three probe sequences Cy5-KA, Cy3-FA and FAM-MA were introduced into the CGO buffer, and the concentration of each probe sequence was 0.1 μM. The mixture was incubated at room temperature for 180 min. After incubation, the mixture was washed twice with the same buffer to remove the unbound probe sequences, and then redispersed in the buffer to prepare a fluorescent aptamer sensor (PCGO-KFM) based on the physical adsorption strategy.

[0077] Build effect analysis:

[0078] In order to verify the construction effect of the multiple aptamers prepared by the present invention for detecting novel psychoactive substances, we characterized the multiple aptamers prepared in Example 1 by a series of analytical techniques to illustrate its structure and physicochemical properties, as follows:

[0079] Atomic force microscopy was used to obtain high-resolution topographic images, which helped to evaluate the surface morphology and nanoscale features of CGO-KFM. The average size of the original CGO was about 100 nm and the thickness was about 1.0 nm, as shown in Figure 2. Figure 1 After mixing the three aptamers (dsCy5-KA, dsCy3-FA, and dsFAM-MA), the lateral size of the constructed CGO-KFM remained the same as that of the original CGO, however, a significant increase in thickness to 3.4 nm ( Figure 1 b). This indicates the effective modification of CGO by the aptamer cocktail.

[0080] In addition, dynamic light scattering (DLS) was used to further characterize the size distribution of CGO before and after chemical grafting, e.g. Figure 1 c and Figure 1 As shown in Figure d, aptamer grafting leads to a measurable increase in the diameter of CGO nanosheets from 136 nm to 170 nm. This significant enlargement, together with the increase in thickness observed by atomic force microscopy, confirms the size expansion of CGO after aptamer modification, confirming the construction effect of CGO-based aptamer sensors.

[0081] Figure 2Characteristic UV-visible spectra of CGO-KFM samples are shown, where spectra of original CGO and three double-stranded aptamers (dsCy5-KA, dsCy3-FA, and dsFAM-MA) were also measured for a more intuitive comparison. In original CGO, a peak at 257 nm was clearly observed, indicating a "π-π" transition, as well as a shoulder peak at 300 nm, corresponding to an "n-π" transition. In the spectral analysis of dsCy5-KA, dsCy3-FA, and dsFAM-MA samples, a common DNA absorption peak at 255 nm was observed, as well as characteristic absorption peaks of the respective dyes at 647 nm, 546 nm, and 459 nm. GO-KFM spectral analysis revealed distinct characteristic peaks of Cy3 at 546 nm and Cy5 at 647 nm. However, the expected absorption peak of FAM at 459 nm was not observed, probably due to its overlap with the obvious absorption profile of CGO, which is consistent with the literature report (Nanoscale, 2018, 10, 7067-7076.). In addition, the disappearance of the shoulder peak at 300 nm indicates the consumption of carboxyl groups on CGO during the amide reaction, further supporting the effective chemical grafting of the aptamer on the CGO surface. This observation is also supported by Figure 3 The increase in zeta potential after the introduction of these aptamers into CGO indicated a decrease in the number of negatively charged carboxyl groups, as supported by the zeta potential measurements shown. Overall, these double-stranded aptamers were able to be effectively grafted onto the CGO surface via a classical amide reaction.

[0082] In order to further characterize the chemical composition and functional groups of the CGO-KFM, Fourier transform infrared spectroscopy was used. Figure 4 It is shown that compared with the original CGO, the characteristic carboxyl peak at 1720 cm - 1 disappeared, and 1649cm - A new peak appeared at 1, corresponding to the formation of amide bonds in the CGO-KFM aptamer sensor.

[0083] Detection effect analysis:

[0084] In order to further verify the detection effect of the multiple aptamers prepared by the present invention for detecting novel psychoactive substances, the following experiment was designed:

[0085] Experimental group: The CGO-KFM multi-aptamer sensor prepared in Example 1 was used to detect three NPS (KET, FET, and MET);

[0086] Blank control group: no NPS added;

[0087] Result analysis: Figure 5As shown, in the absence of NPS, the dyes Cy5, Cy3 and AFM labeled on the three aptamers are very close to the CGO surface, so the fluorescence resonance energy transfer (FRET) mechanism will severely inhibit the fluorescence of these dyes. On the contrary, in the presence of target NPS, the probe sequences of the three labeled dyes preferentially bind to their respective corresponding NPS to form a more stable complex. In this case, the probe sequences of the three labeled dyes will be away from the CGO surface, and the simultaneous recovery of the fluorescence of Cy5, Cy3 and AFM dyes is observed, indicating that the CGO-KFM multi-aptamer sensor constructed by the present invention can successfully detect KET, FET and MET without cross-reaction.

[0088] Sensitivity determination:

[0089] The corresponding single-modified CGO-aptamer sensors (CGO-KA, CGO-FA, and CGO-MA, which were modified with dsCy5-KA, dsCy3-FA, and dsFAM-MA, respectively) were constructed with the same construction process as the multiple CGO-aptamer sensors, and the detection sensitivity of the corresponding three NPS single-modified CGO-aptamer sensors was evaluated.

[0090] Result analysis: Figure 6 As shown in a, 6c and 6e, as the concentration of the respective target NPS increased from 0 to 100 ng / mL, the fluorescence of these aptasensors was observed to be significantly enhanced. The maximum fluorescence intensity peaks of the CGO-KA, CGO-FA and CGO-MA aptasensors were at 665 nm, 565 nm and 520 nm, respectively. By plotting the fluorescence change ratio (F-F0) / F0 as the Y-axis and the NPS concentration as the X-axis ( Figure 6 b, 6d, and 6f), the three corresponding single-modified CGO-aptamer sensors showed strong linear relationships (correlation coefficient R 2 >0.9990), as shown in the inset, F0 and F represent the peak fluorescence intensity before and after interaction with the target NPS, respectively. This illustrates the detection effect of the corresponding single-modified CGO-aptamer sensor on the respective NPS and the versatility of this method.

[0091] Then, the sensitivity of the multi-aptamer sensor CGO-KFM constructed by the present invention for simultaneously detecting ketamine KET, fentanyl FET and methadone MET was measured. After measurement, the final sensitivities of KET, FET and MET were 0.178, 0.642 and 0.467 ng / mL, respectively.

[0092] Coupling efficiency:

[0093] At the same time, we measured the coupling efficiency of three aptamers dsCy5-KA, dsCy3-FA and dsFAM-MA on the CGO-KFM multi-aptamer sensor constructed in Example 1. The experimental results are as follows: Figure 7 shown.

[0094] Result analysis: The coupling efficiencies of KA, FA and MA were 64%, 55% and 83% respectively.

[0095] Comparison of the effects of the aptamer sensor of Example 1 of the present invention and the aptamer sensor of the control group 1:

[0096] In order to verify the effects of the chemically coupled CGO-KFM aptamer sensor of Example 1 of the present invention and the physically coupled PCGO-KFM aptamer sensor of Control Group 1, we set up a control experiment, and the specific results are as follows.

[0097] Result analysis: The experimental results showed that the CGO-KFM aptasensor could detect three specific types of NPS: KET, FET, and MET through unique fluorescence signals at 665 nm, 565 nm, and 520 nm, e.g. Figure 8 a; the presence of a single NPS activated the fluorescence of the corresponding aptamer, while the other two aptamers remained non-fluorescent; when two NPSs coexisted, the fluorescence of the two target aptamers recovered, while the fluorescence of the third aptamer remained unchanged; in the presence of all three NPSs, the fluorescence of all aptamers was simultaneously activated, matching the fluorescence intensity observed during single-target detection ( Figure 8 b) These findings strongly confirm the remarkable specificity of the CGO-KFM multiplex aptasensor in discriminating different NPSs without cross-reactivity, thus ensuring the reliable simultaneous detection of all three NPSs.

[0098] In contrast, PCGO-KFM, which consists of three aptamers physically adsorbed onto the CGO surface, showed obvious cross-reactivity ( Figure 8 c), light-colored positive and negative signs indicate that false positive / false negative results have occurred. This comparison intuitively shows the superiority of the chemical coupling method in the context of a multiplex detection platform.

[0099] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0100] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A multi-aptamer sensor for detecting novel psychoactive substances, characterized in that: The multiple aptamer sensor includes a two-dimensional nanocarrier and a plurality of double-stranded aptamers grafted onto the two-dimensional nanocarrier through a liquid phase amide reaction; The two-dimensional nanocarrier is carboxylated; The double-stranded aptamer is composed of a probe sequence for a specific novel psychoactive substance labeled with a fluorescent dye and a complementary sequence with a terminal modified amino group through base pairing, and the fluorescent dyes used in multiple double-stranded aptamers are different.

2. The multi-aptamer sensor for detecting novel psychoactive substances according to claim 1, characterized in that: After the multiple aptamer sensors are grafted with multiple double-stranded aptamers on the two-dimensional nanocarrier, a passivating agent may be used to passivate the surface of the two-dimensional nanocarrier.

3. The multi-aptamer sensor for detecting novel psychoactive substances according to claim 2, characterized in that: The multiple double-stranded aptamers target different types of new psychoactive substances, including ketamine, fentanyl, methadone, synthetic cannabinoids, methamphetamine, phenethylamine, cathinone, piperazine, fluamine, siloxane and dimethyltryptamine; the two-dimensional nanocarrier is carboxylated graphene oxide CGO, graphene oxide, MoS2, MnO2 nanosheets; the fluorescent dye is Cy3, Cy5, FAM, rhodamine, fluorescein, coumarin, NBD-amines, naphthylimide, BODIPY, cyanine dyes, thiazine or oxazine.

4. The multi-aptamer sensor for detecting novel psychoactive substances according to claim 3, characterized in that: When the new psychoactive substances detected are ketamine KET, fentanyl FET and methadone MET, their two-dimensional nanocarriers are carboxylated graphene oxide CGO; their passivators are salmon sperm DNA; their double-stranded aptamers are dsCy5-KA, dsCy3-FA and dsFAM-MA, respectively, and the specific sequences are as follows: KA probe sequence: 5'-GGGGGGACGGGGCGGGACGTGGTGTGTGGTT CGTGTCCCC-3', SEQ ID NO.1; the 5' end of the sequence is labeled with Cy5; cKA complementary sequence: 5'-ACACACCACGTCCCGCCCCGTCCCCTCAAAAAA-3', SEQ ID NO.2; the 3' end of the sequence is labeled with NH2; FA probe sequence: 5'-CTTACGACGAGCGCGTGTGGCCGGCGTGAGGGA GGTGAGTCG-3', SEQ ID NO.3; the 5' end of the sequence is labeled with Cy3; cFA complementary sequence: 5'-GCCACACGCGCTCGTCGTATGAAAAAA-3', SEQ ID NO.4; the 3' end of the sequence is labeled with NH2; MA probe sequence: 5'-ACCTTAAGTGGGGTTCGGGTGGAGTTTATGG GGT-3', SEQ ID NO.5; the 5' end of the sequence is labeled with FAM; cMA complementary sequence: 5'-CCGAACCCCACTTAAGGTAAAAAA-3', SEQ ID NO.6; the 3' end of the sequence is labeled with NH2.

5. A method for preparing a multi-aptamer sensor for detecting novel psychoactive substances according to claim 4, characterized in that: The steps include: (1) Carboxylation of graphene oxide: adding graphene oxide CGO into MES buffer containing EDC and NHS to activate the carboxyl group; (2) Liquid phase amide reaction: Add three double-stranded aptamers, dsCy5-KA, dsCy3-FA and dsFAM-MA, to the carboxylated graphene oxide solution of step (1) at the same time, incubate with shaking at room temperature, centrifuge and filter to obtain the precipitate after the incubation, and wash the precipitate with buffer to obtain the initial product of the multiple aptamer sensor; (3) Passivation: The initial product of the multiple aptamer sensor in step (2) is placed in a buffer solution containing SSD for incubation. After the incubation, the precipitate is collected by centrifugation and washed with a buffer solution containing SSD to prepare a multiple aptamer sensor for detecting novel psychoactive substances.

6. The preparation method according to claim 5, characterized in that: In step (1), the final concentrations of EDC and NHS in the MES buffer are both 1-100 mM, the final concentration of MES is 25 mM, and the final concentration of graphene oxide is 1-300 μg / mL; the pH of the MES buffer is 3-9; and the time for carboxyl activation is 5-60 min.

7. The preparation method according to claim 5, characterized in that: Step (2) The final concentration of the three double-stranded aptamers dsCy5-KA, dsCy3-FA and dsFAM-MA in the carboxylated graphene oxide solution is 0.01-10 μM; the incubation time is 10-600 min; the buffer composition is as follows: 1-200 mM HEPES, 1-300 mM Na + and 0.01-100 mM Mg 2 + , pH is 5-10; the number of washing times of the precipitate is ≥2.

8. The preparation method according to claim 5, characterized in that: The composition of the buffer in step (3) is as follows: SSD, 1-200 mM HEPES, 1-300 mM Na + and 0.01-100 mM Mg 2+ The final concentration of SSD in the buffer solution is 0.1-100 mg / mL, and the pH is 5-10; the incubation time is 1-120 min; and the number of washing times of the precipitate is ≥2.

9. Use of the multi-aptamer sensor according to any one of claims 1 to 4 in detecting new psychoactive substances.

10. Use of the multi-aptamer sensor prepared by the method according to any one of claims 5 to 8 in the simultaneous detection of ketamine KET, fentanyl FET and methadone MET.

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