Composition for detecting opioid-based drug, comprising opioid receptor nanodisc

Abstract

The present invention relates to a composition for detecting an opioid-based drug, the composition comprising an opioid receptor nanodisc. Specifically, the opioid receptor nanodisc according to the present invention detetcs an opioid-based drug with high sensitivity and selectivity, and thus may find useful application in diagnosing opioid-based drug addiction or detecting an opioid-based drug.

Description

Composition for detecting opioid-based drugs comprising opioid receptor nanodiscs

[0001] The present invention relates to a composition for detecting opioid-based drugs comprising opioid receptor nanodiscs.

[0002]

[0003] Narcotics are a type of psychotropic drug that acts on the nervous system upon administration to induce hallucinations, alertness, anesthesia, and analgesic effects, and are sometimes used for medical purposes. However, long-term use of such substances leads to addiction, causing withdrawal symptoms when the medication is discontinued. Recently, the number of narcotic users in Korea has been increasing daily, and consequently, there is a demand for methods to suppress the importation and distribution of narcotics into the country, as well as methods to accurately identify narcotics using simple means.

[0004] Currently, drug detection kits used in the field operate on the principle of identifying drugs by detecting color changes through contact with reagents such as sulfuric acid, formaldehyde, hydrochloric acid, and chloroform. These kits present problems in that they require mixing the included reagents in a specific sequence, and since these reagents contain substances harmful to the human body, they are not easy to apply in the field and may endanger the user's health. Meanwhile, a method involving the separation of drug components using thin-layer chromatography followed by the application of colorimetric reagents is also used for detection; however, this method suffers from the issues of complex procedures and the need for advanced technical skills. Therefore, there is a pressing need to develop a drug detection method that is economical and easy for anyone to use in the field.

[0005] In this regard, Korean registered patent No. 10-2823864 relates to a composition for detecting narcotics, a kit for detecting narcotics including the same, and a method for detecting narcotics using the same. The composition comprises a strip, a sample tube for holding a collected sample, and a means for collecting the sample. The strip comprises a membrane having an amine functional group, a sample pad, and an absorbent pad. The membrane comprises a potassium hexachloroplatinate solution adsorbed on its surface. When a sample is introduced into the sample pad portion of the strip and spread to the membrane portion, the narcotic component contained in the sample reacts with the potassium hexachloroplatinate present on the surface of the strip to determine whether narcotics are mixed within one minute, thereby disclosing that the presence of narcotics contained in food products such as alcohol or beverages can be detected quickly and simply.

[0006]

[0007] The object of the present invention is to provide a composition for detecting opioid-based drugs comprising opioid receptor nanodiscs.

[0008]

[0009] To achieve the above objective, the present invention provides a composition for detecting opioid-based drugs comprising opioid receptor nanodiscs.

[0010] In addition, the present invention provides a kit for detecting opioid-based drugs comprising an opioid receptor nanodisc.

[0011] In addition, the present invention provides a biosensor for detecting opioid-based drugs comprising an opioid receptor nanodisc.

[0012] In addition, the present invention provides a method for providing information for diagnosing opioid-based drug addiction, comprising the step of reacting the detection composition, the detection kit, or the biosensor with a sample.

[0013] In addition, the present invention provides a screening method for opioid-based drug addiction treatment drugs, comprising the step of treating a candidate drug with the detection composition, the detection kit, or the biosensor treated with the opioid-based drug.

[0014] Furthermore, the present invention provides a method for detecting opioid-based drugs comprising the step of reacting the detection composition, the detection kit, or the biosensor with a sample.

[0015]

[0016] The opioid receptor nanodisc according to the present invention can be usefully employed for the diagnosis of opioid-based drug addiction or drug detection by detecting opioid-based drugs with high sensitivity and selectivity.

[0017]

[0018] Figure 1 is a graph showing the results of confirming that the opioid receptor nanodiscs prepared in one embodiment of the present invention were purified by size exclusion chromatography.

[0019] Figure 2 is a photograph (A) of an opioid receptor nanodisc prepared in one embodiment of the present invention observed with a transmission electron microscope and a graph (B) of the particle size analysis results.

[0020] Figure 3 is a diagram showing the results of confirming the opioid receptor in the opioid receptor nanodisc prepared in one embodiment of the present invention.

[0021] Figure 4 is a diagram showing the result of confirming that an opioid receptor nanodisc is immobilized on a biosensor manufactured in one embodiment of the present invention.

[0022] FIG. 5 is a graph showing the results of confirming electrical stability in a biosensor manufactured in one embodiment of the present invention in a state where the gate electrode is fixed (A) or the drain-source voltage is fixed (B).

[0023] Figure 6 is a graph showing the results of confirming the detection effect of the biosensor manufactured in one embodiment of the present invention on fentanyl (A) and endorphin (B).

[0024] Figure 7 is a graph showing the results of confirming the opioid-based substance screening effect of a biosensor manufactured in one embodiment of the present invention.

[0025] Figure 8 is a graph showing the result of confirming the selectivity of opioid-based substances of a biosensor manufactured in one embodiment of the present invention.

[0026]

[0027] The present invention will be described in detail below.

[0028] The present invention provides a composition for detecting opioid-based drugs comprising opioid receptor nanodiscs.

[0029] As used herein, the term "opioid" refers to a type of narcotic also known as an opioid analgesic. Opioids bind to opioid receptors and exert effects in the brain such as pain relief, alertness, sedation, and anesthesia. Therefore, while opioid-based drugs are used as anesthetics or pain relievers, they can also cause side effects such as itching, sedation, nausea, respiratory depression, constipation, and euphoria; furthermore, long-term use may lead to tolerance, physical dependence, and withdrawal symptoms.

[0030] Meanwhile, the term "opioid receptor" is a protein identified primarily in the central and peripheral nervous systems and the gastrointestinal tract that interacts with opioids, and is one of the Class A G-protein coupled receptors. This opioid receptor exerts an analgesic effect by binding to opioids, inhibiting adenylyl cyclase to reduce intracellular cAMP levels, and simultaneously inhibiting calcium channels in presynaptic neurons while suppressing the opening of potassium channels in postsynaptic neurons.

[0031] The above-mentioned opioid receptor may include all types of opioid receptors known in the ordinary art. In one embodiment of the present invention, the opioid receptor may be a polypeptide composed of the amino acid sequence described in SEQ ID NO. 2. Additionally, the opioid receptor may include a variant in which one or more amino acid residues are substituted, deleted, or inserted from the polypeptide composed of the amino acid sequence described in SEQ ID NO. 2, provided that the protein activity is not altered. Additionally, the opioid receptor may have 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology with the polypeptide composed of the amino acid sequence described in SEQ ID NO. 2.

[0032] In addition, the polynucleotide encoding the opioid receptor may also include any sequence known in the ordinary art as a polynucleotide encoding an opioid receptor. The polynucleotide may be composed of the nucleotide sequence described in SEQ ID NO. 1, and may include variants in which one or more nucleotides in the nucleotide sequence are substituted, deleted, or inserted, provided that the activity of the protein produced therefrom is maintained. In addition, the polynucleotide may have 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology with the nucleotide sequence described in SEQ ID NO. 1.

[0033] As used herein, the term "nanodisc" refers to a structure in which the sides of a lipid bilayer formed of phospholipids are surrounded by hydrophobic bonds by membrane scaffold proteins. The nanodisc exhibits a flattened disc shape in which the hydrophilic groups of the phospholipids are oriented outward and the hydrophobic groups are oriented inward, and may contain hydrophobic drugs or other substances within the disc shape. That is, the opioid receptor nanodisc according to the present invention may be composed of an opioid receptor, lipids, and a membrane scaffold protein (MSP).

[0034] In this case, the lipid may be a phospholipid. The term "phospholipid" refers to a lipid in which glycerol is bonded to two fatty acids and one phosphate group, or to a compound containing a phosphate group and nitrogen (e.g., choline). The phospholipid comprises a hydrophilic phosphate group and glycerol in the head portion and a hydrophobic long fatty acid chain in the tail portion. The phospholipid may include all types of phospholipids known in the ordinary art, and specifically, the phospholipid may be one or more selected from the group consisting of phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol.

[0035] 더욱 구체적으로, 상기 포스파티딜콜린은 DOPC(1,2-Dioleoyl-sn-glycero-3-phosphocholine), DLPC(1,2-Dilauroyl-sn-glycero-3-phosphocholine), DMPC(1,2-Dimyristoyl-sn-glycero-3-phosphocholine), DPPC(1,2-Dipalmitoyl-sn-glycero-3-phosphocholine), POPC(1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DDPC(1,2-Didecanoyl-sn-glycero-3-phosphocholine), DSPC(1,2-Distearoyl-snglycero-3-phosphocholine), DEPC(1,2-Dierucoyl-sn-glycero-3-phosphocholine), DLOPC(1,2-Dilinoleoyl-snglycero-3-phosphocholine), EPC(Egg phosphatidylcholine), MSPC(1-Myristoyl-2-stearoyl-sn-glycero-3-phosphocholine), PMPC(1-Palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine), PSPC(1-Palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine), SMPC(1-Stearoyl-2-myristoyl-sn-glycero-3-phosphocholine) 및 SPPC(1-Stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine)로 구성된 군으로부터 선택되는 어느 하나 이상일 수 있다.In addition, the phosphatidyl glycerol is DMPG (1,2-Dimyristoyl-sn-glycero-3(Phospho-rac-(1-glycerol))), DPPG (1,2-Dipalmitoyl-sn-glycero-3(Phospho-rac-(1-glycerol))), DSPG(1,2-Distearoyl-sn-glycero3(Phospho-rac-(1-glycerol))), POPG(1-Palmitoyl-2-oleoyl-sn-glycero-3(Phospho-rac-(1-glycerol))), DEPG(1,2-Dierucoyl-sn-glycero-3(Phospho-rac-(1-glycerol))), DLPG (1,2-Dilauroyl-sn-glycero-3(Phosphorac-(1-glycerol))), It may include one or more selected from the group consisting of DOPG (1,2-Dioleoyl-sn-glycero-3(Phospho-rac-(1-glycerol))) and DSPG (1,2-Distearoyl-sn-glycero-3(Phospho-rac-(1-glycerol))). In addition, the phosphatidylethanolamine may include one or more selected from the group consisting of DMPE (1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine), DPPE (1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine), DSPE (1,2-Distearoyl-sn-glycero-3-phosphoethanolamine), DOPE (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine), DEPE (1,2-Dierucoyl-sn-glycero-3-phosphoethanolamine), DLPE (1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine) and POPE (1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine).In addition, the phosphatidylserine may include one or more selected from the group consisting of DOPS (1,2-Dioleoyl-sn-glycero-3-phosphoserine), DLPS (1,2-Dilauroyl-sn-glycero-3-phosphoserine), DMPS (1,2-Dimyristoyl-sn-glycero-3-phosphoserine), DPPS (1,2-Dipalmitoyl-sn-glycero-3-phosphoserine), DSPS (1,2-Distearoyl-sn-glycero-3-phosphoserine) and POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine). Furthermore, the phosphatidylinositol may include one or more selected from the group consisting of phosphatidylinositol-4-phosphate, phosphatidylinositol-4,5-bisphosphate, and phosphatidylinositol-3,4,5-bisphosphate.

[0036] The above term, "membrane scaffold protein," is a protein having a helix structure and exhibiting amphiphilicity, which serves to surround the sides of a lipid bilayer. Specifically, the membrane scaffold protein may be an apolipoprotein (Apo), and more specifically, the apolipoprotein may be one or more selected from the group consisting of apolipoprotein A1 (ApoA-1), apolipoprotein A2 (AproA-2), apolipoprotein B (ApoB), apolipoprotein C (ApoC), apolipoprotein E (ApoE), MSP1 (membrane scaffold protein 1), MSP1D1, MSP1D2, MSP1E1, MSP1E2, MSP1E3, MSP1E3D1, MSP2, MSP2N1, MSP2N2, and MSP2N3.

[0037] The above opioid receptor nanodiscs may be formed by mixing opioid receptors and membrane scaffold proteins in a ratio of 1:1 to 10, 1:1 to 8, 1:1 to 6, 1:1 to 4, 1:2 to 10, 1:2 to 8, 1:2 to 6, 1:2 to 4, 1:4 to 10, 1:4 to 8, or 1:4 to 6. Meanwhile, the above-mentioned opioid receptor nanodisc comprises an opioid receptor and lipids in a ratio of 1:1 to 700, 1:1 to 650, 1:1 to 600, 1:1 to 550, 1:1 to 500, 1:1 to 450, 1:1 to 400, 1:1 to 350, 1:1 to 300, 1:1 to 250, 1:50 to 700, 1:50 to 650, 1:50 to 600, 1:50 to 550, 1:50 to 500, 1:50 to 450, 1:50 to 400, 1:50 to 350, 1:50 to 300, 1:50 to 250, 1:100 to 700, 1:100 to 650, 1:100 to 600, 1:100 to 550, 1:100 to 500, 1:100 to 450, 1:100 to 400, 1:100 to 350, 1:100 to 300, 1:100 to 250, 1:150 to 700, 1:150 to 650, 1:150 to 600, 1:150 to 550, 1:150 to 500, 1:150 to 450, 1:150 to 400, 1:150 to 350, 1:150 to 300, 1:150 to 250, 1:200 to 700, 1:200 to 650, 1:200 to 600, 1:200 to 550, 1:200 to 500, 1:200 to 450, 1:200 to 400, 1:200 to 350, 1:200 to 300, 1:200 to 250, 1:250 to 700, 1:250 to 650, 1:250 to 600, 1:250 to 550, 1:250 to 500, 1:250 to 450, 1:250 to 400, 1:250 to 350,It can be formed by mixing in a ratio of 1:250 to 300, 1:300 to 700, 1:300 to 650, 1:300 to 600, 1:300 to 550, 1:300 to 500, 1:300 to 450, 1:300 to 400, 1:300 to 350, 1:350 to 700, 1:350 to 650, 1:350 to 600, 1:350 to 550, 1:350 to 500, 1:350 to 450, or 1:350 to 400.

[0038] In addition, the opioid receptor nanodisc according to the present invention has a diameter of 50 nm or less, 0.1 to 50 nm, 0.1 to 45 nm, 0.1 to 40 nm, 0.1 to 35 nm, 0.1 to 30 nm, 0.1 to 25 nm, 0.1 to 20 nm, 0.1 to 15 nm, 1 to 50 nm, 1 to 45 nm, 1 to 40 nm, 1 to 35 nm, 1 to 30 nm, 1 to 25 nm, 1 to 20 nm, 1 to 15 nm, 3 to 50 nm, 3 to 45 nm, 3 to 40 nm, 3 to 35 nm, 3 to 30 nm, 3 to 25 nm, 3 to 20 nm, 3 It may have a diameter of up to 15 nm, 5 to 50 nm, 5 to 45 nm, 5 to 40 nm, 5 to 35 nm, 5 to 30 nm, 5 to 25 nm, 5 to 20 nm, 5 to 15 nm, 8 to 50 nm, 8 to 45 nm, 8 to 40 nm, 8 to 35 nm, 8 to 30 nm, 8 to 25 nm, 8 to 20 nm, 8 to 15 nm, 10 to 50 nm, 10 to 45 nm, 10 to 40 nm, 10 to 35 nm, 10 to 30 nm, 10 to 25 nm, 10 to 20 nm, or 10 to 15 nm.

[0039] The above-mentioned opioid class drugs may include all types of drugs known in the ordinary art. For example, the above-mentioned opioid class drugs may be one or more selected from the group consisting of morphine, oxycodone, hydrocodone, hydromorphone, fentanyl, tramadol, and methadone.

[0040] In addition, the above composition may include a ligand capable of specifically binding to an opioid receptor nanodisc according to the present invention. The ligand may be a conjugate labeled with a detector such as a chromogenic enzyme, a fluorescent substance, a radioisotope, or a colloid, and a ligand treated with streptavidin or avidin. In addition to the reagents described above, the detection composition of the present invention may further include distilled water or a buffer solution that maintains the structure of the reagents stably.

[0041]

[0042] In addition, the present invention provides a kit for detecting opioid-based drugs comprising an opioid receptor nanodisc.

[0043] The opioid receptor nanodisc included in the kit for detecting opioid-based drugs according to the present invention may have the characteristics described above.

[0044] In addition, the kit may be bound to a solid substrate to facilitate subsequent steps, such as washing the opioid receptor nanodiscs included therein or separating the complex. In this case, the solid substrate may be a synthetic resin, nitrocellulose, a glass substrate, a metal substrate, a glass fiber, a microsphere, or a microbead. Additionally, the synthetic resin may be polyester, polyvinyl chloride, polystyrene, polypropylene, PVDF, or nylon.

[0045] In addition, the above kit may be manufactured by a conventional manufacturing method known to those skilled in the art and may further include a buffer, a stabilizer, an inert protein, etc.

[0046]

[0047] In addition, the present invention provides a biosensor for detecting opioid-based drugs comprising an opioid receptor nanodisc.

[0048] The opioid receptor nanodisc included in the biosensor for detecting opioid-based drugs according to the present invention may have the characteristics described above.

[0049] In addition, the biosensor may have an opioid receptor nanodisc according to the present invention immobilized on a substrate by a linker. Specifically, the substrate may include any type of substrate known in the ordinary art to be used for manufacturing biosensors, and for example, the substrate may be one or more selected from the group consisting of graphene, carbon nanotubes (CNT), and conducting polymers. Meanwhile, the linker may also include any type of linker that can be used in the ordinary art. For example, the linker may be a 1-pyrenebutyric acid N-hydroxysuccinimide ester (PBASE) or a peptide linker. In addition, the peptide linker may be composed of tryptophan and phenylalanine, etc.

[0050] In addition, the biosensor may further include a device capable of amplifying the current generated by the binding of an opioid receptor nanodisc immobilized on a substrate with a target substance. The device may include a working electrode, a reference electrode, a counter electrode, and an electrode protection layer.

[0051]

[0052] In addition, the present invention provides a method for providing information for diagnosing opioid-based drug addiction, comprising the step of reacting the detection composition, the detection kit, or the biosensor with a sample.

[0053] A detection composition, detection kit, or biosensor used in the method for providing information for diagnosing opioid-based drug addiction according to the present invention may include an opioid receptor nanodisk having the characteristics described above.

[0054] In addition, any type of sample may be used as long as it is for diagnosing opioid-based drug intoxication; for example, the sample may be of animal origin. Specifically, the animal may be a mammal, and more specifically, the mammal may be a human. Furthermore, the sample may be one or more selected from the group consisting of blood, saliva, tears, urine, synovial fluid, mucus, cells, and tissues.

[0055] The above sample may be pretreated according to methods well known in the art before being used in the information provision method according to the present invention. For example, the pretreatment may include methods such as anion exchange chromatography, affinity chromatography, size exclusion chromatography, liquid chromatography, serial extraction, centrifugation, and gel electrophoresis.

[0056]

[0057] In addition, the present invention provides a screening method for opioid-based drug addiction treatment drugs, comprising the step of treating a candidate drug with the detection composition, the detection kit, or the biosensor treated with the opioid-based drug.

[0058] A detection composition, detection kit, or biosensor used in the screening method for opioid-based drug addiction treatments according to the present invention may include an opioid receptor nanodisk having the characteristics described above.

[0059] Any substance that is expected to inhibit the binding of opioid-class drugs to opioid receptors may be applied as the above candidate substance. For example, the above candidate substance may include compounds, DNA, RNA, proteins, aptamers, enzymes, extracts, antibodies, aptamers, polypeptides, exosomes, cell lysates, cells, fermented products, and cell culture media.

[0060] In the above screening method, a substance that inhibits the interaction between an opioid-class drug and an opioid receptor nanodisk by treatment with a candidate drug can be identified as an opioid-class drug addiction treatment.

[0061]

[0062] Furthermore, the present invention provides a method for detecting opioid-based drugs comprising the step of reacting the detection composition, the detection kit, or the biosensor with a sample.

[0063] A detection composition, detection kit, or biosensor used in the method for detecting opioid-based drugs according to the present invention may include an opioid receptor nanodisk having the characteristics described above.

[0064] In addition, any type of sample may be used as long as it is a sample for detecting opioid-based drugs, and it may be pretreated according to methods well known in the ordinary art as described above before being used in the detection method.

[0065]

[0066] The present invention is described in detail below by way of the following examples. However, the following examples are merely illustrative of the present invention and do not limit the present invention. Any configuration having substantially the same structure as the technical concept described in the claims of the present invention and achieving the same functional effect is included within the technical scope of the present invention.

[0067]

[0068] Example 1. Fabrication of opioid receptor nanodiscs-(1)

[0069] Nanodisks containing opioid receptors were fabricated using the following method.

[0070] First, the opioid receptor gene fused with the 6×His tag (SEQ No. 1) was cloned into the pET-DEST42 expression vector and transformed into Rosetta strains using conventional methods. The Rosetta strains were cultured overnight at 37°C and 150 rpm using six flasks containing 1 L of culture medium. OD of the culture medium 600When the value reached 0.45 to 0.5, 1 ml of 1 M IPTG (Isopropyl β-D-1-thiogalactopyranoside) was added, and the culture was continued for 4 more hours under the same conditions. After the culture was finished, the culture medium was centrifuged at 4°C and 7,000 rpm for 10 minutes to obtain a pellet, and lysis buffer was added to lyse the cells. The cell lysate was centrifuged under the conditions described above to obtain the supernatant, and the opioid receptor was purified from the obtained supernatant using a nickel column by a conventional method. Purified opioid receptors, MSP1E3D1 protein, and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) were mixed in a volume ratio of 1:3:390 at equal molar concentrations and reacted for 16 hours under conditions of 20°C and 80 rpm to obtain nanodiscs containing opioid receptors. SM2 resin (Bio-Rad) was added to the obtained opioid receptor nanodiscs to remove detergent substances, and the nanodiscs were purified by size exclusion chromatography. As a result, the purified opioid receptor nanodiscs were identified at an absorbance of 280 nm, and the results are shown in Figure 1.

[0071] As shown in Figure 1, it was confirmed that the opioid receptor nanodiscs were purified. Specifically, the purified opioid receptor nanodiscs were identified by a maximum peak observed at a retention volume of approximately 11 ml.

[0072]

[0073] Experimental Example 1. Verification of Nanodisk Size

[0074] The size of the above-described opioid receptor nanodiscs was confirmed using a transmission electron microscope and a particle size analyzer. Specifically, the size of the opioid receptor nanodiscs was confirmed by negative staining with 2% uranyl acetate followed by imaging with a transmission electron microscope. Meanwhile, particle size analysis was performed using a zeta sizer. As a result, the image observed with a transmission electron microscope is shown in Figure 2A, and the results of the particle size analysis are shown in Figure 2B.

[0075] As shown in Figure 2, the nanodiscs manufactured in the above example exhibited an average size of 13.28 nm and were formed to a suitable size.

[0076]

[0077] Experimental Example 2. Confirmation of Opioid Receptors

[0078] The presence of opioid receptors in the fabricated opioid receptor nanodiscs was confirmed as follows using the Western blot method.

[0079] Specifically, an equal amount of 2× sample buffer was added to the opioid receptor nanodisc prepared in Example 1, and the sample was placed in boiling water for 10 minutes. Using this as a sample, electrophoresis was performed by a conventional method, and the proteins electrophoresed on the acrylamide gel were transferred to a nitrocellulose membrane. The nitrocellulose membrane was pretreated with 5% skim milk for 1 hour, and an anti-histidine antibody was added as the primary antibody and reacted overnight at 4°C. After the reaction was complete, the cellulose membrane was washed, and an anti-mouse secondary antibody was added and reacted at room temperature for 1 hour. After the reaction was complete, the cellulose membrane was treated with an ECL solution to check the bands, and the results are shown in Figure 3.

[0080] As shown in Figure 3, the manufactured opioid receptor nanodiscs contained opioid receptors of approximately 50 kDa in size.

[0081]

[0082] Preparation Example 1. Biosensor fabrication

[0083] A biosensor was fabricated using the above-mentioned opioid receptor nanodiscs in the following manner.

[0084] First, 1 μl of 1 mM 1-pyrenebutyric acid N-hydroxysuccinimide ester (PBASE) was applied to the surface of a carbon nanotube (CNT) and reacted for 1 hour. After the reaction was complete, the surface of the CNT was washed three times with methanol and dried using N2 gas. 10 μl of the opioid receptor nanodisc prepared in Example 1 was added to the dried CNT surface and reacted to immobilize the nanodisc on the CNT surface. After 3 hours of reaction, the CNT was washed with PBS buffer to remove unimmobilized opioid receptor nanodiscs, and the results of confirming whether the opioid receptor nanodisc was immobilized on the CNT surface using a scanning electron microscope are shown in Figure 4.

[0085] As shown in Figure 4, a biosensor with opioid receptor nanodiscs immobilized on its surface was obtained.

[0086]

[0087] Experimental Example 3. Confirmation of Electrical Characteristics of the Biosensor

[0088] The electrical characteristics of the biosensor manufactured above were verified by the following method.

[0089] Specifically, a probe station was used to contact the source electrode and the drain electrode. Meanwhile, the gate electrode plate was placed at the bottom and the gate probe was contacted. Subsequently, with the gate electrode fixed, the drain-source current (Ids) was measured while varying the drain-source bias (Vds) from -0.5 to 0.5 V, and the graph of the drain-source current measured while varying the gate voltage from -10 to 2 V with the drain-source voltage fixed is shown in FIG. 5A, and the graph of the drain-source current measured while varying the gate voltage from -10 to 2 V is shown in FIG. 5B.

[0090] As shown in Fig. 5A, it was confirmed that a linear relationship is maintained in the sensor with the nanodisk fixed, exhibiting constant electrical characteristics even when the drain-source bias is varied while the gate electrode is fixed. Meanwhile, as shown in Fig. 5B, when measurements were taken while fixing the drain-source voltage and varying the gate voltage, the transition curve showed a tendency for Ids to decrease as Vg increased. This indicates that the sensor of the present invention can stably detect changes in surface charge distribution that occur when a target substance binds to a sensor with an opioid receptor nanodisk fixed thereon, which is typical p-type behavior of semiconducting carbon nanotubes.

[0091]

[0092] Experimental Example 4. Analysis of the detection function of a biosensor

[0093] The reaction of the biosensor manufactured above to fentanyl, an opioid drug, was confirmed as follows.

[0094] First, fentanyl or endorphin was prepared as a sample by diluting it in PBS to concentrations of 100 fM, 1 pM, 10 pM, 100 pM, or 1 nM. Meanwhile, the biosensor was operated using a probe station under source-drain voltage of 0.1 mV and gate conditions of 1 mV, and the prepared sample was added in 1 µL increments to a 9 µL buffer zone composed of 0.1×V for analysis. As a result, the change in the electrical characteristics of the biosensor caused by fentanyl is shown in Figure 6A, and the change in the electrical characteristics of the biosensor caused by endorphin is shown in Figure 6B.

[0095] As shown in Figure 6, the electrical response increased in a dependency of the treatment concentration of fentanyl, an opioid drug, and endorphin, an endogenous opioid substance.

[0096] From the above results, it was found that the opioid receptor nanodisc according to the present invention has an opioid-based drug detection effect.

[0097]

[0098] Experimental Example 5. Analysis of the screening function of a biosensor

[0099] It was confirmed as follows whether the biosensor manufactured above exhibits a screening function depending on the type of opioid drug. The experiment was performed in the same manner as described in Experimental Example 4 above, and the results obtained by repeating this three times were converted into an opioid concentration-dependent graph and shown in Fig. 7.

[0100] As shown in Fig. 7, K derived using the hill-pilot in the result graph d The values ​​were approximately 0.15 pM for fentanyl and approximately 1.36 pM for endorphin. In other words, from the above results, it was found that the opioid receptor nanodisc according to the present invention has the effect of selectively targeting each substance of the opioid family.

[0101]

[0102] Experimental Example 6. Evaluation of Biosensor Selectivity

[0103] It was confirmed as follows whether the biosensor prepared above selectively detects opioid-based drugs. The experiment was performed in the same manner as described in Experimental Example 4 above, except that dopamine, glycine, aspartic acid, or fentanyl (100 fM or 1 pM) was used instead of fentanyl or endorphin. As a result, the change in the electrical characteristics of the biosensor according to the treatment substance is shown in Figure 8.

[0104] As shown in Fig. 8, dopamine, glycine, or aspartic acid, excluding fentanyl, did not induce an electrical response in the biosensor. Therefore, from the above results, it was found that the opioid receptor nanodisc according to the present invention can specifically detect opioid-based substances.

Claims

1. A composition for detecting opioid-based drugs comprising opioid receptor nanodiscs.

2. A composition for detecting opioid-type drugs according to claim 1, wherein the opioid-type drug is one or more selected from the group consisting of morphine, oxycodone, hydrocodone, hydromorphone, fentanyl, tramadol, and methadone.

3. A composition for detecting opioid-based drugs according to claim 1, wherein the opioid receptor is a polypeptide composed of the amino acid sequence described in SEQ ID NO.

2.

4. The composition for detecting opioid-based drugs according to claim 1, wherein the opioid receptor nanodisc is composed of an opioid receptor, lipids, and a membrane scaffold protein (MSP).

5. A composition for detecting opioid-type drugs according to claim 4, wherein the lipid is a phospholipid.

6. A composition for detecting opioid-type drugs according to claim 5, wherein the phospholipid is one or more selected from the group consisting of phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol.

7. A composition for detecting opioid-based drugs according to claim 4, wherein the membrane scaffold protein is an apolipoprotien.

8. A composition for detecting opioid-based drugs according to claim 1, wherein the opioid receptor nanodisc is formed by mixing an opioid receptor and a membrane scaffold protein in a ratio of 1:1 to 10.

9. A composition for detecting opioid-based drugs according to claim 1, wherein the opioid receptor nanodisc is formed by mixing an opioid receptor and a lipid in a ratio of 1:1 to 700.

10. A kit for detecting opioid-based drugs containing opioid receptor nanodiscs.

11. Biosensor for detecting opioid-based drugs comprising opioid receptor nanodiscs.

12. A method for providing information for diagnosing opioid-based drug addiction, comprising the step of reacting a sample with a detection composition according to claim 1, a detection kit according to claim 10, or a biosensor according to claim 11.

13. A screening method for opioid-based drug addiction treatment drugs comprising the step of treating a candidate drug in a detection composition according to claim 1, a detection kit according to claim 10, or a biosensor according to claim 11, wherein the candidate drug is treated with an opioid-based drug.

14. A method for detecting opioid-based drugs comprising the step of reacting a sample with a detection composition according to claim 1, a detection kit according to claim 10, or a biosensor according to claim 11.

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