Composition for detecting fentanyl and detection method therefor

Abstract

The present invention relates to a composition for detecting fentanyl and a detection method therefor. The composition according to the present invention comprises a peptide in which fentanyl-binding peptides TM3 and TM6 are linked via polyethylene glycol (PEG), thereby enabling binding to fentanyl through the folding of the corresponding peptide, and thus can effectively and sensitively detect only fentanyl mixed in a sample such as a beverage.

Description

Composition for detecting fentanyl and method for detecting the same

[0001] The present invention relates to a composition for detecting fentanyl and a method for detecting the same.

[0002] Fentanyl is a powerful opioid analgesic with an analgesic effect more than 100 times stronger than morphine, another opioid, and about 50 times stronger than heroin. Due to its mild respiratory depressant effect, it is used as an adjuvant anesthetic or analgesic, with an effect lasting 1 to 2 hours. It is a narcotic analgesic primarily prescribed to patients experiencing persistent pain, such as terminal cancer patients. It is also widely used as a skin patch, with the dosage controlled to a very fine level.

[0003] Fentanyl was first created by Paul Janssen in 1959, and its medical use was approved starting in the 1960s, where it began to be used as an intravenous anesthetic under the name 'Sublimaze'. In October 2002, during the Moscow theater hostage crisis, a mixture of halothane and fentanyl was used to subdue terrorists, resulting in many deaths. Due to its high profitability—being much more potent and easier to transport than other opioid drugs such as heroin—smuggling and abuse have recently been increasing in the United States. It is known that most of the drugs leaked from fentanyl-related factories in China undergo illegal processing in various countries around the world before being smuggled into the U.S. through Mexican cartels.

[0004] Fentanyl is a highly addictive and dangerous drug that can lead to death in even healthy individuals with just a minute amount of 2 mg. Recently, as it has spread as a recreational drug in some countries, cases of misuse and abuse have been surging; in the United States in 2021, fentanyl was the drug responsible for the highest number of overdose deaths, with over 70,000 people reported to have died from fentanyl overdose in that single year. Consequently, the need for effective preventive measures and effective treatment methods for habitual users is increasing day by day.

[0005] Recently, mass dosing of fentanyl among teenagers in Korea has emerged as a major social issue. In particular, because fentanyl has physicochemical properties that allow it to be administered through all routes of the body and can produce effects with trace amounts, those illegally manufacturing recreational fentanyl in the black market produce it not only in pill form but also in various forms of food such as beverages. Given the reality that all products are distributed online, it is very difficult to control the influx of illegally manufactured fentanyl, and there is a constant need to develop a method to identify fentanyl more simply and accurately.

[0006] One object of the present invention is to provide a fusion polypeptide comprising a peptide of SEQ ID NO. 1 from the N-terminus, a PEG (polyethylene glycol) having at least 3 to 10 ethylene glycol units connected thereto, and a peptide of SEQ ID NO. 2 connected thereto.

[0007] Another object of the present invention is to provide a nanostructure comprising: a fusion polypeptide consisting of a peptide of SEQ ID NO. 1 from the N-terminus, a PEG (polyethylene glycol) having at least 3 to 10 ethylene glycol units connected thereto, a peptide of SEQ ID NO. 2 connected thereto, and a labeled ligand connected thereto; and an anti-ligand magnetic particle that recognizes the labeled ligand.

[0008] Another objective of the present invention is to provide a composition for detecting fentanyl or fentanyl derivatives comprising the above-mentioned fusion polypeptide or the above-mentioned nanostructure.

[0009] Another objective of the present invention is to provide a kit for detecting fentanyl or fentanyl derivatives comprising the above-mentioned fusion polypeptide or the above-mentioned nanostructure.

[0010] Another object of the present invention is to provide a method for detecting fentanyl or a fentanyl derivative, comprising: (a) treating a sample with the above-mentioned fusion polypeptide; (b) adding an anti-ligand-magnetic particle that recognizes a labeled ligand to obtain a complex to which fentanyl or a fentanyl derivative is bound; and (c) measuring the spectroscopic properties of the obtained complex.

[0011] Another object of the present invention is to provide a method for detecting fentanyl or a fentanyl derivative, comprising: (i) treating a sample with the above-mentioned nanostructure; (ii) obtaining a complex to which fentanyl or a fentanyl derivative is bound; and (iii) measuring the spectroscopic value of the obtained complex.

[0012] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions provided below.

[0013] Furthermore, the terms used in this invention are for illustrative purposes only and should not be interpreted as intended to be limiting. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this invention, terms such as “comprising” or “having” are intended to specify the existence of the features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, and should be understood as not precluding the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof.

[0014] Furthermore, unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by those skilled in the art to which the embodiments pertain. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and should not be interpreted in an ideal or overly formal sense unless explicitly defined in this application.

[0015] Furthermore, to prevent clutter caused by overlapping content, redundant details have been omitted below. In other words, the content of the invention is not limited solely to the following description, and should be interpreted in accordance with the overall context of the invention.

[0016] The inventors of the present invention evaluated the interaction between fentanyl and μ-opioid receptors and developed a fusion polypeptide based on this. Having confirmed that the developed fusion polypeptide can detect fentanyl in beverages with sensitive specificity and sensitivity, the present invention aims to provide a fusion polypeptide for fentanyl detection, a nanostructure containing the same, and an application for fentanyl detection using these.

[0017] As one preferred example for achieving the above-mentioned purpose, the present invention provides a fusion polypeptide comprising, from the N-terminus, the peptide of SEQ ID NO. 1, a PEG (polyethylene glycol) having at least 3 to 10 ethylene glycol units connected thereto, and the peptide of SEQ ID NO. 2 connected thereto. The specific sequences of the peptides of SEQ ID NO. 1 and 2 are shown in Table 1 below.

[0018] Sequence (N term → C term) Sequence Number TM3 PeptideDISL1TM6 PeptideFWH(3-Me)2

[0019] (3-Me: 3-methyl)

[0020]

[0021] According to one embodiment of the present invention, in the fusion polypeptide according to the present invention, when aspartic acid (D) of peptide No. 1 (μ-opioid receptor TM3 peptide) attracts fentanyl and is protonated to histidine (H) Nd of peptide No. 2 (μ-opioid receptor TM6 peptide), fentanyl penetrates deeply into the peptide through hydrogen bonding. At this time, fentanyl strongly binds to the receptor through the action of the piperidine ring of fentanyl, tryptophan (W) and phenylalanine (F) of peptide No. 2, and isoleucine (I), serine (S), and leucine (L) of peptide No. 1. At this time, the tryptophan strongly binds through interaction with the piperidine ring of fentanyl, and the hydrophobic interaction is more stably enhanced by the interaction of phenylalanine with the phenethyl group of fentanyl, and isoleucine, serine, and leucine also participate in the hydrophobic interaction. Furthermore, the fusion polypeptide according to the present invention is designed so that a proton is fixed at the Nd position of peptide histidine of SEQ ID NO. 2 and a deprotonated state is maintained at the Ne position, thereby enabling specific binding to fentanyl. According to a specific embodiment, it is designed to maintain specific binding to fentanyl by maintaining deprotonation through substitution with a 3-methyl group in the imidazole ring of histidine. That is, the N residue corresponding to position 3 in the imidazole ring of histidine may be substituted with a methyl group. In addition, the fusion polypeptide according to the present invention can form a four-dimensional structure by connecting a PEG (polyethylene glycol) having at least 3 to 10 ethylene glycol units between the peptides of SEQ ID NOs 1 and 2, thereby forming a structure suitable for capturing fentanyl. The application of a PEG shorter or longer than this is undesirable as it forms a structure unsuitable for capturing fentanyl.

[0022] For smooth detection identification, a labeled ligand may be attached to the C-terminus of the above-described fusion polypeptide. Any labeled ligand known in the art may be applied without limitation, but as a preferred example, the labeled ligand may be any one selected from the group consisting of biotin, digoxigenin, aptamer, peptide, fluorescent compound, oligonucleotide, polysaccharide, amino acid residue having a thiol group, and amino acid residue having a mercaptoacetyl group attached.

[0023] At this time, the labeled ligand may be bound to the fused polypeptide directly or through a linker, and as an example, the linker may be any one selected from the group consisting of PEG (polyethylene glycol) having 2 to 5 ethylene glycol units, C3 Spacer, C6 Spacer, C9 Spacer, C12 Spacer, C18 Spacer, Hexanediol, Thiol-Modifier C6 SS, 1',2'-Dideoxyribose (dSpacer), glyceryl CPG, Cholesteryl-TEG CPG, 2-Aminopurine, and PC (Photo-Cleavable) Spacer.

[0024] As a more desirable example, the fusion peptide has the structure of Chemical Formula 1 below.

[0025] [Chemical Formula 1]

[0026]

[0027] As another preferred example for achieving the above-mentioned purpose, the present invention provides a nanostructure comprising: a fusion polypeptide consisting of a peptide of SEQ ID NO. 1 from the N-terminus, a PEG (polyethylene glycol) having at least 3 to 10 ethylene glycol units connected thereto, a peptide of SEQ ID NO. 2 connected thereto, and a labeled ligand connected thereto; and an anti-ligand magnetic particle that recognizes the labeled ligand.

[0028] As a preferred example, the anti-ligand that recognizes the labeled ligand may be one selected from the group consisting of avidin or avidin analogs, antibodies, receptors, and lectins, and the avidin analog may be streptavidin, nuetravidin, or captavidin.

[0029] The above magnetic particles are a magnetic material and are not particularly limited to commonly used magnetic particles, but specifically may include one or more selected from the group consisting of Au, Ag, Fe(III), Mg, Mn, Ni, Zn, and Co.

[0030] As another preferred example for achieving the above-mentioned purpose, the present invention provides a composition for detecting fentanyl or fentanyl derivatives comprising the above-mentioned fusion polypeptide or the above-mentioned nanostructure.

[0031] Fentanyl is a narcotic analgesic that exerts an analgesic effect by acting on opioid receptors in the central nervous system to inhibit the transmission of pain. Fentanyl is a highly addictive and dangerous drug that can lead to death in even healthy individuals with just a tiny amount of 2 mg.

[0032] In the present invention, the fentanyl may be selected from the group consisting of, for example, alfentanil, sufentanil, remifentanil, 3-methylfentanyl, and carfentanil, and the fentanyl derivative may have, for example, the structure of Chemical Formulas 2 to 21 below. The fentanyl and fentanyl derivatives all have structural similarity and interact with the same site of the μ-opioid receptor.

[0033] That is, the above-mentioned fentanyl and fentanyl derivatives are all docked to the aspartic acid (D) of the peptide of SEQ ID NO. 1 (μ-opioid receptor TM3 peptide) and, when protonated to the histidine (H) Nd of the peptide of SEQ ID NO. 2 (μ-opioid receptor TM6 peptide), are hydrogen-bonded to the fentanyl or fentanyl derivative and pulled deeply into the peptide. At this time, fentanyl strongly binds to the receptor through the action of the piperidine ring of fentanyl, tryptophan (W) and phenylalanine (F) of the peptide of SEQ ID NO. 2, and isoleucine (I), serine (S), and leucine (L) of the peptide of SEQ ID NO. 1. At this time, fentanyl can be effectively detected by strongly binding the fentanyl or fentanyl analog through the strong hydrophobic action of the above residues.

[0034] [Chemical Formula 2]

[0035]

[0036] [Chemical Formula 3]

[0037]

[0038] [Chemical Formula 4]

[0039]

[0040] [Chemical Formula 5]

[0041]

[0042] [Chemical Formula 6]

[0043]

[0044] [Chemical Formula 7]

[0045]

[0046] [Chemical Formula 8]

[0047]

[0048] [Chemical Formula 9]

[0049]

[0050] [Chemical Formula 10]

[0051]

[0052] [Chemical Formula 11]

[0053]

[0054] [Chemical Formula 12]

[0055]

[0056] [Chemical Formula 13]

[0057]

[0058] [Chemical Formula 14]

[0059]

[0060] [Chemical Formula 15]

[0061]

[0062] [Chemical Formula 16]

[0063]

[0064] [Chemical Formula 17]

[0065]

[0066] [Chemical Formula 18]

[0067]

[0068] [Chemical Formula 19]

[0069]

[0070] [Chemical Formula 20]

[0071]

[0072] [Chemical Formula 21]

[0073]

[0074] Accordingly, in another preferred embodiment, the present invention provides a kit for detecting fentanyl or fentanyl derivatives comprising the above-described composition.

[0075] The above kit is intended for a sample, and the sample according to the present invention refers to a biological sample of a subject suspected of having ingested fentanyl or a fentanyl derivative, or any food sample suspected of being mixed with fentanyl or a fentanyl derivative.

[0076] The biological sample may be any tissue or body fluid obtained from the subject. The biological sample includes, but is not limited to, the subject's urine, blood, and saliva. The biological sample may be obtained in a manner that does not cause harm to the subject.

[0077] The above food samples may include, but are not limited to, foods in the form of jelly, beverages, or candy. However, if the above food samples are in a solid form, they may be applied to detection by eluting them into a liquid form for smooth detection.

[0078] In particular, the composition and / or kit according to the present invention has excellent detection efficacy, and specifically enables the detection (diagnosis) of fentanyl and fentanyl derivatives mixed in beverages.

[0079] The above kit may further include a reagent capable of eluting a solid sample into a liquid phase, specifically, water or an organic solvent, etc.

[0080] In addition, the optimal amount of reagent used in a specific reaction can be easily determined by a person skilled in the art who has acquired the disclosures in this specification. Typically, the kit of the present invention is manufactured as a separate package or compartment containing the aforementioned components. The kit may also further include instructions for use describing optimal reaction conditions, and other tools or equipment necessary for detection.

[0081] Meanwhile, in another preferred embodiment, the present invention provides a method for detecting fentanyl or a fentanyl derivative, comprising: (a) treating a sample with the above-mentioned fusion polypeptide; (b) adding an anti-ligand-magnetic particle that recognizes a labeled ligand to obtain a complex to which fentanyl or a fentanyl derivative is bound; and (c) measuring the spectroscopic value of the obtained complex.

[0082] In addition, as another preferred embodiment, the present invention provides a method for detecting fentanyl or a fentanyl derivative comprising: (i) treating a sample with the above-mentioned nanostructure; (ii) obtaining a complex to which fentanyl or a fentanyl derivative is bound; and (iii) measuring the spectroscopic value of the obtained complex.

[0083] At this time, the sample may include the aforementioned “biological sample” or “any food sample,” but more preferably, it may be one or more liquids selected from the group consisting of ion drinks, water, alcohol, and carbonated drinks suspected of being mixed with fentanyl or fentanyl derivatives.

[0084] The nanostructure according to the present invention is a multifunctional particle at the nanometer scale level that possesses magnetic properties influenced by a magnet and simultaneously possesses the property of specifically separating and detecting fentanyl or fentanyl derivatives. By utilizing this, it is possible not only to qualitatively detect the presence or absence of fentanyl or fentanyl derivatives in various food and clinical samples, such as liquids and solids, in which various other components are mixed, but also to simultaneously apply it to the separation of fentanyl or fentanyl derivatives.

[0085] The above magnetism can be applied through a permanent magnet or an electromagnet. The magnetic particles to which the fusion polypeptide is attached can target fentanyl or fentanyl derivatives present in a sample by immobilizing the fusion polypeptide on the surface of the magnetic particles, and can be easily collected and separated using magnetic separation.

[0086] When magnetic particles to which the fusion polypeptide is attached are treated with the biological sample or food sample of the subject, the fusion polypeptide attached to the magnetic particles binds to fentanyl or a fentanyl derivative present in the sample. That is, a fentanyl or fentanyl derivative-fusion polypeptide-magnetic particle complex is formed. Since the complex can be easily collected through magnetic separation, non-specific reactions can be reduced and detection efficiency can be maximized.

[0087] Meanwhile, the above spectroscopic measurement can preferably be performed by utilizing surface plasmon resonance. That is, the step of measuring the spectroscopic measurement may be a method performed through surface plasmon resonance. A surface plasmon refers to the quantized vibration of free electrons propagating along the surface of a conductor, such as the surface of a magnetic particle. Such surface plasmons are excited by incident light that passes through a dielectric medium, such as a prism, and is incident on a magnetic particle at an angle greater than the critical angle of the dielectric medium, thereby causing resonance; this is called surface plasmon resonance (SPR).

[0088] When using monochromatic incident light, the angle of incidence (resonance angle) and the wavelength at which resonance occurs are highly sensitive to changes in the refractive index of a material near a magnetic particle. SPR sensors utilize this property to analyze whether a sample is detected based on changes in the refractive index of the material near the magnetic particle, i.e., the sample.

[0089] The composition according to the present invention includes a peptide in which peptides TM3 and TM6 that bind to fentanyl are connected by polyethylene glycol (PEG), and thus can bind to fentanyl through the folding of the peptide, thereby enabling effective and sensitive detection of only fentanyl mixed in a sample such as a beverage.

[0090] Figure 1 is the result of an alpha fold simulation to confirm the fentanyl binding structure.

[0091] Figure 2 shows the results of confirming the fentanyl detection efficacy using peptide 3.

[0092] Figure 3 shows the results of a condition experiment performed to derive an optimized signal according to peptide binding conditions.

[0093] Figure 4 shows the results of deriving baseline and pure drug peak standards for detecting fentanyl in beverages using surface enhanced Raman spectroscopy (SERS).

[0094] Figure 5 shows the results of confirming the efficacy of detecting fentanyl in various beverages using magnetic particles surface-modified with peptide 3.

[0095] The present invention will be described in more detail below through examples. These examples are solely for illustrating the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not to be interpreted as being limited by these examples.

[0096] Example 1. Preparation of a peptide for fentanyl detection

[0097] To establish an effective fentanyl detection method, the binding mechanism between the μ-opioid receptor and fentanyl was analyzed.

[0098] As a result, it was confirmed that D147 of the μ-opioid receptor TM3 peptide attracts fentanyl, and when protonated to H297 Nd of the TM6 peptide, fentanyl penetrates deep into the peptide through hydrogen bonding.

[0099] At this time, fentanyl was strongly bound to the receptor through the action of the piperidine ring of fentanyl and W293, F289 of TM6, and I155, S154, and L158 of TM3. W293 strongly binds through interaction with the piperidine ring of fentanyl, and F289 strengthens the hydrophobic interaction more stably through interaction with the phenethyl group of fentanyl, and it was confirmed that I155, S154, and L158 are also involved in the hydrophobic interaction.

[0100] However, a proton was fixed at the Nd position of TM6 peptide H297, and the deprotonated state had to be maintained at the Ne position.

[0101] Based on this, a peptide structure capable of binding with a minimum length was devised by leaving only the part interacting with fentanyl. In addition, polyethylene glycol (PEG) was applied to the part for forming a quaternary structure in the middle to conceive a foldable form, and its specific sequence is shown in Table 2 below.

[0102] Sequence (N term → C term) SEQ ID NO: Peptide 1H(3-Me)-WF-CC-LSID3Peptide 2H(3-Me)-WF-CKC-LSID4Peptide 3DISL-dPEG6-FW-H(3-Me)-K5

[0103] (3-Me: 3-methyl )

[0104]

[0105] Specifically, peptide 1 was fabricated with a structure having peptides TM3 (LSID) and TM6 (HWF) that bind to fentanyl on both sides, and cysteine ​​with a thiol group capable of binding to an Au surface in the center. Peptide 2 was fabricated with a structure having peptides TM3 (LSID) and TM6 (HWF) that bind to fentanyl on both sides, and cysteine ​​with a thiol group capable of binding to an Au surface and lysine with biotin capable of binding to streptavidin in the center.

[0106] Peptide 3 was made capable of binding to fentanyl by forming a structure in which PEG6 connects the peptides TM3 (DISL) and TM6 (FWH) that bind to fentanyl. In addition, a structure capable of attaching to Au was fabricated by applying lysine (miniPEG2-K(mercaptoacetyl)) with a PEG2-mercaptoacetyl group attached to the C' end.

[0107] Experimental Example 1. Verification of Fentanyl Detection Efficacy

[0108] 1. Alphafold simulation

[0109] To verify the efficacy of the peptide prepared in Example 1 against fentanyl, an alpha-fold simulation was performed.

[0110] However, since structural analysis is difficult when using PEG itself, the PEG portion was replaced with a peptide sequence (hydrophilic amino acid & glycine) having similar spacing and polarity to perform the simulation. The specific modified sequence of peptide 3 is shown in Table 3 below.

[0111] Sequence (N term → C term) Sequence Number Peptide 3-RDISLDEGGDEFWHHWFDEGGDELSID6

[0112] As shown in Figure 1, in particular, the modified peptide of peptide 3 (Peptide 3-R) was found to have a high structural folding prediction rate and a strong binding affinity for fentanyl. However, in the case of peptides 1 and 2, it was determined that they could not bind to fentanyl because a folding structure was not formed.

[0113] 2. Surface plasmon resonance

[0114] To evaluate the fentanyl detection efficacy of the peptide prepared above, surface plasmon resonance (SPR) was performed on an Au chip. Specifically, the peptide prepared above was bound to an Au chip via an Au-thiol coordination bond, and changes in the SPR signal were measured according to the binding ratio at different fentanyl concentrations.

[0115] As shown in Figure 2, it was confirmed that fentanyl was specifically detected only in the peptide combined with peptide 3.

[0116] Accordingly, peptide 3 was selected as the final peptide for fentanyl detection, and its structure is shown in Chemical Formula 1 below.

[0117] [Chemical Formula 1]

[0118]

[0119] Example 2. Synthesis of nanostructures

[0120] We intended to synthesize magnetic nanoparticles with surface enhanced Raman spectroscopy (SERS) enhancement to selectively separate fentanyl from a sample and detect it immediately.

[0121] To this end, a nanostructure having plasmonic enhancement was synthesized by modifying the surface of magnetic nanoparticles with peptide 3.

[0122] Specifically, magnetic material-silica nanoparticles were synthesized by reducing silica on the surface of a magnetic material, and then the silica surface was modified into a silane structure having a thiol group, and gold nanoparticles serving as growth points of 2 nm were attached. Subsequently, ascorbic acid was used as a reducing agent and polyvinylpyrrolidone as a stabilizer, and a HAuCl4 solution was added to synthesize magnetic material-silica-gold nanoparticles.

[0123] A nanostructure was synthesized by modifying the surface of the nanoparticles synthesized in this way with peptide 3 via an au-thiol bond.

[0124] Experimental Example 2. Evaluation of Receptor-Mediated Drug Detection Using Magnetic Separation

[0125] 1. Derivation of optimized signals based on peptide binding conditions

[0126] Experiments were conducted using two types of particles: MNC@PEG@Au particles, in which a MNC@Gold shell was synthesized by directly modifying PEI on a MNC (magnetic nanocluster), and MNC@Si@Au particles, in which a gold shell was synthesized after coating a shell with SiO2 on the surface of the MNC.

[0127] Specifically, peptide 3 was bound to MNC@Si@Au and MNC@PEI@Au particles containing 1 mg / mL MNC, and 50 µL of magnetic solution was added to 1 mL of fentanyl solution to separate and purify the particles, then dried on a Si wafer for sampling.

[0128] As shown in Fig. 3, 1004 cm -1 When quantifying using the scale for the peak, the most distinct signal was obtained from a 1 μg / ml peptide modified sample of MNC@Si@Au particles, and this was confirmed as the subsequent condition.

[0129] 2. Detection of fentanyl in beverages

[0130] In order to confirm the drug detection capability in five types of actual beverages (sports drink, soju, beer, carbonated water, and cider), a baseline was first confirmed in each beverage, and a drug peak was secured to establish a standard.

[0131] Signal detection was performed by adding 100 µl of MNC@Si@Au surface-modified with peptide 3 to 1 ml of beverage solution, as in the previous spike sample, and then sampling from a Si wafer after separation and purification.

[0132] As shown in Figure 4, a baseline was formed on the negative control (MNC@si@Au-fentanyl_pep3+various beverages) (left panel), and the detection results were compared based on the pure drug peak (GNR substrate+ 10 μg / ml drug) (right panel).

[0133] As shown in Fig. 5, fentanyl and zoretil were dissolved in five types of beverages at concentrations of 1 µg / ml or 500 ng / ml, respectively, and the peak shapes were analyzed. As a result, within the beverages containing fentanyl, a peak of 1030 cm⁻¹ was specifically observed only at the fentanyl targeting peptide (peptide 3). -1 By confirming that a second peak occurs distinctly, it was confirmed that the nanostructure according to Example 2 can detect fentanyl in beverages with specific and sensitive sensitivity.

Claims

1. A fusion polypeptide comprising, from the N-terminus, the peptide of SEQ ID NO. 1, a PEG (polyethylene glycol) having at least 3 to 10 ethylene glycol units connected thereto, and the peptide of SEQ ID NO. 2 connected thereto.

2. The fusion polypeptide of claim 1, wherein the fusion polypeptide has a labeled ligand attached to the C-terminus.

3. A fusion polypeptide, wherein the labeled ligand is any one selected from the group consisting of biotin, digoxigenin, aptamer, peptide, fluorescent compound, oligonucleotide, polysaccharide, amino acid residue having a thiol group, and amino acid residue having a mercaptoacetyl group.

4. A fusion polypeptide according to paragraph 2, wherein the labeled ligand is bound to the fusion polypeptide directly or through a linker.

5. A fusion polypeptide according to claim 4, wherein the linker is any one selected from the group consisting of PEG (polyethylene glycol) having 2 to 5 ethylene glycol units, C3 Spacer, C6 Spacer, C9 Spacer, C12 Spacer, C18 Spacer, Hexanediol, Thiol-Modifier C6 SS, 1',2'-Dideoxyribose (dSpacer), glyceryl CPG, Cholesteryl-TEG CPG, 2-Aminopurine, and PC (Photo-Cleavable) Spacer.

6. A fusion polypeptide according to claim 1, wherein the fusion peptide has the structure of the following chemical formula 1. [Chemical Formula 1] 7. A fusion polypeptide comprising, from the N-terminus, the peptide of SEQ ID NO. 1, a PEG (polyethylene glycol) having at least 3 to 10 ethylene glycol units connected thereto, the peptide of SEQ ID NO. 2 connected thereto, and a labeled ligand connected thereto; and A nanostructure composed of anti-ligand magnetic particles that recognize a labeled ligand.

8. A nanostructure according to claim 7, wherein the anti-ligand recognizing the labeled ligand is one selected from the group consisting of avidin or avidin analogs, antibodies, receptors, and lectins.

9. In claim 8, the avidin analog is a nanostructure that is streptavidin, nuetravidin, or captavidin.

10. A composition for detecting fentanyl or fentanyl derivatives comprising any one of the fusion polypeptides selected from claims 1 to 6 or any one of the nanostructures selected from claims 7 to 9.

11. A detection composition according to claim 10, wherein the fentanyl is any one selected from the group consisting of alfentanil, sufentanil, remifentanil, 3-methylfentanyl, and carfentanil.

12. A detection composition according to claim 10, wherein the fentanyl derivative has the structure of the following chemical formulas 2 to 21. [Chemical Formula 2] [Chemical Formula 3] [Chemical Formula 4] [Chemical Formula 5] [Chemical Formula 6] [Chemical Formula 7] [Chemical Formula 8] [Chemical Formula 9] [Chemical Formula 10] [Chemical Formula 11] [Chemical Formula 12] [Chemical Formula 13] [Chemical Formula 14] [Chemical Formula 15] [Chemical Formula 16] [Chemical Formula 17] [Chemical Formula 18] [Chemical Formula 19] [Chemical Formula 20] [Chemical Formula 21] 13. A kit for detecting fentanyl or fentanyl derivatives comprising any one of the fusion polypeptides selected from claims 1 to 6 or any one of the nanostructures selected from claims 7 to 9.

14. (a) A step of treating a sample with any one of the fusion polypeptides selected from claims 1 to 6; (b) a step of obtaining a complex to which fentanyl or a fentanyl derivative is bound by adding an anti-ligand-magnetic particle that recognizes the labeled ligand; and (c) a step of measuring the spectroscopic properties of the obtained complex; a method for detecting fentanyl or fentanyl derivatives.

15. (i) A step of treating a sample with any one of the nanostructures selected from claims 7 to 9; (ii) a step of obtaining a complex to which fentanyl or a fentanyl derivative is bound; and (iii) a step of measuring the spectroscopic properties of the obtained complex; a method for detecting fentanyl or fentanyl derivatives comprising.

16. The method of claim 14 or 15, wherein the sample is one or more liquids selected from the group consisting of ion drinks, water, alcohol, and carbonated drinks suspected of being mixed with fentanyl or a fentanyl derivative.

17. A method according to claim 14 or 15, wherein the step of measuring spectroscopy is performed through surface plasmon resonance.

18. A method according to claim 14 or 15, wherein the fentanyl is selected from the group consisting of alfentanil, sufentanil, remifentanil, 3-methylfentanyl, and carfentanil.

19. Method according to claim 14 or 15, wherein the fentanyl derivative has the structure of the following chemical formulas 2 to 21: [Chemical Formula 2] [Chemical Formula 3] [Chemical Formula 4] [Chemical Formula 5] [Chemical Formula 6] [Chemical Formula 7] [Chemical Formula 8] [Chemical Formula 9] [Chemical Formula 10] [Chemical Formula 11] [Chemical Formula 12] [Chemical Formula 13] [Chemical Formula 14] [Chemical Formula 15] [Chemical Formula 16] [Chemical Formula 17] [Chemical Formula 18] [Chemical Formula 19] [Chemical Formula 20] [Chemical Formula 21]

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