Article Synthesis of Quenchbodies for One-Pot Detection of Stimulant Drug Methamphetamine Hee-Jin Jeong 1,2, Jinhua Dong 3,4,5, Chang-Hun Yeom 2 and Hiroshi Ueda 5,* 1 Department of Biological and Chemical Engineering, Hongik University, 2639 Sejong-ro, Jochiwon-eup, Sejong-si 30016, Korea; [email protected] 2 Department of Chemical System Engineering, Hongik University, 2639 Sejong-ro, Jochiwon-eup, Sejong-si 30016, Korea; [email protected] 3 Key Laboratory of Biological Medicines in Universities of Shandong Province, Weifang Key Laboratory of Antibody Medicines, School of Bioscience and Technology, Weifang Medical University, Shandong 261053, China; [email protected] 4 World Research Hub Initiative, Institute of Innovative Research, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8503, Japan 5 Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8503, Japan * Correspondence: [email protected]; Tel.: +81-45-924-5785 Received: 7 May 2020; Accepted: 9 June 2020; Published: 11 June 2020 Abstract: The problem of illicit drug use and addiction is an escalating issue worldwide. As such, fast and precise detection methods are needed to help combat the problem. Herein, the synthesis method for an anti-methamphetamine Quenchbody (Q-body), a promising sensor for use in simple and convenient assays, has been described. The fluorescence intensity of the Q-body generated by two-site labeling of Escherichia coli produced anti-methamphetamine antigen-binding fragment (Fab) with TAMRA-C2-maleimide dyes increased 5.1-fold over background in the presence of a hydroxyl methamphetamine derivative, 3-[(2S)-2-(methylamino)propyl]phenol. This derivative has the closest structure to methamphetamine of the chemicals available for use in a laboratory. Our results indicate the potential use of this Q-body as a novel sensor for the on-site detection of methamphetamine, in such occasions as drug screening at workplace, suspicious substance identification, and monitoring patients during drug rehabilitation. Keywords: Quenchbody; antibody; methamphetamine; illicit drug; in-situ immunoassay 1. Introduction The issue of drug abuse is increasing worldwide and has been associated with various social problems. Methamphetamine (MA) is a highly addictive psychostimulant that causes adverse biological effects, such as acute toxic effects on the cardiovascular system, acute renal failure, altered behavioral and cognitive functions, and permanent brain damage [1,2]. Therefore, illicit use of MA is prohibited in many countries. An accurate and rapid detection system for MA is required for clinical diagnostics and criminal forensics, such as drug screening at the workplace and monitoring patients during drug rehabilitation. One of the current methods for quantitating MA is the Duquenois-Levine colorimetric test, in which an indicator reacts with MA and is compared with standardized color charts visually [3]. Such a colorimetric approach is economic and simple, and can be performed without complicated training. However, the visual interpretation affects the accuracy of results, which is occasionally presumptive and limits the quantification. Immunochromatography, which uses a colloidal gold-labeled antibody or antigen as a detection agent provides a simple analytical procedure for drug screening Methods Protoc. 2020, 3, 43; doi:10.3390/mps3020043 www.mdpi.com/journal/mps Methods Protoc. 2020, 3, x FOR PEER REVIEW 2 of 15 Methods Protoc. 2020, 3, 43 2 of 15 or antigen as a detection agent provides a simple analytical procedure for drug screening in biological specimens [4]. However, as the test result is typically interpreted by the observation of a color band onin biologicalthe membrane, specimens the determination [4]. However, of as thea positive test result or isnegative typically result interpreted is also bysubjective the observation. Another of method,a color bandthin-layer on the chromatograph membrane, they (TLC) determination has been ofused a positive to detect or MA negative. However, result TLC is also has subjective. limited accuracyAnother when method, used thin-layer on complex chromatography samples due to (TLC) low resolution has been usedof separation; to detect thus MA., However,the method TLC is typicallyhas limited used accuracy for preliminary when used identification on complex samples[3]. Chromatographic due to low resolution methods of, separation;including thus,gas chromatogrthe methodaphy is typically and liquid used chromatography for preliminary, identificationcan be used in [ 3combination]. Chromatographic with mass methods, spectrometry including to providegas chromatography high precision and results liquid [5,6 chromatography,]. However, these can methods be used involve in combination long sample with pre massparation spectrometry times, complicatedto provide high experimental precision resultsprocedure [5,6].s that However, require these a high methods-level of involve scientific long knowledge sample preparation and advanced times, skillcomplicateds and expensive experimental instruments procedures. Enzyme that-linked require immunosorbent a high-level of scientificassay (ELISA knowledge) has been and used advanced as a primaryskills and tool expensive for the qu instruments.antification Enzyme-linkedof MA [7]. However, immunosorbent ELISA involves assay multiple (ELISA) hassteps been, including used as severala primary washing tool for and the incubating quantification steps, of MAwhich [7]. require However, one ELISA or two involves days multipleto complete steps, the including entire procedureseveral washing. Thus, anda high incubatingly accurate steps, assay which with require easy sample one or treatment two days tois completeneeded for the rapid, entire onsite procedure. MA detection.Thus, a highly accurate assay with easy sample treatment is needed for rapid, onsite MA detection. AAntibody-basedntibody-based reagentless reagentless fluorescence fluorescence immu immunoassaysnoassays offer offer the the advantages advantages of of only only taking taking minutesminutes to completecomplete byby eliminating eliminating washing washing steps steps as well as aswell the as use the of secondaryuse of secondary antibodies. antibodies. However, However,these types these of types assays of oassaysffer a fewoffer challengesa few challenges in the in developmental the developmental stage, stage, such such as di asff erentdifferent dye dyeconjugation conjugation efficiencies efficienc toies individual to individual antibodies antibodies and non-specific and non-specific binding tobinding nontarget to nontarget molecules. moleculesFor example,. For example, labeling anlabeling N-hydroxysuccinimide an N-hydroxysuccinimide ester-conjugated ester-conjugated dye to the dye lysine to the (Lys) lysine R-group (Lys) Ramines-group isamines advantageous is advantageous due to thedue numerous to the numerous Lys residues Lys onresidues the antibody on the surface.antibody However,surface. However,this method this hasmethod the disadvantage has the disadvantage such that such a good that numbera good number of Lys residues of Lys residues are distributed are distributed all over allthe over antibody, the antibody, making making it difficult it difficult to exactly to quantifyexactly quantify a target a antigen target antigen due to thedue random to the random labeling labelingof the of fluorescent the fluorescent dye, dye which, which is not is not consistently consistently producing producing a a conjugate conjugateproduct product with a a known known dye:antibodydye:antibody conjugation conjugation ratio ratio.. Another Another potential potential issue issue is is that that the the Lys Lys residues residues in in the the antigen antigen binding binding regionregion might might be be conj conjugatedugated to to the the dye, dye, which which could could hamper hamper the the antigen antigen-binding-binding activity activity.. T Too increase increase thethe site site-specific-specific dye dye-antibody-antibody conjugation conjugation efficiency, efficiency, we we developed developed a a new new antibody antibody-labeling-labeling method method usingusing a acysteine cysteine (Cys) (Cys)-containing-containing peptide peptide tag tag (Cys (Cys-tag;-tag; MSKQIEVNCSNET MSKQIEVNCSNET [8] [8,], aa mimic mimic of of ProX ProX-tag-tag (MSKQIEVN*SNET,(MSKQIEVN*SNET, * == amberamber codoncodon [ 9[9]]) that) that was was developed developed for thefor incorporationthe incorporation of unnatural of unnatural amino aminoacids), acids) and applied, and applied it to the it generation to the generat of variousion of Quenchbodiesvarious Quenchbodies (Q-bodies). (Q- Abodies). Q-body A is Q an-body innovative is an innovativeimmunosensor immunosensor that fluoresces that uponfluoresces antigen-dependent upon antigen removal-dependent of theremoval fluorophore of the quenchingfluorophore [9]. quenchingIn the absence [9]. Inof the an antigen,absence theof an fluorescence antigen, the of fluorescence the dye is quenched of the dye by is a photo-inducedquenched by a electronphoto- inducedtransfer electron from the transfer tryptophan from (Trp)the tryptophan residues in (Trp) the nearby residues antigen-binding in the nearby antigen sites of-binding antibody. sites When of antibodyan antigen. When binds an to antigen an antibody, binds theto an binding antibody stabilizes, the binding the conformation stabilizes the of theconformation