Chromogenic and Fluorogenic Probes for the Detection of Illicit Drugs

DOI:10.1002/open.201800034

Chromogenic and Fluorogenic Probes for the Detection of Illicit Drugs Eva Garrido+,[a] Luis Pla+,[a] Beatriz Lozano-Torres,[a] Sameh El Sayed,[a, b] RamónMartínez-MµÇez,*[a, b, c] and FØlix Sancenón[a, b, c]

ChemistryOpen 2018, 7,401 –428 401 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim The consumption of illicit drugs has increased exponentially in sary to develop highly rapid, easy,sensitive,and selective recent years and has become aproblem that worries both methods for their detection. Currently,the most widely used governments and international institutions. The rapid emer- methods for drug detection are based on techniques that re- gence of new compounds, their easy access,the low levels at quire large measurement times, the use of sophisticated equip- which these substances are able to produce an effect, and ment, and qualified personnel. Chromo-and fluorogenic meth- their short time of permanence in the organism makeitneces- ods are an alternative to those classical procedures.

1. Introduction euro), ecstasy(22 million euro), opiates (18 millioneuro), and cocaine(17 million euro). According to their pharmacologic be- The use of novel illicit drugs and the constant appearance of havior,drugs can be divided into depressants, stimulants, hal- new psychoactive substances have rapidlygrown in the last lucinogenic, and opioids. years, and reports of the availability and manufacture of such Because of their harmfulnesstohealth,the ability to detect substances have increased.[1] Illicit drugs are those for which and quantify “drugsofabuse” in afast, easy,and reliable way nonmedicaluse has been prohibited by internationalcontrol is crucial. The United Nations Office on Drugs and Crime treaties because theyare believed to presentunacceptable (UNODC)established 28 million years of healthylife lost world- risks of addiction to users. According to the FDA, these com- wide in 2015 because of premature death and disability pounds can also be defined as “drugsofabuse”, substances caused by drug use.[3] Apart from the health problemsgenerat- that are used in amanner or amount inconsistentwith the le- ed by illicit drug consumption, UNODC exposes the existence gitimate medicaluse.[2] The problematic use of these illicit of continue evolvingorganized crime/terrorism related with drugs is associated with considerable mortality and morbidity. drug market. The European homologue,European Monitoring During the last 30 years, international control has been extend- Centrefor Drugs and Drug Addiction (EMCDDA), assumes ed from plant-based drugs (e.g. heroin, cocaine, and cannabis) there exists an important internet market dedicated to the sale to synthetic drugs (e.g. amphetamines,3,4-methylenedioxyme- of illicit substancesprotected by anonymization services.[4] Last thamphetamine, etc.) and pharmaceutical drugs (e.g. bupre- annualinforms of both the UNODCSand EMCDDAemphasized norphine, methadone, benzodiazepines, etc.). Specifically,syn- the necessity to control the production and distribution of thetic drugs are proliferating at an unprecedented rate and are new psychoactive substances(NPSs), as there are no assuran- posing significant public health challenges. The majority of ces to the consumers of the compounds contained,and the these addictive substances, directly or indirectly,attack the need to reinforce the development of new detection systems brain’s rewardsystem, mainly serotonin and dopamine recep- with the aim to decrease and eliminate the abuse of traditional tors, which regulate movement,emotion, motivation, and, and synthetic emerging drugs.[5] The development of reliable above all, the feelings of pleasure and euphoria associated methodsfor their detection and quantification is timely and ur- with the consumption of narcotics. Nowadays, amidst all illicit/ gently required. abuse substances,cannabis is the most consumed drug world- Nowadays, illicit drug detection is performed mostly through wide with 183 millioneuro incautions, followed by ampheta- chromatographic techniques, as can be seen by the large mines and derivatives (37 million euro), opioids (35 million number of research articles published in this field, because of the excellent features of chromatographictechniques, which [6] [a] E. Garrido,+ L. Pla,+ B. Lozano-Torres,Dr. S. El Sayed, include great versatility,robustness, and sensibility. Several [7] [8] [9] Prof. R. Martínez-MµÇez,Dr. F. Sancenón electrochemical, infrared, Raman spectroscopy, and mag- Instituto Interuniversitario de InvestigacióndeReconocimientoMolecular y netic resonance[1] methods also exist for the detection of sever- Desarrollo Tecnológico (IDM), Universitat Politcnica de Valncia al drugs, and these techniques show excellent results in terms Universitat de Valncia, Camí de Vera s/n,46022 Valncia (Spain) E-mail:[email protected] of selectivity and sensibility.However,they presentsome short- [b] Dr.S.ElSayed, Prof. R. Martínez-MµÇez,Dr. F. Sancenón comings such as high cost, the use of trained personnel, tedi- CIBER de Bioingeniería ous sample pretreatment, and so on. As an alternative to these Biomateriales yNanomedicina (CIBER-BBN) traditional techniques, the design of molecular probesfor the [c] Prof. R. Martínez-MµÇez,Dr. F. Sancenón undemandingchromo- and fluorogenic detection of illicit Departmento de Química, Universitat Politcnica de Valncia drugs can be of importance.Both chromogenic and fluorogen- Camí de Vera s/n, 46022Valncia (Spain) E-mail:[email protected] ic probes provideseveral advantages over other traditional an- [+] These authors contributed equally to this work. alytical techniques, such as their chemical simplicity,ease of The ORCID identification number(s) for the author(s) of this article can use, rapid response suitable forreal-time on-sitedetection, be found under:https://doi.org/10.1002/open.201800034. easy detection to the naked eye, and so on.[10] Despite these 2018 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA. remarkable features, the development of chromo- and fluoro- This is an openaccessarticleunder the termsofthe Creative Commons genic sensors for the selective andsensitive detection of cer- Attribution-NonCommercial-NoDerivs License, which permits useand tain illicit drugsisstill poorly explored. In this review,athor- distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are ough overview of chromogenicand fluorogenic sensing sys- made. tems for the detection of most consumed drugs is provided.

ChemistryOpen 2018, 7,401 –428 www.chemistryopen.org 402 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim The review is organized by the description of probes for differ- Table 1contains asummary of the different strategies for ent drugs classifiedaccording to their pharmacologic behavior, the chromo- and fluorogenic detection of illicit drugs used in that is, depressants, stimulants, hallucinogenic, and opioids. the examples described below.

Eva MaríaGarrido graduated in Sameh El Sayed received his Masters chemistry from the University of Sevilla Degree in chemistry from the Poly- (US) in 2015 and received her Masters technic University of Valencia (Spain) Degree (Masters in Food Technology in 2012 and his Ph.D. degree from the and Industry) from the University of same university in 2015 with Professor Seville (US) in 2016. She is aPh.D. stu- RamónMartínez-MµÇez. After apost- dent at the Polytechnic University of doctoral period at University of Pavia Valencia (UPV), and her area of interest (Italy), he joined Centro de Investiga- is the development of chemical sen- ciónBiomØdica en Red (CIBER) under a sors and probes based on the use of Juan de la Cierva postdoctoral fellow- gated nanomaterials. ship for two years. His research areas of interest fall in the fields of chromo- and fluorogenic sensors and molecular probes for the detection of anions, cations, and neutral species, in addition to the development of nanomaterials for sensing and smart-delivery applications.

Luis Pla graduated in chemistry from Ramon Martínez-MµÇez was born in the University of Valencia (UV) in 2013 Valencia, Spain. He received his Ph.D. and received his Masters Degree (Mas- degree in Chemistry from the Universi- ters in Experimental and Industrial Or- ty of Valencia in 1986 and was apost- ganic Chemistry) from the University doctoral fellowship at Cambridge Uni- of Valencia (UV), University of Barcelo- versity,U.K. Presently,heisafull pro- na, Polytechnic University of Valencia, fessor in the Department of Chemistry Cardenal Herrera University (CEU), and at the Polytechnic University of Valen- University of the Balearic Islands in cia and is also the director of the IDM 2014. He is aPh.D. student at the Poly- Research Institute at the Polytechnic technic University of Valencia (UPV), University of Valencia and the Biomedi- and his area of interest is the develop- cal Research Networking center in Bio- ment of chemical sensors and probes based on the use of gated engineering, Biomaterials, and Nanomedicine (CIBER-BBN). He is nanomaterials. the coauthor of more than 400 research publications and 20 pat- ents. His current research interest involves designing gated hybrid materials for on-command delivery applications. He is also involved in developing new sensing methods for different chemicals of interest.

Beatriz Lozano-Torres graduated in Felix Sancenónwas born in 1968 in chemistry from the Complutense Uni- Manises, Valencia, Spain, and graduat- versity of Madrid (CUM) in 2013 and ed in Chemistry in 1991. He received received her Masters Degree (Masters his Ph.D. degree in 2003. Afterwards, in Organic Chemistry) from the Com- he worked with Professor L. Fabbrizzi plutense University of Madrid (CUM) in at the Universitµdi Pavia on the syn- 2014. She is aPh.D. student at the Pol- thesis of chromogenic receptors for ytechnic University of Valencia (UPV), ion pairs. Then, he joined the Depart- and her main area of interest lies in ment of Chemistry at the Polytechnic developing chemical probes based on University of Valencia with aRamon y gated nanomaterials for biomedical Cajal contract. He became alecturer in applications. 2006. He is the coauthor of more than 240 research publications. His current research interest comprise the use of hybrid materials for the development of sensors and for the design of gated materials.

ChemistryOpen 2018, 7,401 –428 www.chemistryopen.org 403 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim Table 1. Summary of the different chromo- and fluorogenic methodsfor illicit drugsdetection.[a]

Analyte Recognition SolventLOD Interferent Ref. 1 mechanism [mg mLÀ ] DEPRESSANTDRUGS GBL BODIPY fluorescent sensorwater and alcoholic 3–[12] drinks GHB BODIPY fluorescent sensorbeverages ––[14] (alcoholic, nonalco- holic, colored,and colorless) GHB colorimetric sensor water –GBL, 1,4-butanediol, propionic acid, butyric [15] acid, and common salts GHB iridium(III) chemosensor water,black tea, 0.15 –[16] soda, orangejuice, red wine, whisky, and milk 3 CPH spectrophotometric methodwater and pharma- 1.7 10À –[17] ceutical formulations ketamine hydrochloride colorimetric test water and pharma- 5–[18] ceutical formulations ketamine colorimetric test water 100 AMP,MAMP,MDMA, cocaine,EPH, scopola- [20,21] mine, caffeine, acetaminophen,ibuprofen,

acetylsalicylic acid, starch, sugars, and MgSO4 9 drugs containingcyclic fluorometricmethodpharmaceutical 310À to –[22] 5 a-methylene carbonyl preparations and 210À groups biofluids 3 dextromethorphanspectrophotometric methodpharmaceuticals 3.5 10À –[23] preparations 6 morphine and codeineAuNPs human serum, 4.85 10À and cations, anions, ascorbicacid, glucose, urea, [24] 6 urine samples, and 2.69 10À cysteine, glutamic acid, asparagine,leucine, pharmaceutical for- valine,proline, phenylalanine, tramadol,AMP, mulations and MAMP STIMULANTDRUGS

3 III MA, EPH,and MeEPH formationofcolored product urine 3.7510À , CaCl2,NaCl, MgCl, NaNO3,CH3COONa, Al , [26,27] 3 III II II + 4.13 10À ,and Fe ,Ni,Co,NH4 ,urea, uric acid, creatinine, 3 4.48 10À and hippuricacid AMP,MA, MDMA,and NPs basicbuffered 2–5 cocaine, cannabinol, cannabidiol, THC, sco- [28] MDA aqueous polamine, EPH, and procaine cocainebiomimetic material (AuNPs) absorbent pad –Benzoylecgonine, nicotine, cotinine, codeine, [29] THC and MA 2 MA Simon’s reagentapplied to applicationinstalled 1.10 10À to –[30,31] 2 mobile phonetechnology on amobile phone 4.4 10À 5 MDMA bis-diarylurea-basedprobe ecstasy tablets2.45 10À EPH,PEPH,AMP,MDA, and MDMA[32] 3 MA molecular recognition (thiophene vapor1.9 10À and –[33] 3 heterocycles, fluorene, and 6.4 10À polymer) 6 MA molecular recognition (perylene vapor5.5 10À amines, organic solvents, water,apple [34] bisimidefluorophore, and two pomace cholesterol subunits) MDMA NPs water0.95 cocaine, heroin, methadone, and morphine[35] 5 MA molecular recognition (amine- THF 2.5 10À pethidine and EPH [36] functionalized polyfluorene) MDMA NPs water–AMP,glucose, glycine, and sarcosine [37] 6 AMPH,METH, MDMA; molecular recognition H2O/MeOH (7:3, v/v, 7.4 10À , –[38] 6 and DA (macrocyclic) pH 7.4) 1.3 10À , 6 8.0 10À ,and 5 6.7 10À MDMA and MDA colorimetric reaction sulfuric acid aque- 0.19 –[39,40] ous (75%, w/v) MA NPs toluene and vapor – o-toluidine, hexylamine, diethylamine,dibu- [41] tylamine, allylamine, trimethylamine, and aniline catecholaminemolecularrecognition water–biogenicamines, sugars, neurotransmitters, [42] and aminoacids phenothiazinedrugs spectrophotometric methodsulfuric acid ––[43]

ChemistryOpen 2018, 7,401 –428 www.chemistryopen.org 404 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim Table 1. (Continued)

STIMULANT DRUGS

4 PEA UV n-hexane10À phenylethanolamine [44] d-and l-a-phenylethyla- fluorescencechiral sensors MeCN ––[45] mines PEPH hydrochloride spectrofluorometricmethodPBS(pH 7.8) 0.5 additiveorexcipients present in the pharma- [46] ceuticalsformulations cocaine, MDMA/MDA, spectralfluorescence signature water ––[47] and heroine/morphine (SFS) measurements 4 MA aptasensor combined with AuNPs human urine 1.2 10À pethidine, triazolam, barbital, morphine, keta- [49] (colorimetric assay) mine, and diazepam 9 MA aptasensorand oxidationreaction water 7.4 10À ketamine,norketamine, morphine, metha- [50] of afluorophore done, cocaine, mephedrone, cathinone, methcathinone, 3-trifluromethylphenyl piperazine,1-3-trifluromethylphenyl piperazine, 3,4-methylenedioxy pyrovalerona, MDA, MDMA, EDDP,and mCPP 4 cocaineaptamernanomachine (FRET pro- water 1.5 10À –[51] cessallowed or not depending on the analyte) 5 cocaineaptamernanomachine and ampli- water 3.0 10À for –[52] fied aptamer nanomachine normal and 17 (quenching off/on process) 3.5 10À for amplified process 6 cocaineaptamernanomachine (quench- water,saliva, serum, 1.7 10À –[53] ing off/on process) and urine 7 cocaineaptasensorcombined with CQDs water 6.1 10À benzoylecgonine and ecgonine methyl ester [54] (quenching off/on process) 5 cocaineaptasensorcombined with CQDs water 1.5 10À [55] (quenching off/on process) 9 cocaineaptasensor combined with silica water or serum6.3 10À and chloramphenicol, propranolol, diazepam, and [56] 9 NPs and AuNPs(quenching off/on 8.9 10À morphine process) 6 cocainefluorophore quenched by apta- water,urine, and 6.1 10À , benzoylecgonine [57] 5 merand recognition (quenching saliva 1.4 10À ,and 5 off/on process) 1.5 10À 9 cocaineaptasensorcombined with CQDs buffered solution 4.2 10À benzoylecgonine, MA, 3-acetamidophenol, [58] and NPs (quenching off/onpro- and codeine cess) 5 cocaineaptameric moleculargate Tris solution 1.5 10À morphine and heroine [59,61] (fluorogenic or SERS detection) (pH 7.5) and saliva 5 cocaineaptasensor(quenching off/on buffered solution 3.0 10À benzoylecgonine and ecgonine methyl ester [62] process) 4 cocaineaptasensor(quenching off/on serum 3.0 10À benzoylecgonine and ecgonine methyl ester [63] process) 5 cocaineaptasensor(molecular displace- buffered solution 6.1 10À benzoylecgonine and ecgonine methyl ester [64] ment of dye and increase in absorptionband) 4 cocaineaptasensor(conformation buffered solution 1.5 10À theophylline, caffeine,BSA, IgG,arginine, [65] changes after molecular recogni- phenylalanine, and aspartic acid tion of analyte, highfluorescence anisotropy) 3 cocaineaptasensor(conformation water 3.0 10À ecgonine, pethidine, and methadone [66] changes after addition of analyte in presence of CuNPs) 3 cocaineaptasensorcombined with GO plasma and serum 3.0 10À pethidine and methadone [67,68] and AuNPs (quenching off/on process) 5 cocaineaptasensorcombined with AuNPs water 6.1 10À –[69,70] (changesinabsorption band due to aggregation of AuNPs) cocaineaptasensorcombined with AuNPs urine and serum 30.3 –[71] (changesinabsorption band due to aggregation of AuNPs)

ChemistryOpen 2018, 7,401 –428 www.chemistryopen.org 405 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim Table 1. (Continued)

STIMULANT DRUGS

7 cocaine aptasensorbased on G-quadru- serum 1.5 10À caffeine, morphine, and theophylline [72] plex/ruthenium complex interaction (decrease in fluorescence in presence of analyte) 6 cocaine aptasensorgraftedonto optical serum 5.0 10À kanamycin, amikacin, and ibuprofen [73] fiber (quenching off/on process) 3 cocaine aptasensorcombined with AuNPs buffered solution 2.5 10À ephedrine, codeine, ketamine,AMP,mor- [74] (naked-eye detection system) phine, MA, benzoylecgonine, and ecgonine methyl ester 3 cocaine aptasensorcombined with AuNPs water 1.5 10À –[75] (changesinabsorptionband due to aggregation of AuNPs) 6 cocaine aptasensorcombined with UCNPs saliva3.0 10À benzoylecgonine and ecgonine methyl ester [76] and AuNPs (quenching off/on process) 5 cocaine aptasensorcombined with GO urine 5.8 10À adenosine, caffeine, theophylline, and mor- [77] and afluorophore (quenching phinehydrochloride off/on process) 6 cocaine aptasensorcombined with CD serum 4.0 10À MA, codeine, THC, nicotine, cotinine,and [78] and QDs (quenching on/off benzoylecgonine process) 7 cocaine and MA aptasensorcombined with buffered solution 1.5 10À for co- ketamine,norketamine, morphine, cathinone, [79] Au@AgNPs (changes in absorp- caine and 14.9 methacathinone, 3-trifluoromethyphenylpi- tion band due to aggregation of for MA perazine, and MDA NPs) 7 cocaine aptasensorbased on G-quadru- buffered solution 9.1 10À ATP, adenosine,warfarin,and suramin [80] plex/iridium complex interaction (switch-on luminescence) 5 cocaine aptasensorcombined with MNPs buffered solution 1.5 10À ecgonine, pethidine, and methadone [81] 6 cocaine aptamernanomachinecombined serum 6.1 10À ecgonine and benzoylecgonine [82] with AgNPs(changes in fluorescence band due to aggregation of AgNPs) 6 cocaine aptasensor(conformation buffered solution 6.1 10À benzoylecgonine and ecgonine methyl ester [83] changes after molecular recognition of analyte,enhanced fluorescence intensity) HALLUCINOGENIC DRUGS

4 LSD fluorescence water and NaOH 2.6 10À mg mescaline, DOM, psilocin,psilocybin, bufote- [85] nin, ibogaine, and phencyclidine OPIODS

1 morphine, codeine, oxy- AuNPs water 5 mmol LÀ –[87] codone, noroxycodone, thebaine, tramadol, and methadone 8 heroine, morphine, oxy- fluorescence water and urine 710À for mor- –[88] codone, and their main phine and 5 metabolites 8.25 10À and 6 2.78 10À for M3G and Nmor in water 5 2.75 10À , 4 9.72 10À ,and 5 7.1310À for morphine, M3G, and Nmor in urine

[a] LOD:limit of detection;GBL: g-butyrolactone;GHB: g-hydroxybutyric acid;CPH:chlorpromazine hydrochloride;AMP:amphetamine; MA:methamphetamine; MDMA: 3,4-methylenedioxymetamphetamine; MDA:3,4-methylenedioxyamphetamine;EPH:ephedrine;MeEPH:methylephedrine;THC:tetrahydrocannabinol;PEPH:pseudoephedrine;DOM:2,5-dimethoxy-4-methylamphetamine;AMPH:(+)-amphetamine sulfate;METH: (+)-methamphetamine hydrochloride;DA: dopamine;PEA:2-phenylethylamine;EDDP:2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine; mCPP: meta-chlorophenylpiperazine;IgG: immunoglobulin G; BSA:bovine serum albumin.

ChemistryOpen 2018, 7,401 –428 www.chemistryopen.org 406 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim 2. Depressant Drugs l=557 nm and emission at l=574 nm. The addition of GHB induces moderate emission quenching (2.2-fold in the pres- 1 Depressant drugs slow down the nervous system and brain ac- ence of 10 mg mLÀ of the drug)ofthe band at l= 574 nm. tivity and thus have asedating effect.[11] Most depressants This behavior can be ascribed to the formation of ahydrogen affect one of the brain’s neurotransmitters, that is, g-aminobu- bond between the phenolic hydrogen atom of the probe and tyric acid (GABA), which upon activation produces asense of the carboxylate moiety of the drug. As aconsequence of the calm and relaxation. GABA is the chief inhibitory neurotrans- coordination,aphotoinduced electron-transfer (PET) process is mitter in the mammalian centralnervous system (CNS), and its activated, which induces the observed emission quenching. principal role is to reduce neuronal excitability.Depressant Moreover,the authors demonstrate that probe 4 can be used drugs have the particularity that often leads to “drug toler- to detectGHB easily in severalbeverages (alcoholics, nonalco- ance”, which makes the organism ask for higher doses until de- holics, colored, and colorless). For this purpose, 4 is dissolved pendence or overdosearises. Coma, respiratory diseases,and in DMSO and then mixed with the beverages spiked with death are the potentialconsequences of their continuous con- known amounts of GHB (1:1, v/v,final solution). Irradiation of sumption.Depressant drugs comprise barbiturates, benzodia- the prepared samples with ahand-held lamp (l=365 nm) zepines, alcohol, and g-hydroxybutyrate. allows differences in the fluorescence intensities of GHB-free In 2013, Chang and co-workers reported boron–dipyrrome- and GHB-spiked beverages to be observed clearly by the thene (BODIPY)fluorescentsensor 3 for g-butyrolactone (GBL) naked eye. detection in water and in different alcoholicdrinks Garcia andco-workers have developed acolorimetric sensor (Figure 1).[12] GBL is aprodrug of g-hydroxybutyric acid (GHB), array for the detection of GHB.[15] The array is composed of three columns and six rows. The first column has six tricyclic dyes (methylene blue, thionine, oxonine, pyronine, acridine orange, and proflavine), the second column is formed by the same dyes complexed with cucurbit[7]ril (CB[7]), and the third columncontains the dyes complexed with cucurbit[8]ril 5 (CB[8]). The addition of GBH (in the 0.1–10À m range) to the sensor array inducesremarkable color changes in some plates due to the dethreading of CB[7] and CB[8] complexes with the dyes after coordination of the macrocycles with the drug. The colors obtained are ascribed to the different stabilityconstants for the dye–CB complexes and tothe ability of GBH to disrupt the formed supramolecular ensembles. Moreover, the chromogenic response of the array to GHB is quite selective, and Figure 1. Structures of probes 3 and 4 for the detection of GBL (1)and GHB (2), respectively. other possible interferents, such as GBL, 1,4-butanediol, propionic acid, butyric acid, and common salts present in water, are unable to induce color changes. and its relevance is due to the strong regulation to which GBH Ma and co-workersoutline the synthesis of along-lived iri- is subject, but this is not applicable to GBL. GBL overdose dium(III)chemosensor,the luminescence emission of whichat causes irrational behavior,severe sickness, coma, and death.[13] l=570 nm is quenchedinthe presence of GHB (Figure 2).[16] Sensor 3 shows amarked absorption band centered at l= Due to its long-lived luminescence, time-resolved emission 569 nm in water.Besides,upon excitation at l= 569 nm, a spectroscopy (TRES) allows the detection of GBH. Aquenching small emission band at l= 582 nm (with alow quantum yield effect of 30%luminescence emission is observed 10 safter the 1 of 0.05) is observed. The addition of GBL to an aqueous solu- addition of alow concentration of GBH (0.15 mgmLÀ )toaso- tion of 3 induces an emissionenhancement at l=582 nm ( lution of the complex. This quenching is clearly seen by the 10-fold). Alimit of detection for GBL by using 3 is reported naked eye under UV illumination and is not pH dependent. 1 to be 3mgmLÀ .The off–on emission response is related with Real samples of water,black tea, soda, orange juice, red wine, the hydrophobic character of the probe.The authors indicate that probe 3 forms nonemissive aggregates in water that are progressively broken upon coordination with GBL with asubsequent enhancement in emission. In addition, probe 3 can be used to detect GBL in drink samples after asimple extraction process.The presence of GBLissimply assessed by irradiation with agreen laser pointer;this induces the appearance of orange fluorescence, whichisnot observed in samples without the drug. The same authors also report on the preparation of BODIPY probe 4 (Figure 1) for the fluorogenic detection of GHB.[14] Eth- anol/water (1:1, v/v)solutions of 4 show an absorption band at Figure 2. Chemical structure of iridium probe 5.

ChemistryOpen 2018, 7,401 –428 www.chemistryopen.org 407 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim whisky,and milk previously spiked with GHB have been evalu- solutionsofthe drug. In the presenceofketamine, apurple ated, which assuresthe suitabilityofthis procedure for GHB precipitate on the surface of the matrix polymer is formed. drug detection over awide range of real-lifebeverages. However,after afew minutes, this precipitatedisappears, and Al-Kaffiji and co-workers have developed aspectrophoto- the solid changes from brown to purple because of the diffu- metric methodfor the opticaldetection of chlorpromazine hy- sion of ketamineinside the polymer and the formation of a drochloride(CPH). CPH pertains to the family of drugs com- complexbetween Co(SCN)2 and the drug. The best response is monly known as neuroleptic tranquilizers (used as sedatives, observed for asolid containing 0.3%Co(SCN)2 at l= 625 nm. 1 antihistamines, and anesthetics) but nowadays is commonly Alimit of detection of 100 mgmLÀ for ketamineisreported. mixed with narcotic drugs.[17] The method is based on the reac- Besides, no interference is observed if the sensing materialis tion of p-aminoacetanilide (7)with CPH in the presenceofan exposed to amphetamine, methamphetamine, 3,4-methylene- oxidizingagent (i.e. ferric chloride hexahydrate). This reaction dioxymethamphetamine (MDMA), cocaine, ephedrine, scopola- yields an intense violet product (see structure 8 in Scheme1) mine, caffeine, acetaminophen, ibuprofen, acetylsalicylic acid, with an intense absorption band at l= 590 nm. Probe 7 has starch, saccharose,glucose, lactose, and magnesium sulfate. successfully been applied to the determination of CPH in aque- The material has been tested in real samples with fine results. ous solutionsand pharmaceutical formulations with high accu- Elokely and co-workersdescribe the designofanew fluoro- racy and shows alinear range between 4and 32 ppm with a metricmethodfor the detection of drugs containing cyclic a- limit of detection of about 1.7 ppm. methylene carbonyl groups in pharmaceuticals, preparations, and biofluids by using N-methylnicotinamide chloride (NMNCl) as afluorogenic reagent (Scheme 2).[22] In basic media, N-meth-

Scheme1.Oxidation reaction with p-aminocetanilide (7)and ferric chloride for the detection of chlorpromazine drug 6.

Morrishas developedacolorimetrictest for ketamine hydro- chlorideonthe basis of atypical cocaine detectionassayby Scheme2.Chemical structures of drugs 9–11 containingcyclic a-methylene [18] using Co(SCN)2. Ketamine is amedication mainly used for carbonyl groups, and syntheticpathwayfor the preparationoffluorophore startingand maintaining anesthesia, but it is also adrug for 15. recreational purposes. Unregulated use has side effects such as high bloodpressure, abnormal heart rhythms,vomiting, double and tunnelvision, anaphylaxis, airway obstruction, and ylnicotinamide chloride forms neutral and highly reactive a- dependence.[19] The test consists of the basification of the sam- carbinol compound 12,which reacts with drugs containing ples (with sodiumhydroxide) followed by the addition of cyclic a-methylene carbonyl groups (such as 9, 10,and 11)to

Co(SCN)2.This two-stepprocedure results in the formation of a yield fluorescent derivative 15.The test is quite sensitive with 1 lavender to purple precipitate in the presence of ketamine, limits of detection in the 0.003–20 ng mLÀ range. The authors whereas ablue to green precipitate is obtained in the absence also demonstrate the possible use of N-methylnicotinamide for 1 of ketamine. Sensibility as high as 5mgmLÀ can be achieved, the detection of drugs 9, 10,and 11 in plasma samples with which is good enough for the detection of this drug in com- nearly the same limits of detection obtained upon using water. mercial productsfor medical applications(contents ranging Elmosallamyand Amin have developed anew spectrophoto- 1 from 50 to 100 mgmLÀ ). Selectivity studies show that other metric method for the detection of dextromethorphan (16) controlled substances and related chemicals yield anegative (Figure 3) in pharmaceutical preparations.[23] The spectrophoto- response. metric protocol involves the reaction between dextromethor- Herrµez-Hernµndez and co-workers follow asimilar reaction phan hydrobromide with eriochrome black T(17)inacetate for the colorimetric sensing of ketamine by immobilizing differ- buffer (pH 2.8), which resultsinthe formation of an ion-pair ent amounts of the Co(SCN)2 reagentonto apolydimethylsil- complex with an absorption band at l=520 nm. This method [20] 5 oxane (PDMS) matrix. For the preparation of the materials, for the detection of 16 is lineal in the 7.37–73.7 10À m range 5 the authors disperse Co(SCN)2 in an aqueous solutioncontain- with alimit of detection of 1.2910À m. ing tetraethylorthosilicate (TEOS) andexpose the resulting dis- Mohseni and Bahram outline the use of melamine-coated [21] persion to PDMS to embed Co(SCN)2 in the polymer matrix. gold nanoparticles (AuNPs) for the detection of morphine and Once the sensing material is prepared, pure ketamine detec- codeine in human serum, urine samples, andpharmaceutical tion can be performed by immersing the sensorin0.1m NaOH formulations.[24] The presence of melamine molecules in the

ChemistryOpen 2018, 7,401 –428 www.chemistryopen.org 408 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim otine are the most common. On the other hand, cocaine, crack, amphetamine, and their derivatives are found among illegal stimulant drugs. Sakai and Ohno describe aprocedure for the sensitive and selective detection of methamphetamine, ephedrine, and methylephedrineinurine (Scheme3).[26, 27] The method consists in the extraction of basified urine containing the drugs with 1,2-dichloroethane. Then, the organic phase, whichcontains

Figure 3. Chemical structures of the drug dextromethorphan (16)and erio- chromeblack T(17). outer sphere of the AuNPs prevents aggregation,and aqueous solutionsofthe nanoparticles present as awine-red color (absorptionband centered at l=675 nm). In the presenceofmor- phine and codeine, amarked color change to blue is observed (absorbance band at l=690 nm) due to the aggregationof the AuNPsinduced by the formation of hydrogen bonds be- Scheme3.Tetrabromophenolphthalein ethyl ester 18 transformation into tween melamine and the drugs. The proposed method exhib- red-colored product 19 in the presence of methamphetamine, ephedrine, its lineal ranges of 0.07 to 3 mm for morphine and 0.03 to and methylephedrine in urine. 0.8 mm for codeine with limits of detections of 17 and 9nm,respectively.Interference studies show that cations, anions, as- the drugs, is added to a1,2-dichloroethane solution of tetra- corbic acid, glucose,urea, cysteine, glutamic acid, asparagine, bromophenolphthalein ethyl ester (18). After afew minutes, leucine, valine, proline, phenylalanine, tramadol, amphetamine, colored product 19 is formed. The absorption maximaare and methamphetamine are unable to induce color modula- found at l=570 nm for methamphetamine, l= 555 nm for tions. Besides, melamine-coated AuNPs have also been used ephedrine, and l=550 nm for methylephedrine. The proce- for the detection of morphine and codeine in biological fluids dure described presentshigh selectivity,and calcium chloride, and pharmaceutical formulations with fine results. sodium chloride, magnesium chloride, sodium nitrate, sodium The probes described in this sectionare based on different acetate, AlIII,FeIII,NiII,CoII,ammonium ions, urea, uric acid, crea- chemicalreactions and the formation of complexes with de- tinine,and hippuric acid are all unable to induce any chromo- pressant drugs.Also, the analyte-induced aggregation of genic response. In addition, the limits of detection are 1 1 AuNPs hasbeen used to detect morphine and codeine. High 3.75 mgmLÀ for methamphetamine, 4.13 mgmLÀ for ephe- 1 selectivity andlow detection limits are achieved with mostof drine, and 4.48 mgmLÀ for methylephedrine. the probesexposed above,and thus, they are good alterna- Herrµez-Hernµndez and co-workers embed 1,2-naphthoqui- tives for traditional chromatographic methods. Naked-eye de- none-4-sulfonate 20 (Figure 4) into polydimethylsiloxane/tet- tection, short analysis times, and no need for overqualified per- raethylorthosilicate/silicon dioxide sonnelfor their daily use are some of the advantages of these nanoparticles and use this mixture sensing probes. Besides, some probes have been tested in real to identify amphetamine (AMP), human body fluids,and thus, some of these reported exam- methamphetamine (MAMP), 3,4- ples could have potential applications for the development of methylenedioxymethamphetamine realistic systems for the detection of depressant drugs. (MDMA), and 3,4-methylenedioxyamphetamine (MDA) chromogeni- [28] 3. Stimulant Drugs cally. Basic buffered aqueous suspensions of the nanoparticles Figure 4. Chemical structure Stimulant drugs accelerate the activity of the central nervous show alight yellow color due to of 1,2-napthoquinone-4-sulfo- system.Ifhigh doses are taken,elevated blood-pressure levels the presence of abroad absorp- nate (20)used for the detection of amphetamine-based and increased heart and respiration rates may ensue, with the tion band at around l= 460 nm. drugs. potentialrisk for cardiovascular failure;however,thesedrugs Upon the addition of ampheta- can also make consumersfeel angry and/or paranoid.[25] mines, clear colorchanges to Normally,these drugs inhibit the reuptakeofserotonin, nor- orange (for MAMP and MDMA, epinephrine, anddopamine, which resultsingreater concen- which are secondary amines)orgray-brown (for AMP and trationsofthese three neurotransmitters in the brain, and this MDA, which are primaryamines) are observed. Limits of detec- 1 leads to multiple effects on the state of mind of the individual tion in the 0.002–0.005 gmLÀ range are found. The prepared user.There are anumber of stimulant drugs,including those sensing materialdisplays better selectivity for amphetamines that are legal and prescribed by doctors and those that are than for other drugs such as cocaine, cannabinol, cannabidiol, considered illegal. In the group of legal drugs, caffeine and nic- tetrahydrocannabinol(THC), scopolamine, ephedrine, and pro-

ChemistryOpen 2018, 7,401 –428 www.chemistryopen.org 409 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim caine. The only two drugs that interferewithamphetamine de- the detection of alkaloids(containing secondary amines). The tection are ephedrine and prococaine. The application of this amine moiety of the alkaloids reacts with acetaldehyde to sensing system to real ecstasy samples allows the percentage yield an enamine, which subsequently reacts with sodium ni- of amphetamine contained in the pills to be calculated. Com- troprusside to generate an immonium salt. Finally,this is hy- paring these results with those obtained by LC methods, no drolyzed to abright cobalt-blue product (known as the significant differences are found. Simon–Awe complex)that allows for the identification of Yagci and co-workersdelineate astrategy involving the use drugs containing secondary amines. Chooduma and co-work- of anoncompetitive assayformatbyusing abiomimetic mate- ers use this reagent to sense methamphetamine by using rial, poly(phenylene)b-cyclodextrin poly(ethylene glycol)(PPP- mobile phone technology.[30] In their study,the samples (con- CD-g-PEG), to recognize cocaine due to the affinity of b-CD taining methamphetamine) are kept in an opaqueblack corru- toward this drug (Figure 5).[29] The authors prepare test strips gated box and are treated with Simon’s reagent;the intensity of the colored product inside the microtubeisthen detected by using the ColorAssistapp (FTLapps,Inc.) for an iPhone 4.0 in the flash-off mode. The authors relate the RGB values obtained to the concentration of methamphetamine andthen compare the data with the gas chromatography data. The authors note that the correlation between both sets of data, in terms of methamphetamine concentration,isremarkable. Besides, one year later,the same authors report the entrapment of Simon’s reagent into asol–gel matrix and include it in the corrugated box.[31] For sample analysis, the solid sensor is pierced and methamphetamine is putinside to allow the reactionbetween the analyte and the reagent. The samples are then directly analyzed by using the same app by studying the RGB values. Torroba and co-workersdescribe the synthesis of new bis-diarylurea-based probe 21 (Figure 6) for the detection of MDMA

Figure 5. Schematic representation of atest stripe functionalized with PPP- CD-g-PEG and Anti-IgG antibodiesfor the detection of cocaine. composed of apolyvinyl chloride (PVC) backing card, the sample pad, the conjugate release pad, anitrocellulose mem- brane,and an absorbent pad. In the absorbent pad, there is a conjugated pad [containing absorbed antibenzoylecgonine antibody/AuNPs (Anti-BE/AuNPs)],acontrol line (containing absorbed Anti-IgG antibody), and atest line (containing absorbed Figure 6. Chemical structure of bis-diarylurea-based probe 21 for MDMA PPP-CD-g-PEG). Upon applying aliquid sample without cocaine detection. to the sample pad and upon reaching the conjugate pad, the Anti-BE/AuNPs migrate forward together with the liquid sample to give ared negative line. If cocaineispresentinthe from ecstasytablets.[32] DMSO solutions of probe 21 show a sample, cocainemolecules interact with the b-CD residues in weak emission band centered at l= 517 nm upon excitationat the test line and another red line appears in the test zone as a l=288 nm. The addition of aqueous solutionsofephedrine, result of the attachment of the Anti-BE/AuNPs to cocaine. The pseudoephedrine,amphetamine, 3,4-methylenedioxyampheta- test resultscan also be analyzed by using amobile phone ap- mine, and 3,4-methylenedioxymethamphetamine induces a plication by photographing the strips and evaluating the color marked emission enhancement at l= 517 nm due to the for- group and color percentage in the image. Agood linear trend mation of a1:1 stoichiometry complex of acharge-transfer 1 in the 0.01–1.0 mgmLÀ range is observed. Moreover,the au- nature.Inspite of the fact that the fluorogenic response of thors find that the sensorisselectivefor cocaine in the pres- probe 21 towardselected drugs is unselective, principal com- ence of benzoylecgonine, nicotine, cotinine,codeine,tetrahy- ponent analysis (PCA) score plots allow their discrimination. drocannabinol, and methamphetamine. Finally,the authors Besides, MDMA has been detected in asimulated tablet of ec- demonstrate the validity of the test stripes by determining co- stasy.Nointerference is observedinthe presence of excipients caine in synthetic saliva with nearly the same accuracy as that (e.g. sucrose, chalk, or caffeine) in ecstasy tablets. achievedbyusing chromatographic methods. Cheng and co-workers give details on the preparation of Simon’s reagent (a mixture of sodium nitroprusside, sodium molecular probe 22 containing thiophene heterocyclesand a carbonate, and acetaldehyde) is achromogenictest used for fluorene moiety (Figure7)and also polymer 23 containing the

ChemistryOpen 2018, 7,401 –428 www.chemistryopen.org 410 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim Figure 7. Structures of thiophene-containingfluorene derivatives 22 and 23 for MA detection.

same functional groups for methamphetamine(MA) vapor detection.[33] Films of the molecular probe and the polymer are Scheme4.Schematicrepresentation of MDMAdetectionbyusing silica obtainedbyspin coating. The prepared films are weakly fluo- nanoparticles and the [CBPQT][PF6]4 macrocycle. rescent,but in the presence of MA, amarked emission enhancement is observed for both sensing materials. The weak emission of the films is ascribed to aheavy-atom effect from MDMAinwater (Scheme 4).[35] In afirst step, the external sur- the sulfur atom of the thiophenetothe fluorene fluorophore. face of the MSNs is functionalized with anaphthalene deriva- In the presence of MA, the reactionofits amino groups with tive, and this is followed by the encapsulationoffluorescein the thiophenerings of compounds 22 and 23 induces a inside the porousnetwork of the inorganic scaffold. In a marked reduction in the heavy-atomeffect with asubsequent second step, the loaded MSNs are capped upon the addition emission enhancement. Limits of detection of 1.9 and 6.4ppm of the cyclobis(paraquat-p-phenylene) hexafluorophosphate for MA by using films containing 22 and 23 have been mea- ([CBPQT][PF6]4)macrocycle, which forms inclusion complexes sured. Besides, exposure of the films to vapors of propylamine, with 1-naphtol subunitsattached onto the externalsurface of aniline,ortoluidine induces negligible changes in the emission the nanoparticles. Aqueous suspensionsofthe nanoparticles at intensity. pH 7.0 show negligible fluorescein release, whereas in the Following asimilar concept, Fang and co-workers design a presence of MDMA amarked emission band at l=520 nm perylenebisamide-containingfilm for methamphetamine (lex = 495 nm) in the solution is found. Theobserved emission vapor detection.[34] Molecular probe 24 (Figure 8), containinga band can be ascribed to the release of fluorescein from the perylenebisimide fluorophore and two cholesterol subunits, is nanoparticles due to the preferential coordination of MDMA able to form self-assembly fluorescent films on aglass surface. with the CBPQT4+ macrocycle, which results in pore opening Abroad emission band at l=678 nm is observed for the film and cargo delivery.Besides, the prepared material is selective that is absent in the probe alone.This emission can be as- to MDMA, and other common drugs (e.g. cocaine, heroin, cribed to the formation of fluorescent aggregates of the probe methadone, and morphine) are unable to induce the release of within the film (through p–p interactions).This emission band fluorescein. Alimit of detection of 4.9mm forMDMA in water of the film is markedly quenched in the presence of metham- has been determined. phetamine vapor with alimit of detection of 5.5 ppb. Vapors He and co-workersdetail the synthesis and characterization of aniline,xylidine, o-toluidine, and 1,4-diaminobenzenealso of an amine-functionalizedpolyfluorene for the detection of induce moderate emission quenching. Moreover,the authors methamphetamine.[36] THF solutions of the prepared polyfluor- demonstrate that the film can be easily reusedbypurging ene show two broad emissionbands at l= 417 and 440 nm with cold air. (upon excitation at l=388 nm) due to the fluorene core. The Lozano-Torres and co-workers recountthe preparation of addition of methamphetamine induces amarked quenching of mesoporoussilica nanoparticles (MSNs)capped with pseudoro- the emission bands (80 %upon the addition of 40 mm drug), taxanesfor the selectiveand sensitive fluorogenic detection of which can be ascribed to an aggregation process of the poly-

Figure 8. Perylene bisamidebased probe 24 for MA detection.

ChemistryOpen 2018, 7,401 –428 www.chemistryopen.org 411 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim mer.Fromthe emission titration profiles, alimit of detection of ride (MDMA), and dopamine (DA).[38] Awater/methanol (7:3, 1 25 ng mLÀ of methamphetamine hydrochlorideisnoted. v/v,pH7.4) solution of probe 27 shows typical fluorene emis- Other drugs such as pethidine and ephedrine also induce sion bands at l=377,396, and 416 nm. The addition of AMPH, marked quenching of the emission. METH, MDMA, and DA to asolution of 27 induces amarked Prodi, Dalcanale, and co-workers report the preparation of emissionenhancement that is nearly of the same order forthe silica nanoparticles with pyrene derivative 25 insertedand four drugs and ascribed to the formation of 1:1adducts. In with ahydrophilic externalsurface due to the presence of a contrast, more selectivebehavior is found with naphthalene- poly(ethylene glycol)shell for MDMA detection in water bearing probe 28.Inthis case, awater/methanol (7:3, v/v, (Figure 9).[37] Aqueoussuspensions of the prepared nanoparti- pH 7.4) solution of 28 shows the typical naphthalene emission band that is enhanced only upon the addition of MDMA and DA. Ishii and co-workers presentanoptimized version of aprevi- ously published method[39] for the detection of MDMA,MDA, and relateddrugs that consists in arapid colorimetric reaction with chromotropic acid (CTA) in 75 %(w/v)aqueous sulfuric acid at room temperature (Scheme 5).[40] In this protocol, form-

Figure 9. Schematic representation of MDMA detection by interaction with pyrene derivative 25. cles present the typicalmonomer/excimer emission spectrum of the pyrene fluorophore with bandsatabout l=395 and 475 nm. Upon the incorporation of cavitand 25 into the hydrophobic compartment of the nanoparticles, both pyrene units are in closespatial proximity,which allows the formation of an excimer (band centered at l=475 nm). The addition of in- Scheme5.MDMAdetection through the generation of formaldehyde. creasingquantities of MDMA induces progressive and marked Formed formaldehyde reacts with CTAtoyieldred-violet product 34. quenching of only the excimer band. The authors ascribe this fact to the inclusion of the amine moiety of MDMA into the cavitand, which induces spatial separation of both pyrene fluo- aldehyde (33)isgenerated by treating MDMAwith sulfuric rophores anddisrupts initial excimer 26.The addition of 3-fluo- acid. Then, formaldehyde induces the couplingoftwo CTA romethamphetamine (FMA) also induces excimer emission molecules to yield red-violet product 34 that can be examined quenching but to alesser extent than that observed for visually or by measuringits absorbance at l=570 nm. The MDMA. Amphetamine, glucose, glycine, and sarcosine are band at l=570 nm gradually increases with an increase in the unable to induce any emission change. drug concentration. The optimal concentration of CTAand the García-EspaÇaand co-workers report the synthesis of macro- optimal reactiontime are 1.0 mm and 30 min, respectively, cyclic probes 27 and 28 (Figure 10) containing fluorene and whereas the drug concentration should not be greater than naphthalene fluorophores and their sensing behaviors towards 100 mm to avoid nonspecific color development. Finally,seized amphetamine sulfate (AMPH), methamphetamine hydrochlo- tablets containing well-known concentrations of both MDMA ride (METH), 3,4-methylenedioxymethamphetamine hydrochlo- and MDA have been processed and analyzed by using the proposed methodology. Haghgoo and Rouhanidescribe the synthesis of 1,8-naphtali- mide–thiophene derivative 35,which is further attached to [41] silica nanoparticles to give 35@SiO2. Toluene solutions of 35 present abroad emission band centered at l= 435 nm, which shifts to l= 473.5 nm if grafted onto silica nanoparticles. The addition of methamphetamine to asolution of probe 35 (Figure 11)ortoasuspension of the nanoparticles (using toluene in both cases) induces marked emission enhancements

(7.3 and 15.4-fold for free 35 and 35@SiO2,respectively). These emission enhancements can be ascribed to inhibition of the Figure 10. Chemical structuresofmacrocyclic probes 27 and 28 for the de- heavy-atomeffect (from the thiophenering) upon coordina- tection of drugs 29, 30,and 31. tion of the probeswith methamphetamine. In contrast, negligi-

ChemistryOpen 2018, 7,401 –428 www.chemistryopen.org 412 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim Figure 11. MA detection process with functionalized nanoparticles 35@SiO2.

ble fluorescencechanges are observed for other amines such as o-toluidine, hexylamine, diethylamine, dibutylamine, allylamine, and trimethylamine, whereas slight quenching is found for aniline. Furthermore, methamphetamine vapor sensitivity has successfully been evaluated by exposing analyte-saturated 1 1 vapors (6.7 mmolLÀ to 0.27 mmol LÀ concentration) over

35@SiO2 on glass, which shows reversibility after vapor remov- al. Finally,the proposed probe has been validated with HPLC, and there is satisfactory agreement between the results obtained with both methods. Maue and Schrader outline the design and synthesis of a new family of catecholamine probes(i.e. compounds 36–38) combining bisphosphonate recognitionmoieties (for amino al- cohols) and boronic acid groups (for the recognitionofcate- Figure 12. Chemical structures of catecholamine probes 36–38 and recogni- chols) in one chemical structure.[42] The addition of alizarine tion mechanism. Alizarinered-S is displaced in the presence of noradrenaline red-S to aqueous solutions of the three probes induces a due to higher affinity with the probe. marked color change from deep red (free dye with an absorbance band at l 520 nm) to orange (band centered at l= 460 nm) due to the formation of 1:1complexes (Figure 12). In oxidation of the drugs in sulfuric acid mediumbyaknown the presence of noradrenaline, amarked decrease in the band excess amountofchloramine-T.Then, the excess amount of at l=460 nm is observed together with an increaseinthe ab- chloramine-T reacts with 4-N-methylaminophenolinthe pres- sorbance at l=520 nm (color change from orange to deep ence of an aromatic amine to produce apurple-red product red). These changes in color can be ascribed to preferentialco- with an absorption maximum at l= 520 nm. ordination of noradrenaline with the probesthat displace ali- Inwang and Mosnaim report the development of an efficient zarine red-S from the sensing ensemble to the solution.Several and specific extraction procedurefor the UV spectrophotomet- potentialinterferents have been examined, and no positive re- ric detection of 2-phenylethylamine (PEA) through reaction [44] sponse for simple biogenicamines (creatinine), sugars (e.g. with ceric sulfate(CeSO4)toform afluorescent probe. Sam- glucosamine, glucose, fructose, saccharose), neurotransmitters ples are treated with CeSO4 and HCl in n-hexane, and this is (e.g. g-aminobutyric acid, serotonin, acetylcholine), and amino followed by filtration and extraction of the aqueous layer.This acids (e.g. serine, phenylalanine, alanine, histidine, lysine,tryp- procedure has also been evaluated for biological samples, in tophan, glycine) is observed. addition to real human, cat, and rabbit organs as well as urine. Basavaiah and co-workersdesign aspectrophotometric Alinear relationship is found between the concentration of methodfor the determination of six phenothiazine drugs (i.e. PEA and the UV absorbance at l=287 nm, with alimit of de- 1 chlorpromazine hydrochloride, promethazine hydrochloride, tection of 0.1 mgmLÀ . trifluoromazine hydrochloride, trifluoperazine hydrochloride, Wang and co-workers discussthe development of two fluo- thioproperazine mesylate, and prochlorperazine mesylate) in rescence chiral sensorsfor the enantiomeric recognition of d- pharmaceutical preparations.[43] This method is based on the and l-a-phenylethylamines (Figure 13).[45] The author prepared

ChemistryOpen 2018, 7,401 –428 www.chemistryopen.org 413 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim Poryvkina and co-workersvalidate anew procedure for the detection of several common street drugs such as cocaine, MDMA/MDA, and heroine/morphine on the basis of spectral fluorescencesignature (SFS) measurements and artificial neuron networks (ANNs).[47] SFS analyses generateatwo-dimensional spectra matrix for which the intensity depends on the excitation/emission wavelengths and the concentrationof the analyte. For cocainedetection, the fluorescence emission

intensity at l=315 nm can be evaluated (lex =235 and 273 nm) with an enhancement and quenching in the presence of cocaine salt. On the other hand, the MDMA fluorescence

emission intensity at l=320 nm (lex = 235 and 285 nm) is en- Figure 13. Chemical structuresoffluorescentdimeric and oligomeric probes hanced if the drug is present. To differentiate MDMA from 39 and 40 for l-a-phenylethylamines. MDA, the samples can be treated with o-phthalaldehyde-N- acetylcysteine (OPA-NEC), which forms selectively an isoindole complex with MDA with amaximum emission at l=425 nm dimericand oligomeric binaphthol derivatives 39 and 40,re- (lex = 335 nm). On the other hand, heroin analysisisbased on spectively,for detection through ahost–guest interaction be- the detection of morphine in the sample due to the great simi- tween 39 or 40 with the chiral amines. It is suspectedthat the larity of their corresponding structures. The fluorescenceof chiral amines interact with the host through p–p interactions heroin in water is not strong enough to confirmthe presence between the naphthyl moieties and the amino phenylgroup. of heroin in multicomponent samples, but the presence of These interactions produce changes in the emission intensity morphine can be detected because of its high quantumyield of the probesdepending on the concentration of the guest of fluorescence. For this purpose, samples are treated with hy- molecule. As most amines do not have chromophores, the typ- drochloricacid to convert morphine into the morphine salt. ical emission at l= 410 nm for the naphthalene ring of the Growth of the fluorescencesignal is observed at l=345 nm host structures can be used to follow changes in the fluores- (lex = 285 nm) after treatment.The designed protocol shows cence intensity.With the addition of different concentrations excellentselectivity and limits of detection. Besides, 98.7%of of the chiral guest phenylethylamines, the fluorescencequan- samples are correctly identified, assuringthe robustness of the tum yield of the solutions can be measured, and fluorescence method.Due to the great amount of data generated, the use emission reversion behavior is observed. The fluorescence re- of ANN as atool for pattern recognition of SFS is required, sponses of these two developed chiral sensors are influenced which includes alearningstep and posterior validation. by the concentration of the target chiral amines. The recogni- One of the most extensive strategies for the fluorogenic and tion can be confirmed by 1HNMR spectroscopymeasurements. chromogenic detection of drugs is based on aptasensors, El-Didamonyand Gouda report on the development of a which are aclass of biosensor systemsbased on the use of spectrofluorometric method for the detection of pseudoephe- DNA or RNA aptamersasrecognition sites. Aptamers can rec- drine (41)incommercial pharmaceuticalformulations in pure ognizeselectively certaintarget molecules through the forma- and dosage forms through its derivatization with 4-chloro-7-nition of aspecific three-dimensional site obtained due to nu- trobenzofurazan (42)(Scheme6).[46] Compound 42 itself is non- cleotide chain folding.[48] Aptasensors usually show very high emissive but upon reactionwith thiols or amines,itforms sensitivity,specificity,and reproducibility against awidevariety highly fluorescent product 43.Inphosphate-buffered saline of targets. (PBS, pH 7.8), 41 reacts with 42 to yield astrongly emissive de- As an alternative to traditional chemosensors for metham- rivativewith aband at l=532 nm. The authors optimize sever- phetamine (MA) detection, Shi and co-workers present a al parameters such as pH, buffer,concentration, solvent, and simplecolorimetric assay for the sensitive and visual detection temperature finally to proposeamethod that can measure of this drug in both aqueous solution andhuman urine.[49] The 1 concentrations of 41 down to 0.5 mgmLÀ with good accuracy proposed strategy comprises the advantages of using aselec- and precision. tive methamphetamine aptamer (as the recognition element) and unmodified easy-to-prepare AuNPs (as an indicator). In a first step, citrate-coated AuNPsare prepared, and then, the cocaine aptamer is adsorbed onto the externalsurfaceofthe nanoparticles. An aqueous solution of the aptamer-coated AuNPs shows the characteristic plasmon absorption band at l=525 nm (red color), which is indicative of the presence of nonaggregated individual nanoparticles. The addition of in- creasingamounts of methamphetamine induces the progressive appearance of an absorptionband centered at l=660 nm Scheme6.Detection of 41 by derivatization with 4-cloro-7-nitrobenzofura- with asubsequent color change from red to blue. This change zan (42). The reaction results in the formation of fluorescentadduct 4. in color can be ascribed to coordination of methamphetamine

ChemistryOpen 2018, 7,401 –428 www.chemistryopen.org 414 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim with the coating aptamer,which induces aggregation of the edrone, cathinone, methcathinone, 3-trifluromethylphenyl pi- AuNPs (Figure 14). Using acalibration curve,the reported apta- perazine, 1-(3-trifluromethylphenyl)piperazine, 3,4-methylene- sensor is able to detect methamphetamine in aqueous solu- dioxypyrovalerona,MDA, MDMA, 2-ethylidene-1,5-dimethyl- tions at concentrations ranging from 2to10mm with alimit of 3,3-diphenylpyrrolidine, and meta-chlorophenylpiperazine) has detection of 0.82 mm.The aptasensor is also able to detect been evaluated, yet none of them show asignificant UV/Vis methamphetamine in urine with recoveries ranging from 96.0 response. to 107.0%and with high selectivity against potentialcompet- Willner andco-workersreport the design of an aptamer- ing alkaloids such as pethidine, triazolam, barbital, morphine, based nanomachine for the fluorogenic detection of cocaine.[51] ketamine, and diazepam. The nanomachine contains three regions: 1) acocaineaptamer;2)a nicking site for endonuclease (Nt.BbvCI); 3) atrans- ductionregion (Figure 16). Initially,the aptamer recognizes the

Figure 14. Schematic representation of MA colorimetric detectionconsisting in the preferential interaction of the aptamerwith the drug ratherthan with AuNPs.

Li et al. outline the construction of acolorimetric metham- Figure 16. Fluorogenicaptasensorfor the detection of cocaine:a)cocaine recognition, b) complementary strand synthesis, c) nicking with Nt.Bbv.Cl fol- phetamine probe consisting of aG-quadruplex–hemin DNA- lowedbypolymerization,and d) hybridization of the synthesized strand zyme molecular beacon, adrug aptamer,and acolorimetric with fluorescentconjugatedhairpin structure that avoids the FRET process substrate (Figure 15).[50] In the presence of methamphetamine, and allows carboxyfluorescein emission. the DNAzyme dissociates because of strong interactions between the drug and its aptamer,and this resultsinthe formation of aG-quadruplex–hemin complex with peroxidase activi- cocainemolecule, and then,inthe presence of polymerase ty that catalyzes the oxidation reactionof2,2’-azinobis(3-ethyl- and 2’-deoxynucleosidetriphosphates (dNPTs), the complemen- benzothiozoline)-6-sulfonic acid to yield alight-green deriva- tary strand is synthesized (see Figure 16 b). Then, region 2is tive with an absorbance band at l= 415 nm. The proposed nickedbyNt.BbvCI endonuclease, and this is followed by a aptamer-based probe shows excellent linearity with the meth- cyclic processbywhichthe complementary region is repeated- amphetamine concentration and alimit of detectionof0.5 nm. ly polymerized with the aid of the polymerase enzyme. The re- The selectivity with 15 other illicit drugs or metabolites (i.e. leased transduction region (see Figure 16 d) hybridizes with its ketamine, norketamine, morphine, methadone, cocaine, meph- complementary strand, which separatesthe two fluorophores (i.e. carboxyfluorescein and 5-carboxytetramethylrhodamine) and forbids the fluorescenceresonance energy transfer (FRET) process to which they were submitted. Finally,alimit of detec- 6 tion of 510À m for cocainecan be achieved. The same authors have also created an amplified aptamer sensorfor cocaine (Figure 17).[52] The developed system is formed by three domains:1)adomain that corresponds to the cocaineaptamer, whichisblockedbythe complementary strand;2)a domain containing anicking strand;3)the trans- ductionregion.Inpresence of cocaine, the system begins the replication and nicking processes;this releases domain 3, which is complementary to the strand containing afluorophore and aquencher (off state).Then, the DNAzyme cleaves the fluorophore/quencher to yield the fluorophore-labeled strand (separatedfrom the quencher) that provides the read- out signal for the analysisofcocaine (on state). The limit of de- Figure 15. Colorimetric methamphetamine aptasensor based on the perox- tection for cocaine is 0.1 mm.Finally,amethod for amplified idase activity of the formed G-quadruplex–hemin complex resultinginthe oxidation of 2,2’-azinobis(3-ethylbenzothiozoline)-6-sulfonic acid to alight- cocainedetection has been developed by the coupling of a green-colored derivative. ssDNA =single-strandedDNA. second replication/nicking system that includes an autono-

ChemistryOpen 2018, 7,401 –428 www.chemistryopen.org 415 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim to afluorescenceenhancement. The limit of detection for cocaine is 20 nm.Finally,the authors also evaluatethe systems in the presence of benzoylecgonine and ecgonine methyl ester. Neither of the tested interferents induce emission changes. Johnson and co-workers discuss the development of an aptameric cocaine sensorbased on FRET between quantum dots (QDs) and the fluorophore Cy5 (Figure 19).[55] For this purpose,

Figure 17. Schematic representation of the cocaine aptasensors. mous self-replication process. The limit of detection for the 18 4 amplified system is 110À m;this is roughly 10 -fold more sensitivethan other DNA-sensing platforms. Wang andco-workersvalidate the use of afluorescence label-free aptasensor for detecting cocaine in complex biofluids.[53] The system consists of an aptamer and two fluorophores,2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND) Figure 19. Cocaine aptasensoroperating scheme. In the presence of co- and SYBR Green I(SGI), whichserve as asignal reporter and a caine,the aptamer changes to ahairpin conformation and avoids the FRET process and suppresses Cy5 emission. CQDs = carbon quantum dots. built-in reference, respectively.Both fluorophores interact strongly with the aptamer,and as aconsequence, their fluorescence is quenched. However,ifcocaineisadded, an enhance- QDs coated with streptavidin are functionalized with the ment in the fluorescenceemission of ATMND is observed. This single-stranded DNA sequence 5’-TCA CAG ATGAGT-biotin-3’. emission enhancement can be ascribed to the fact that Then, the cocaineaptamer is sandwiched between this oligo- ATMND coordinates with the binding cavity of the aptamer mer and 5’-GTC TCC CGA GAT-Cy5-3’.Upon excitation of the and is displaced in the presence of cocaine. The authors report QDs at l=605 nm in an aqueous suspension of the sensor,the alimit of detection of 56 nm.The system has been tested in bi- typical emission band of the Cy5 fluorophore at l 675 nm ofluids (e.g. saliva, serum, and urine) with fine results. appearsdue to the presenceofaFRET process between the Huang and co-workers detail the design of anew system nanoparticles and the fluorophore. However, in the presence based on astrand-displacementpolymerization reaction by of cocaine, the random-coil aptamer changes to ahairpin con- using aptamer recognitionfor the detection of cocaine formation,which releases the Cy5 fluorophore brand with sub- (Figure 18).[54] The system consists of ahairpin probe labeled sequent suppression of the FRET process. As aconsequence, with Cy5 (acceptor fluorophore) on its 3’-end and containing the emissionofCy5 is progressivelyquenched depending on the cocaine aptamer sequence, aprimer derivatized on its 5’- the amountofcocaine added. The limit of detection for co- end with carboxy fluorescein(adonor fluorophore), and a caine is 0.5 mm,asdeterminedbyatitration profile. Besides, polymerase (which induces DNA sequence synthesis). If co- the authors modify the probe to obtain an emission enhance- caine is present, the hairpin probe recognizes the drug and ment as output.For this purpose, they include aCy5 quencher the polymerase induces DNA synthesis;this resultsinproximi- into the probe structure. The surfaceofthe QDs is functional- ty between the donor and acceptorfluorophore, which leads ized with 5’-Cy5-TCACAG ATGAGT-biotin-3’.The cocaine aptamer is again sandwiched between this oligomer and 5’-GTC TCC CGA GAT-Iowa Black RQ-3’ containing aquencher of Cy5. The presence of the quencher inhibits the FRET process between the QDs and Cy5. In the presence of cocaine, the quencher oligomer is released due to achange in the aptamer to the hairpin conformation and the FRET process occurswith asubsequentturn-on response. The limit of detection for cocaine is 0.5 mm,asdetermined by atitration profile. Abnous and co-workersmerge two different types of nanoparticles to construct an aptasensor for the fluorogenic detection of cocaine.[56] The authors first prepared silica nanoparticles coated with streptavidin and functionalized with 5 -fluo- Figure 18. Schematic representation of acocaine aptasensor based on the ’ actuationofpolymerase and interaction of two fluorophores in the presence rescein-ATT GAA GGA TTT ATCCTT GTC TCC CTATGC TTC AAT- of the drug. biotin-3’ (complementarysequence to that of the cocaine

ChemistryOpen 2018, 7,401 –428 www.chemistryopen.org 416 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim aptamer). On the other hand, they also prepare AuNPsfunc- 1.66 mm,respectively.The aptasystem is selective for cocaine tionalized with the 5’-CCA TAGGGA GAC AAG GATAAA TCC and benzoylecgonine in the presence of methamphetamine, 3- TTC AATGAA GTG GGT CTC CC-thiol-3’ sequence containing acetamidophenol, and codeine.Finally,upon analyzing syn- the cocaine aptamer.Upon mixing both nanoparticles, the thetic urine samples containing cocaineand benzoylecgonine complementary strandshybridize, and the emission of fluores- with the sensing material and comparing the results with cein is quenchedbecause the fluorophore is in close proximity those obtained by traditional HPLC methods, no significant dif- to the AuNPs. The addition of cocaine induces dehybridization ferences are found. of the double-stranded DNA fragment duetodrug–aptamer Martínez-MµÇez and co-workersimplement “molecular coordination.This process detaches both types of nanoparti- gates” onto nanoporous anodic alumina (NAA) to prepare a cles, and as aconsequence, fluorescein emission is restored. sensingmaterial for cocaine detection (Figure 22).[59] The NAA Besides, the limits of detection of cocaine in water (209 pm) and serum (293 pm)have been determined. Theauthors find that chloramphenicol, propranolol, and diazepam are unable to induce any emission enhancement, whereas morphine yields the same response as cocaine. Xiao et al. reportalabel-free aptamer–fluorophore ensemble to detectcocaine.[57] The authors observe that the typical aptamer used to recognize cocaine is able to complexthe fluorophore 2- amino-5,6,7-trimethyl-1,8-naphthyridine (44)(Figure 20). Due to com- plexation, fluorescenceof44 is quenched. However,inthe presence of cocaine, the complex be- Figure 20. Structure of 2- tween the aptamer and the fluoro- amino-5,6,7-trimethyl-1,8- naphthyridine (44). phore is disrupted, and an emission enhancement is observed. The authors note alimit of detection of 200 nm in water.Besides, Figure 22. Schematic representation for cocaine detection by using silica the sensing ensemble allows the selective detection of cocaine nanoparticles with afluorescentdye encapsulated and an aptamerasmolec- in the presence of its metabolite benzoylecgonine. In urine ular gates. and saliva, the aptasensorpresents limits of detection of 460 and 520 nm,respectively. support, obtainedaccording to reported procedures,[60] is Coskunol et al. create an aptamer-folding-based sensing loaded with rhodamine Band functionalized with (3-isocyana- device for cocainedetection.[58] Poly-l-lysine-coated microwells topropyl)triethoxysilane. Then, the amino-terminal DNA se- are used as inorganic supports andQDs functionalized with quenceNH2-(CH2)6-5’-AAA AAA CCC CCC-3’ is covalently linked carboxylic acidgroupsare grafted onto the surfacebyusing to the surface by the formation of ureabonds. In afinal step, N-hydroxysuccinimide (NHS)/1-ethyl-3-(3-dimethylaminopro- the pores are capped with the 5’-TTT TGGGGG GGG GAG ACA pyl)carbodiimide (EDC). On the other hand, AuNPs functional- AGG AAA ATCCTT CAA TGA AGT GGG TCT CCA GGG GGG TTT ized with the cocaine aptamer sequence 5’-NH2-C6-AGACAA T-3’ sequence, which specifically hybridizes with the short DNA GGA AAA TCC TTC AATGAA GTG GGT CG-C3-SH-3’ are anch- fragment attached to the externalsurfaceofNAA and contains ored to the QDs also by using NHS/EDC. In the absence of co- the cocaineaptamer.Ifthe sensing materialisimmersed in Tris caine, strong fluorescencefrom the QDs is observed, whereas solution (pH 7.5) without cocaine, negligible dye releaseisob- in the presence of the drug, this emission is quenched served.However,inthe presence of the drug, marked rhoda- (Figure 21). Thisemission quenching can be ascribed to coordi- mine Bdelivery is found (reaching 100%after 50 min). Using a nation of the aptamer to cocaine, which brings the QDs close calibration curve obtained upon adding increasing amounts of 7 to the AuNPs with subsequent fluorescence deactivation. The cocaine, alimit of detection of 5.010À m is calculated. Be- same response is observed for benzoylecgonine. The limits of sides, the material presentsgood selectivity for cocaine, and detection for cocaine and its metabolite are 0.138 nm and no response is observed by exposing it to other drugs such as morphine and heroine. Finally,the sensorhas been validated in saliva. The same research group has developed ahomolo- gous nanostructured system for the detectionofcocaineby surface-enhanced Ramanspectroscopy (SERS).[61] Landry and co-workersoutline the development of acocaine sensorbased on the assembly of fluorophore-labeled heterodi- [62] Figure 21. Schematic illustration for cocaine detection by using an aptamer meric aptamersinthe presence of the analyte. The authors and AuNPs. preparetwo oligonucleotides containing the cocaineaptamer

ChemistryOpen 2018, 7,401 –428 www.chemistryopen.org 417 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim sequence. One of the oligonucleotides is labeled with a5’-car- and quencher come into close proximity.Asaconsequence, boxyfluorescein fluorophore, whereas the other is functional- the emission of fluorescein is quenched. The aptamer allows ized with the well-known fluorescence quencher dabcyl at the the detection of cocaineinthe 10 mm to 2.5 mm range. Finally, 3’-position. In the presence of cocaine in abuffered solution the authors demonstrate that the sensor is highly selectivefor and with optimal oligonucleotide chain concentration, hetero- cocaine and is useful for the screening of cocaine hydrolases. dimerization of both aptameric sequences results in fluorescein In 2002, Landry and Stojanovic developed another cocaine emission quenching at l=518 nm (Figure23). This sensor has sensorthat consists in the displacementofacyanine dyefrom the hydrophobic pocket generated by anticocaine aptamer MNS-4.1 upon bindingwith the drug (Figure 25).[64] The au-

Figure 23. Conformational change in the aptamerinthe presence of cocaine and subsequent fluorescent emission quenching. alimit of detection of 1 mm and has excellent selectivity,asno quenching effectisobserved in the presence of the cocaine Figure 25. Displacementofcyanine dyefrom the hydrophobic pocket gen- metabolites benzoylecgonine and ecgonine methylester. eratedbythe anticocaine aptamerifthe cocaine drug is present. The same authors have also engineered anew method for the detection of cocaine in serum by using adeoxyribonucleo- tide-based aptamer (Figure 24).[63] The proposed method con- thors screen 35 different dyes to find one that displays both sists of an aptamer that is designed taking into account that significant attenuation of absorbance andachange in the ratio binding with cocaine induces amarked change in its secon- of two relative maxima. Thus, the authors select diethylthiotri- dary structure and, as aconsequence, in the emission of a carbocyanide dye and mix it with the aptamerMNS-4.1 in a graftedfluorophore. The authors construct adouble-labeled buffered media. Then, increasing concentrations(2to600 mm) aptamerwith 5’-fluorescein as the fluorophore and 3’-dabcyl of cocaineare added, and adecrease in the absorption at l= as the quencher.Inthe absence of cocaine, both the fluores- 760 nm is observed (whereas the absorption band at l= cein and quencher are far away,and excitation of the fluoro- 670 nm remains unaltered). Negligible changes in the visible phore results in its emission at l=518 nm. However,inthe spectra are found upon adding the cocaine metabolites ben- presenceofcocaine,the aptamer changes its conformation zoylecgonine and ecgoninemethyl ester (up to 2mm). Finally, upon coordination with the drug, and thus, the fluorophore marked color changes (from colorless to blue) are observed upon using higher dye and aptamer concentrations(40 and 20 mm,respectively)inthe presence of 500 mm cocaine. Recently,Liu and Zhao presented an aptasensor labeled on aspecific position at one Gbase with the tetramethylrhoda- mine (TMR) fluorophore that is able to detect cocaineinaque- ous environments.[65] In the absence of cocaine, the labeled aptamer adopts aconformationinwhich TMR interacts with the surrounding guaninenucleotides.Asaconsequence of these interactions, TMR suffersfrom restricted freedom, which is reflected in high fluorescenceanisotropy (unequal intensities along different axes of polarization).However, upon binding of cocaine to the aptasensor there is achange in the conformation andalso an alteration in the TMR–guanine interactions, which is reflectedbyadecrease in the fluorescenceanisotropy (Figure 26). The best fluorescenceanisotropy changes are obtained in the pH range of 7.0 to 8.5 at 108C. Moreover, the pre- Figure 24. Conformational change in the aptamerinthe presence of cocaine sentedaptasensor allows the direct detection of cocainewith and subsequent fluorescent emission quenching. alimit of detection of 5 mm and also enables the detection of

ChemistryOpen 2018, 7,401 –428 www.chemistryopen.org 418 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim done) and the complete hydrolysate form of cocaine (ecgonine) induce negligible responses. Besides, this aptasensor shows agood linear relationship in the presence of cocaine with alimit of detection of 0.1 mm. Li’s group outlines the development of asystem for the de- tectionofcocainebyemploying acocaineaptamerasarecog- nition site, graphene oxide (GO) as the fluorophore, and gold nanoparticles (AuNPs) as the quencher.[67] The cocaineaptamer is divided into two differentfragments,C1and C2, that are co- valentlyimmobilized onto GO and the AuNPs, respectively.In the presence of cocaine, aptamers C1 and C2 hybridize. This induces spatialproximity between the AuNPs and GO with subsequentemissionquenching of the latter (Figure 28). With this simple aptasensor,the limit of detection for cocaine is 0.1 mm.The system works well in biological fluids such as plasma and serum. Moreover,the authors find that 0.1 mm cocaine leads to fluorescence quenching, whereas no effect is Figure 26. Schematic representation of the conformational change in the co- observed in the case of pethidine and methadone, even at caine aptasensorthat diminishes fluorescence emission of the TMR fluorophore attached to aGbase in the presence of cocaine. concentrations as high as 50 mm (500 times the concentration of cocaine). the drug spiked in diluted serum and urine samples. The system has also been tested against theophylline,caffeine, bovine serum albumin,immunoglobulinG,and afew amino acids, including arginine, phenylalanine, and aspartic acid. No remarkable fluorescencechanges are observed, even at high concentrations. Dong and co-workers delineate the development of a simple and easy to prepare colorimetric aptasensor for cocaine detection in aqueous solutions.[66] Double-stranded (ds) DNA can act as an efficient template for the formation of highly Figure 28. Schematic representation for cocaine detection through aptasen- emissive coppernanoparticles (CuNPs) by using low concentra- sors based on graphene oxide and gold nanoparticles. tions of CuSO4.However, the ssDNA template does not support the formation of CuNPs. The design consists of the use of two ssDNAsequences, one of them the cocaine aptamer and AsimilarGO-based aptasensor for cocainedetection by the other its complementary counterpart. Both sequences hy- using cocaine-selective aptamersisalso reported by Zhang bridize to form dsDNA, which acts as atemplate for the forma- and co-workers(Figure 29).[68] tion of fluorescent CuNPs in the presence of CuSO4.However, Fan and co-workerspresent abioassay strategy to detect co- in the presence of cocaine, the dsDNA dehybridizes due to the caine on the basis of AuNPs and modified DNA aptamers.[69] In selectivebinding of the aptamer sequence with the drug, this design, the cocaine aptamer is divided into two pieces which precludes the formation of the emissive CuNPs that present random-coil-like conformations(ACA-1and ACA- (Figure 27). Other analgesic drugs (e.g. pethidine and metha-

Figure 29. Schematic representation for the cocaine-sensing mechanism. In the presence of cocaine, the aptameradopts ahairpin conformation and Figure 27. Schematic illustration for cocainedetection through aptamercon- joins acomplementary strand containingafluoresceindye, which is released formational change. by enzyme Exo-III.

ChemistryOpen 2018, 7,401 –428 www.chemistryopen.org 419 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim 2). In the absence of cocaine, ACA-1 and ACA-2 spontaneously bind with the AuNPs,which stabilizes the AuNPs and precludes them from undergoing aggregation.Consequently,the typical plasmon band centered at l= 520 nm and adark red color are observed. However,inthe presence of cocaine, the solution turns blue (absorption band at l=650 nm) due to the formation of atertiary structure between ACA-1, ACA-2, and the drug, and this results in aggregation of the AuNPs (Figure 30). Using this strategy,cocaineinthe low-micromolar range can be selectively detectedbythe naked eye. Thelimit of detec- Figure 32. Threefragments of the cocaine aptamerare assembled in the tion for cocaine is as low as 2 mm. presence of the drug,allowing AuNP aggregation and acolor change.

is increased from 0to200 mm.Aconcentration of 100 mm cocaine in humanurine or serum is readily detected by using this protocol. Zhang and co-workersdiscuss alabel-free fluorescence aptasensor for cocainedetection on the basis of the high affinity of aG-quadruplex for the ruthenium polypyridyl complex Ru- 2+ [(bpy)2(bqdppz)] (bpy= 2,2’-bipyridine, bqdppz = benzo[j]- quinoxalino[2,3-h]dipyrido[3,2-a:2’,3’-c]-phenazine).[72] The authors modify acocaine-selective aptamer by extending the originalsequence with guaninebases, which can form an in- termolecular G-quadruplex under certain experimental condi- Figure 30. Cocaine detection by using aptamers ACA-1 and ACA-2. In the presence of cocaine, aptamers ACA-1 and ACA-2 adopt acoil-like conformations. The authors note that the Ru complex can be successful- tion, allowing AuNP aggregation and acolor change. ly embedded into the G-quadruplex structure to increasethe fluorescence. However, in the presence of cocaine, the drug binds selectively to the aptamer,which resultsindissociation 2+ Liu and co-workersreport asimilaraptasensor for cocaine of the G-quadruplex and release of Ru[(bpy)2(bqdppz)] ac- detection by using aptamerspreviously immobilized on the companied by significant emission quenching (Figure 33). The surfaceofAuNPs (Figure 31).[70] fluorescenceintensity at l= 600 nm decreases upon increasing the concentration of cocainefrom 12 to 1300 nm,and the detection limit is as low as 5nm.This system is highly selective towardscocaineover other small drugs such as caffeine, morphine, andtheophylline. Furthermore, detection of cocainein human serum can be performed with recoveries in the 94.5– 103.6%range. An example using modified aptamers grafted onto an optical fiberbiosensing platform for the detection of cocaine is reported by He’s group.[73] In this work, acocaineaptamer is split into two fragments, and one of them is modified with afluoro- Figure 31. Cocaine aptamer anchored onto magnetic microbeadsurface. The aptamerchanges its conformation in the presence of cocaine, releasing ssDNA and allowing chemiluminescence detection of thedrug.HRP = horse- radish peroxidase, p-1P = p-iodophenol, CL = chemiluminescence.

Zou and co-workers also use the combination of AuNPs and atriple-fragment aptamer for the selective opticaldetection of cocaine.[71] In the absence of cocaine,the three aptamer fragments interact with the AuNPs,which prevents their aggregation, and as aconsequence, the solution remains red. In the presenceofcocaine, the three aptamerfragments bind with the drug, leaving the AuNPs to form aggregates (Figure32) with acolor change to blue. The system is selective to cocaine, and no response is found in the presence of ecogonine methyl Figure 33. The intramolecular complex formedbetween the G-quadruplex ester and benzoylecgonine. This aptasensor shows agradual 2 + and Ru[(bpy)2(bqdppz)] is disassembled in the presenceofcocaine, allow- color change from red to blue as the concentration of cocaine ing its detection by emission quenching.

ChemistryOpen 2018, 7,401 –428 www.chemistryopen.org 420 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim phore (Cy3) and the other with aquencher (BHQ = black hole quencher). In addition, the fluorophore-containing aptamer fragment is extended by 13 bases that can hybridize with an oligonucleotide previouslyimmobilizedonthe optical fiber platform.Inthe presence of cocaine, the two fragments quick- ly form athree-way junction aroundthe drug, whichpositions the fluorophore and the quencher in close proximity with subsequent emission quenching(Figure 34). At the same time, the tail of the three-way junction hybridizes with the oligonucleotide sequences immobilized on the opticalfibersurface, and changes in the fluorescenceare controlled by an evanescent wave. The aptasensor displays ahighly competitive limit of detection of 165 nm and asensing concentrationrange between 200 nm and 200 mm.The selectivity of this aptasensor has also Figure 35. Schematic representation for cocaine detection by using AuNPs been investigated against severalcompounds, including kana- and aptamers. mycin, amikacin, and ibuprofen. No signal changes are observed,even at high concentrations(100 mm)ofthese compounds.This system can be used to detect cocaineinhuman AuNPs free to aggregate. The authors find that even at low serum. concentrations of cocaine(10 mg) the carriersolutionpasses through the aptamer zonetothe location of the AuNPs and the color changes from pink-red to gray in 5min. In contrast, in the absence of cocaine, the moving solution arrives at the end of the strip and the AuNPs area remains red, which can be noted by the naked eye. The limit of detection for cocaine re- portedfor this system is 2.5 mg. Besides, ahigh selectivity over other illicit drugs such as ephedrine, codeine, ketamine, amphetamine, morphine, and methamphetamine can be achieved.Furthermore, metabolites of cocaine,benzoylecgonine, and ecgonine methyl ester are unable to induce acolor change. Liu and Lu present amethod for the detection of cocaine on the basis of the colorimetric change observed upon disas- sembly of AuNPs functionalized with different thiol-modified DNA sequences connected to acomplementary DNA sequence (linker) and to the corresponding analyte aptamer.[75] In the absence of cocaine, gold nanoparticles form aggregates of thou- sands of nanoparticles (blue color due to an absorbance band Figure 34. Schematic representation for cocaine detectionthroughmodified aptamers graftedonto an optical fiber biosensing platform. centered at l= 700 nm). However,inthe presence of this drug, these nanoparticles separate from each other because of a conformational change in the aptamer upon binding to co- Recently,Wang and co-workers have developed anew cocaine (Figure 36), and this changes the color of the solution to caine naked-eyedetectionmethod (Figure 35) by using paper red (absorbance at l= 522 nm). Characterization by transmis- microfluidic strips (PADs) coupled with gold nanoparticles and two cocaine aptamers(ACA-1 and ACA-2).[74] The PADs are fab- ricated by using acommercial waxprinter to form three cham- bers (with different diameters) on the surface of chromatographic paper Whatmann No.1. More in detail, the chromatographic strip has, at the upper part, achamber that contains 2nmol AuNPs. In the middle chamber,ACA-1 and ACA-2 (0.6 nmol each) are placed.Finally,there is achamber at the bottom of the strip for the samples. After adding the sample in the bottom chamber,the preparedtest strip is placed in a 1 carrier solution containing 2.8m MgCl2/50 mgmLÀ sucrose (300 mL).Itisknown that free aptamerscan bind to the surface of AuNPs, preventing them from aggregating even in the presence of concentrated salts (red color). However,inthe pres- Figure 36. Cocaine detection through aptamerconformation changes in the ence of cocaine, these aptamersbind to cocaine, leaving the presence of the drug.

ChemistryOpen 2018, 7,401 –428 www.chemistryopen.org 421 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim sion electron microscopy after/before cocaineaddiction con- firms the disaggregation of the nanoparticles. Cocainecan be quantified in the range of 50 (LOD) to 500 mm within a10s period. Liu et al. reported in 2016 aportable up-conversion nanoparticle(UCNP)-basedpaper device for cocainedetection.[76] The paper device is functionalized with polyethylenimine (PEI)- coated UCNPs and with the amine-modified ACA-1 fragment of the cocaine aptamer. Besides, another fragment of cocaine aptamer (ACA-2) bearing athiol moietyisgraftedonto the external surface of 16 nm AuNPs (Figure 37). In the presenceof Figure 38. Representation of cocaine aptasensorscombining GO, aptamers, and polymerase activity.

phine hydrochloride is observed. Finally,good linear correlation from 0.5 to 100 mm is achievedfor cocaine-spiked real urine samples (20-folddilution). Yagci and co-workersassess adouble fluorescencemicrowell assay that combinesQDs labeled with cocaine antibodies with apolyphenylene-based polymer(PPP) containing b-cyclodex- trins (CDs) derivatized with poly(ethylene glycol) (PEG) chains Figure 37. Luminescence quenching effect due to energy transfer between (Figure 39).[78] The PPP-CD-g-PEG polymer can be synthesized PEI-UCNPsand AuNPs if cocaine is present. cocaine, both DNA fragments (ACA-1and ACA-2) reassemble, and the luminescence of the UCNPsisquenched by the proximity of the AuNPs.This quenching effect can be attributedto the perfect match of the emission of the UCNPs and the ab- sorptionofthe AuNPs and can be observed by the naked eye for qualitative analysisorrecorded by asmartphonetoconvert data into RGB intensity for quantification. The limit of detection for cocaine is 10 nm,whereas in human saliva the mini- mum amount detected is 0.05 mm.Besides, recoveries in the Figure 39. Schematic representation of the double fluorescenceassay de- 89–103 %range can be measured for cocaine-spiked samples signed for the detection of cocaine. of human saliva. On the other hand, neither ecgoninemethyl ester nor benzoylecgonine induce any emission change. Yu and co-workersreport acocainedetection method based by aSuzukicoupling reaction. In this polymer, the hydropho- on graphene oxide (GO) and on polymerase-aided isothermal bic cavityofthe CD is envisioned as acocaine recognitionsite, circularstrand-displacement amplification (ICSDA) combined whereas the PEG chains provide water solubility.The surfaceof with the use of the dye SYBR Green I(SGI).[77] The authors pre- the microwell is modifiedbygrafting of the PPP-CD-g-PEG pare ahairpin probe consisting of two different DNA frag- polymer.Cocaine forms inclusion complexes with the CD in ments:1)acocaineaptamer;2)aregiondesigned to hybridize the polymericbackbone. Then, the QDs are added, and they with aprimer. In the absence of cocaine,the 3’-phosphorilated coordinate with cocaine through selectiveinteraction of the end of the hairpin probe oligonucleotide chain avoidspoly- drug with the antibodies that coat the nanoparticles. As acon- merase elongation, whereas in the presence of the drug aconsequence, the emission of the QDs centered at l=655 nm formationalchange exposes the 3’-single-stranded tail that hy- (lex = 400 nm) is enhanced. Besides, the intrinsic fluorescence bridizes with the primer and allows initiation of the poly- emissionofPPP-CD-g-PEG at l=410 nm (lex = 330 nm) is merase chain reaction. Thisprocess forms large amounts of quenched by the adsorbed QDs. By using this methodology,a dsDNA,with low affinity towardGO, which intercalates the SGI linear range from 10 to 100 nm of cocaine with alimit of de- dye, and its fluorescenceisobserved. In addition, in the ab- tectionof13.35 nm is obtained. Negligible response is ob- sence of cocaine, dsDNA is not formed, and the hairpin probe tained in the presence of methamphetamine, codeine, tetrahy- and SGI are adsorbed onto GO with subsequentquenching of drocannabinol, nicotine,cotinine, and benzoylecgonine. Finally, the emissionofthe fluorophore (Figure 38). The limit of detec- synthetic serum samples spiked with known cocaineamounts tion forcocaine is estimated to be 190 nm,and the linear have been analyzed, andthe resultsobtained have been com- range is from 0.2 to 100 mm.Weak fluorescence emission in pared to those obtained by using LC–MS/MS;good agreement the presence of adenosine, caffeine,theophylline, and mor- is observed.

ChemistryOpen 2018, 7,401 –428 www.chemistryopen.org 422 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim Hu et al. present anovel colorimetric cocaine biosensor tion of the G-quadruplex structurewith other planar molecules based on Au@Agcore–shell nanoparticles (Figure 40).[79] For such as ochratoxin A, thiazoleorange, protoporphyrin IX, crys- this purpose, Au@Ag nanoparticles are functionalized with a tal violet, thioflavin T, and adenosinetriphosphate has been ssDNA reporter (RP), andthen, magnetic beads (MBs) are pre- studied, and only aweak luminescence response is observed. pared and coatedwith acapturessDNA (CP). RP and CP are Du et al. report asimple and sensitive DNAzyme-based colorimetric sensorfor cocaine detection by using the 3,3,5,5-tet- [81] ramethylbenzidine sulfate (TMB)/H2O2 reaction system. For this purpose, the amino groups of functionalized magnetic nanoparticles (MNPs) are linked covalently to athiolated cocaine aptamer fragment (C2). On the other hand, another cocaine aptamer fragment is extended with aG-enriched strand AG4 (C1-AG4). In the presence of cocaine, the C2 layer on the MNPs hybridizes partlywith C1-AG4 to result in the folding of the aptamer into athree-way junctiononthe surfaceofthe MNPs to form aMNPs-C2–cocaine–C1-AG4complex. Using an externalmagnet, the nanoparticles can be separated from the solution (Figure 42). Then, the addition of hemin to the MNPs-

Figure 40. Schematic illustration of the mechanism for the detection system based on gold nanoparticles core–shell functionalized with aptamers.

designed to present complementarity with the cocaineaptamer.Inthe absence of cocaine and in asolutioncontaining its aptamer, RP-functionalized Au@Ag nanoparticlesand CP- coated MBs aggregate and can be removeduponapplication of an external magnetic field.However,ifcocaineispresent, its selectivecoordination with the aptamerinhibits nanoparticle aggregation, and the solution remainsyellow (absorbance of the nanoparticles centered at l=400 nm). With this protocol, the limit of detection for cocaineis0.5 nm with alinear response of 0.5 to 200 nm.The system does not respond to the presence of other illicit drugs such as ketamine,norketamine, Figure 42. Colorimetric assay for cocaine detection by using aptamers, morphine, cathinone, methacathinone, 3-trifluoromethyphenyl- hemin, and TMB. piperazine, and MDA. Asimilarbiosensor can be prepared for sensing methamphetamine with alimit of detection of 0.1 nm. Leung and co-workers describe alabel-free switch-on lumi- C2–cocaine–C1-AG4 complex promotes the formation of a [80] nescent sensor for the detection of cocaine. The authors DNAzyme, which catalyzes the TMB/H2O2 reaction system to design two DNA oligomers composed of afragment of the co- yield achange in the color of the solution from colorless to caine aptamer and afragment able to form G-quadruplex yellow.Inthe absence of cocaine, the separated MNPs do not structures. In the presence of both cocaineand an iridium include C1-AG4;thus, no background signal can be produced. complex, the two aptamer fragments came together to allow Using these aptasensors,drug concentrations of 100 nm can the formation of aG-quadruplexstructure, which gives alumi- be directly detected by the naked eye, with alimit of detection nescent response. The ability to form the G-quadruplex struc- of 50 nm.Besides, this protocol shows high selectivity for co- ture with different iridium complexes has been studied. The caine over ecgonine, pethidine, and methadone, which do not best response is observed with 45 induce any color changes. (Figure 41), as the size and shape Silver nanoclusters(AgNCs)can be used as an indicator for of the N^N ligand is crucial for cocainedetection in the presence of DNA templates utilizing constructionofthe G-quadruplex the advantages of the nicking endonuclease-assisted signaling structure. The limit of detection of amplification (NEASA)strategy(Figure 43).[82] The reported ap- the aptasensor with complex 45 tasensorconsists of three different DNA sequences, DNA1, for cocaineis30nm.Besides, the DNA2, and DNA3. Thefirst one is designed to contain three aptasensor is selective for cocaine domains: the cocaine aptamer,asequence that is complemen- in the presence of adenosine tri- tary to DNA2, and anicking endonuclease (Nb.BbvCI) enzyme Figure 41. Chemical structure phosphate, adenosine, warfarin, recognition sequence. On the other hand, DNA2 includes two of iridium complex 45. and suramin. Moreover,the forma- AgNCs template segments (coordinates with metallic AgNCs)

ChemistryOpen 2018, 7,401 –428 www.chemistryopen.org 423 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim Figure 43. Schematic representation of fluorescentAgnanocluster formation in the presence of cocaine. Figure 44. Scheme for cocaine detection by using aptamers grafted onto polydimethylsiloxane glass. In the presence of cocaine, the aptamer adopts and the recognition sequence and cleavage site for Nb.BbvCI. aspecific conformation that interactswith Hoechst 33342. This dye restores FITC emission. Finally,DNA3 is designed to be partly complementary to both ends of DNA2. It must be noted that the free AgNC template segments act as ascaffold for the synthesis of fluorescent probesfor stimulant drugs,namely:1)the formation of fluores- silver nanoclusters in the presence of Ag+ through its reduc- cent complexes;2)antibody-based systems; 3) the use of tion with NaBH4.Therefore, in the absence of cocaine, DNA3 nanoparticles;4)derivatization reactions;5)spectral fluores- and DNA2 undergo hybridization to form aclosed circular cent signatures;6)aptamer-based methods. structurethat keeps the AgNC template segment in arigid The formation of fluorescent complexes is one of the most stage that cannotcombine with Ag+ to form the fluorescent exploited strategies.Probes reported within this approach silver nanoclusters. However,the addition of cocainetothe so- change or enhancethe fluorescenceemission almost instanta- lution induces openingofthe structure due to the binding of neously and are easy to handle;someofthem have the ability cocainetoits specific aptamer; thus, hybridizationbetween to detect drugs in the vapor phase, which considerably in- the three DNAs to form aDNA1–DNA2–DNA3 complex is facili- creasestheir potential use as adetection method in realistic tated. Then, selective enzymatic cleavage of the DNA2–DNA3 settings.Besides,some of them can be fixed over surfaces. duplex by Nb.BbvCIresults in the release of free AgNC tem- However,some limits of detection are relativelyhigh, and plate segments, and this induces the synthesis of fluorescent some of the reported examples work in organic solvents or in silver nanoclusters. Theaptasensor shows high selectivity for mixed aqueous solutions. Several examples have high potential cocaineover other commoninterferences such as ecgonine for real applicability.For instance, sensing strips for the naked- and benzoylecgonine. The prepared aptasensor presents a eye detection of cocainebyusing antibodies and associated to linear range from 2nm to 50 mm cocainewith alimit of detec- amobile app for quantificationofthe drug have been de- tion as low as 2nm.Besides, the probe is able to detectco- scribed.Sensory systems based on porous nanoparticles caine in human serum with very high recovery values of loaded with dyes/fluorophores show signalamplification fea- around98to99.8%. tures and allow drug detection in the vapor phase. Chemical Zhou and co-workersoutline the construction of anew opti- derivatization of drugs with the aim to confer fluorescenceto cal aptasensor for cocaine detection.[83] The system is based on the final molecules is another widely followed strategy.The afluorescein isothiocyanate (FITC)-labeledaptamerthat produ- used reactions are fast and reliable, and some of the protocols ces aconformational change from apartial single-stranded described also include the use of mobile technology.Inspite DNA to adouble-stranded T-junction in the presence of co- of these positive facts, none of the described examples based caine (Figure 44). This change facilitates aminor-based energy on this approach can detect drugs in the vapor phase. Other transfer (MBET)between the FITC-labeled 3’-end of the apta- approaches involvethe use of spectral fluorescencesignature mer and Hoechst 33342 bound to the double-stranded T-junc- (SFS) measurements and artificial neuron networks for data tion, and this leads to fluorescenceemission at l=520 nm treatment. This approachpresentssome drawbacks, such as

(lex = 360 nm). The authors test the response of the aptasensor the need for learningand validation stepsand its complicated with benzoylecgonine and ecgonine methyl ester.None of the translation into an in-field system measurement.Moreover,a selected interfering chemicals induce any emission change. large percentage of the published examples dealing with the Furthermore, the limit of detection for cocaine is 0.2 mm. detection of well-known abuse drugs (such as cocaineand Taking into account the previously exposed works, several methamphetamine) are based on the use of aptamers (alone strategies have been used to develop chromo- and fluorogenic or graftedonto organic/inorganic supports). The selectivity

ChemistryOpen 2018, 7,401 –428 www.chemistryopen.org 424 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim achieved with the use of aptamersisvery remarkable. Besides, principally in the central and peripheralnervoussystem and severalaptamer-based detection systemsare coupled with the gastrointestinal tract. The concurrent use of opioids with DNA replication processes for signal amplification, allowing other depressant drugs such as benzodiazepines increases the very low limits of detectioninthe nanomolar range. However, rates of adverseevents and overdose. from atechnological point of view,these aptamer-based sen- Baheriand co-workers propose acolorimetricmethod for sors are usually more expensive and complex than others de- the selectivedetection of opioid-based drugs such as mor- scribed above. phine,codeine, oxycodone, noroxycodone, thebaine, tramadol, and methadone in aqueous media on the basis of color 4. Hallucinogenic Drugs changes produced by the aggregation of AuNPs in the presence of these analytes that act as molecular bridges between Hallucinogenic drugs include cannabis,psilocybin, lysergicacid them (Figure 45).[87] The sensingsystemscomprise four citrate- diethylamide (LSD), peyote, and dimethyltryptamine (DMT), among others.[84] Historically,some of these drugshave been used for religiousrituals. They cause the consumer to see and hear inexistent and unrealthings, which causes the consumers to feel out of control or disconnected from their body and en- vironment. Specifically, some of their most prominenteffects occur in the prefrontal cortex-anarea involved in mood, cogni- tion, and perception, as well as other important regions in reg- ulating arousal and physiological responses to stress and panic. These drugs can be found in some plants and mush- rooms or can be man made. LSD, psilocybin, and DMT produce their effects through interaction with serotonin (5-HT)receptors. Others, the most, affect the N-methyl-d-aspartate receptor (NDMA-receptor), the k-opioid receptor (KOR),and the neuro- transmitter acetylcholine. Figure 45. Schematic representation for the colorimetric detection of opioid Badout and Andre takeadvantage of the intrinsic fluores- drugsthroughAuNPcolor changes produced by nanoparticleaggregation. cent properties of lisergamide (commonly known as LSD) to develop alow-costing and portable device for its detection.[85] LSD presents afluorescenceband in the l=400–530 nm coatedAuNPs with different particlesizes (diameters of 13, 23, range, depending on the solvent and pH. The apparatus is 32, and 43 nm);thesedifferent AuNPspresentdifferent aggre- constructed to allow the naked-eye detection of LSD by com- gation behaviors with selected drugs, whichresultsinafinger- paring the emission of an LSD solution with its corresponding print pattern in the spectra that can be translated by algorith- blank solution upon excitation with amercury lamp by using mic pattern recognition [principal component analysis (PCA) both neutral and 1m NaOH alkali solution as the solvent. If and hierarchical cluster analysis(HCA)] in an array for the clas- LSD solutionsinneutral water are submitted to the mercury sification,identification,and quantification of these analytes. lamp, acharacteristic blue emission is observed. This occurs For each opioid and particlesize, the difference in absorbance also with other hallucinogens such mescaline,2,5-dimethoxy-4- (DA)with the blank is used as aresponse, and the effects of methyl amphetamine (DOM), psilocin, psilocybin, bufotenin, critical parameters, such as pH, ionic strength, time of interac- ibogaine, and phencyclidine. However,ifthey are dissolved in tion, and analyte concentration, are evaluated. After optimiza- 1m NaOH aqueous solution, only with LSD is achange in fluo- tion, discriminationofseven opioids is possible, with limits of 1 rescence emission to green observed. The limit of detection detection of about 5 mmol LÀ (except for noroxycodone), and for LSD samples dissolved in neutral water is 50 ng, whereas experiments with severalpotentialinterfering substances show that for LSD samples in alkali media increases to 260 ng. negligible responses. Finally,color-difference maps are ob- There is only one example reported in this section, anditis tained before/after exposure for each opioid by using repre- anticipated that new chromo- and fluorogenic probes forthe sentativeRGB wavelengths (l=730, 685, and 600 nm), and detection of hallucinogenic drugs will be developed in the they show good accuracy andreproducibility. near future. Anzenbacher and co-workerspresentanew approachfor sensing heroine, morphine, oxycodone, and their main metab- 5. Opiods olites on the basis of the interaction of these molecules with the flexible cavity of fluorescent acyclic cucurbituril (aCBs) de- Opioidsdrugs such as heroin,codeine, morphine, fentanyl, hy- rivatives (Figure 46).[88] ThreeaCBs functionalized with naphtha- drocodone, oxycodone, buprenorphine, and methadoneact lenes (with different degrees of steric hindrance) in the termi- through opioid receptors in the spinal cord and brain.[86] These nal walls are employed in the sensing protocol. The steric hin- compounds have been used forcenturies to treat pain, cough, drance defines the diameterofthe recognitioncavity in the and diarrhea. Nowadays, medical use is to treat acute pain. aCBs. The gueststested (i.e. heroine, morphine, oxycodone) Opioidsact by binding to opioid receptors, which are found share acommon skeleton with small structuraldifferences.

ChemistryOpen 2018, 7,401 –428 www.chemistryopen.org 425 2018 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim 6. Conclusions

The problematicuse of illicit drugs (both plant-based and synthetic derivatives) associated with their harmfulnesstohealth and mortality has urged the scientific community to develop reliable methodologies to detect and quantify these lethal substances.Wehave herein reviewed probesfor the chromo- and fluorogenic detection of illicitdrugs. These probes(which can be simple molecules of more sophisticated systems involving biomolecules or hybrid organic–inorganic materials) provide excellent advantages over traditional analytical techniques, such as their chemical simplicity,ease of use, rapid response suitable for real-time on-site detection, and easy detection (in Figure 46. Fluorescence emission changes observed if morphine, heroine, certaincases to the naked eye). However,inspite of these re- and oxycodoneinteract with their corresponding cucurbituril (aCBs) markable features, the development of chromo- and fluoro- derivatives. genic probes for the selective and sensitive detection of some illicit drugs is still poorly explored. In fact, more than half of the described chromo- and fluorogenic probeshave been de- Host–guest recognition resultsinfluorescence modification of signedfor the recognition/quantification of common plant- the terminal naphthalene emission, and this induces a“turn- based drugs (such as cocaine), yet relatively few works de- on” response for morphine and heroine as aconsequence of scribe the preparation of sensing systems for the detection of the increased rigidity.However,inthe case of oxycodone, and new synthetic illicit drugs.Inaddition, most chromo- and fluo- due to the quencher effect of cyclohexanone on naphthalenes, rogenic detection systems are designed for the detection of il- the fluorescenceemission is diminished. Molecular dynamics licit drugs in solution, and there are very few examples for calculations confirm that the naphthalene walls and the resul- their detection in the vapor phase. Moreover,withthe increas- tant cavity as well as guest uptake are decisive factors for fluo- ingly ubiquitous internet and other methods of communica- rescence detection. The inducedresponses of these organic tion, the presence of illicit drugs has expanded in recent years. sensors with the studied opiates differ in the amplitude and As these synthetic drugs have not been approved for con- saturation of the fluorescencechange, in that the sensors all sumption or medical use, their long-term effects are potentially present different binding isotherms, and this suggeststhat severefor humans.Inthis scenario, the developmentofnew, they act as cross-reactive sensors. Furthermore, these organic rapid,inexpensive, and easy-to-use new probesfor these new probesshow different behaviors for the studied parentdrugs substances is of importance,and new advances in this fieldare (i.e. morphine, heroin,and oxycodone) and their metabolites expected in the near future. (i.e. normorphine, 6-monoacetylmorphine, morphine-3-glucuronide, morphine-6-glucuronide, noroxycodone, and oxymor- phone), which makes it possible to determine both in aspecific, qualitative, and quantitative manner.Aqualitative assayhas Acknowledgements been performedinahigh-throughput fashion by using linear discriminant analysis(LDA) for pattern recognition, and it We thank the Spanish Government [projects MAT2015-64139-C4- shows three clusters consistingofthe parentdrugs and their 1-R and AGL2015-70235-C2-2-R (MINECO/FEDER)] and the Gener- corresponding metabolites. Ageneral calibration makes it pos- alitat Valenciana (project PROMETEOII/2014/047) for support. sible to detect the components of the clusters simultaneously S.E.S thanksthe Ministerio de Economia yCompetitividad for his with asingle fluorescent reading. The limits of detection in Juan de la Cierva contract. B.L.T and E.G. thank the Spanish Gov- aqueous media are 0.07 ppb for morphine and 82.5 and ernment for their predoctoral grants.L.P.also thanks the Univer- 2.78 ppb for the morphine metabolites morphine-3-glucuro- sitat Politcnica de Valncia for his predoctoral grant. nide (M3G) and normorphine(Nmor), respectively.Inhuman urine samples spiked with selected components, the limits of Conflict of Interest detection are 27.5, 972, and 71.3 ppb formorphine, M3G, and Nmor,respectively. The authors declare no conflict of interest. Only afew examples of chromo-and fluorogenic sensors for the detection of individual opioidshavebeen described by now,and new examples in this area are expected to be report- Keywords: chromophores · drugs · emission · fluorescent ed in the future. probes · sensors

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