key: cord-0920806-9p3a5qyg authors: Mao, Kang; Zhang, Hua; Pan, Yuwei; Zhang, Kuankuan; Cao, Haorui; Li, Xiqing; Yang, Zhugen title: Nanomaterial-based Aptamer Sensors for Analysis of Illicit Drugs and Evaluation of Drugs Consumption for Wastewater-Based Epidemiology date: 2020-07-06 journal: Trends Analyt Chem DOI: 10.1016/j.trac.2020.115975 sha: 12a7e0fbbc0531a19ee9c93aca0af3c17e21f63d doc_id: 920806 cord_uid: 9p3a5qyg The abuse of illicit drugs usually associated with dramatic crimes may cause significant problems for the whole society. Wastewater-based epidemiology (WBE) has been demonstrated to be a novel and cost-effective way to evaluate the abuse of illicit drugs at the community level, and has been used as a routine method for monitoring and played a significant role for combating the crimes in some countries, e.g. China. The method can also provide temporal and spatial variation of drugs of abuse. The detection methods mainly remain on the conventional liquid chromatography coupled with mass spectrometry, which is extremely sensitive and selective, however needs advanced facility and well-trained personals, thus limit it in the lab. As an alternative, sensors have emerged to be a powerful analytical tool for a wide spectrum of analytes, in particular aptamer sensors (aptasensors) have attracted increasing attention and could act as an efficient tool in this field due to the excellent characteristics of selectivity, sensitivity, low cost, miniaturization, easy-to-use, and automation. In this review, we will briefly introduce the context, specific assessment process and applications of WBE and the recent progress of illicit drug aptasensors, in particular focusing on optical and electrochemical sensors. We then highlight several recent aptasensors for illicit drugs in new technology integration and discuss the feasibility of these aptasensor for WBE. We will summarize the challenges and propose our insights and opportunity on aptasensor for WBE to evaluate community-wide drug use trends and public health. The abuse of illicit drugs usually associated with dramatic crimes may cause significant 42 problems for the whole society. Wastewater-based epidemiology (WBE) has been demonstrated to 43 be a novel and cost-effective way to evaluate the abuse of illicit drugs at the community level, and 44 has been used as a routine method for monitoring and played a significant role for combating the 45 crimes in some countries, e.g. China. The method can also provide temporal and spatial variation 46 of drugs of abuse. The detection methods mainly remain on the conventional liquid 47 chromatography coupled with mass spectrometry, which is extremely sensitive and selective, 48 however needs advanced facility and well-trained personals, thus limit it in the lab. As an 49 alternative, sensors have emerged to be a powerful analytical tool for a wide spectrum of analytes, 50 in particular aptamer sensors (aptasensors) have attracted increasing attention and could act as an 51 efficient tool in this field due to the excellent characteristics of selectivity, sensitivity, low cost, 52 miniaturization, easy-to-use, and automation. In this review, we will briefly introduce the context, 53 specific assessment process and applications of WBE and the recent progress of illicit drug 54 aptasensors, in particular focusing on optical and electrochemical sensors. We then highlight 55 The abuse of illicit drug is becoming increasingly serious worldwide [1] [2] [3] . For drug addicts biomarkers [13, 14] and even bacteria [29, 30] . The detection of a variety of illicit drugs in 107 different matrices with aptasensors has been reported. 108 In this review, we first demonstrate the process of WBE that can be used in the evaluation of 109 temporal patterns of drug use, showing its potential to provide complementary information with 151 standard technology. It is worth mentioning that our research group is the earliest one in China to 152 have been evaluating of illicit drug use using WBE. In the past few years, our group has 153 performed many works on monitoring of illicit drugs and made a series of contribution for WBE 154 [11, [47] [48] [49] . One of the most important works is that we used this technique to help police track 155 down and arrest drug manufacturers [3] . 156 The commonly used advanced analytical technique for WBE is high-performance liquid 157 chromatography-mass spectrometry because it can identify and quantify the illicit drug targets at 158 very low concentrations in a particular complex sample (raw wastewater). A mass spectrometry is 159 the most widely used technique for illicit drug quantification in WBE because it can quantify the 160 illicit drugs or their metabolic residues in wastewater utilizing an internal standard, such as a 161 deuterated analogue of the target [50] . High-resolution mass spectrometry as a powerful tool has 162 also many applications for WBE, including quantification of drugs at ultralow concentrations, 163 screening of a large number of non-target compounds, identification of new metabolites and 164 degradation/transformation chemicals, investigation of illicit drugs and elucidation of unknowns 165 in wastewater [51] . Although the mass spectrometry is a gold-standard technique for the 166 quantification of target drugs in wastewater due to its excellent stability, selectivity and sensitivity, 167 novel methods with promoted performances will be beneficial to further extend the application of 168 WBE. However, mass spectrometry involves troublesome sample collection and purification, 169 expensive testing, and necessary operation by professional staff become a burden for the 170 evaluation process, especially for the testing in the field. Therefore, there is an urgent need to 171 develop a rapid method (for example, biosensors) to provide real-time monitoring of wastewater profiles [52] . 173 Aptasensors have emerged as an innovative and powerful technique and they have been widely 175 employed in biomedical diagnosis, drug screening, food safety, forensic analysis, and 176 environmental monitoring [53] [54] [55] . Increasing efforts have been made to construct aptasensors for 177 illicit drug analysis, due to its low-cost and excellent stability [56] . The signals from the binding 178 between the aptamer and illicit drug include a colorimetric signal [57] , an electrochemical signal 179 Many optical aptasensors have been developed to detect illicit drugs so far. In the literature, the 186 following illicit drug aptasensors have been classified into colorimetric, fluorescence, SERS, 187 luminescence, and other optical platforms (listed in Tab. 1). In this section, we mainly discuss 188 colorimetric, fluorescence, and SERS aptasensors, which are the most widely used optical sensing 189 platforms in conjunction with aptamers. Apart from a nanomaterial-based colour aptasensor for illicit drug detection, DNAzyme has 233 been used as an example of an allosteric aptamer that has been applied in biosensor construction 234 [76]. In these platforms, DNAzyme could act as either signal readouts or recognition elements. 235 The G-quadruplex structure was composed of repetitive G-rich sections, which could form a 236 G-quadruplex/hemin complex under the existence of hemin that exhibited peroxidase behaviour similar to horseradish peroxidase. Herein, horseradish peroxidase mimicking DNAzymes could be 238 used to fabricate colorimetric aptasensors. In fact, many novel colorimetric illicit drug aptasensors 239 were constructed by using a combined aptamer and peroxidase-mimicking DNAzyme [19, 61, 65, 240 76] . We also designed a simple and unlabelled aptasensor for the detection of methamphetamine 241 through a G-quadruplex-hemin DNAzyme [61] . 242 Similar to a colorimetric aptasensor, a fluorescence aptasensor (listed in Tab. 1) has also been 244 successfully employed in illicit drug analysis. Usually, fluorescence aptasensors require a 245 fluorophore and a fluorescent quencher. The former is usually bound to one terminus of an 246 aptamer, which mainly includes fluorescent dyes, gold/silver nanoclusters (Au/AgNCs), and 247 quantum dots (QDs); the quencher is usually bound to the other terminus and mainly included a 248 fluorescence quenching organic fluorescent molecule (dabcy and BHQ), and nanomaterial 249 (Au/AgNPs and graphene oxide (GO)). The initial aptamer conformation could make the 250 fluorophore and quencher close to each other, leading to low or even no fluorescence. After adding 251 an illicit drug, the conformational change of the aptamer could separate the fluorophore and 252 quencher, leading to high fluorescence intensity (turned on) [77] . Alternatively, the quencher could 253 modify a complementary nucleic acid sequence that gets unbound from the aptamer upon illicit 254 drug binding. When the fluorophore and quencher were spatially apart, the intensity of 255 fluorescence was high. Once the aptamer bound to the target, the conformational change of the 256 aptamer caused the fluorophore and quencher to be close to each other, leading to fluorescence 257 loss (turned off). The fluorescence intensity could be relative to the concentration of the illicit 258 drug target depending on whether the fluorophore and quencher contained nanomaterials or not; this is further discussed in four categories. 260 The first category was that the fluorescent and quenching agents did not contain nanomaterials. 261 For example, Roncancio et al. [70] constructed a simple fluorescence aptasensor for one-step 262 cocaine detection with a minimal amount of sample. They discovered that cocaine aptamer 263 could also bind the fluorescent molecule 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND) 264 and thereby quench its fluorescence. They changed aptamer and engineered a novel aptamer II 265 that exhibited a high affinity for both ligands, which reduced the background signal, thus gaining 266 an increased target signal. Using this aptamer, they successfully constructed a novel detection 267 method that was dependent on the cocaine-mediated displacement of ATMND from aptamer II 268 due to the competitive binding mechanism (as shown in Fig. 2A ). This competitive binding 269 method overcame the limitation of target sensitivity compared with that of the traditional The second category was that the fluorescent agents used nanomaterials, while the quenchers 280 did not. The common fluorescent nanomaterials were Au/AgNCs and QDs [81, 82] , which were The third category was that the fluorescence quenchers used nanomaterials, while the 288 fluorescent agents did not. As a fluorescence quencher, nanomaterials, especially two-dimensional 289 could be adsorbed on the surface of GO to detect cocaine because poly-cytosine DNA could be strongly adsorbed on many common nanomaterials. Moreover, cocaine could be adsorbed on the 300 surface of bare GO to limit further improvements on the sensitivity. Tween 20-protected GO was 301 therefore used to prevent the cocaine from nonspecific binding because Tween 20, as a nonionic 302 surfactant, may strongly interact with GO through its hydrocarbon lipophilic group. Apart from 303 the 2D nanomaterial, other nanomaterials, such as Au/Ag nanomaterials (seen in Fig. 2C ), have 304 also been applied in illicit drug detection [79] . The fourth category was when both the fluorescent and quenching agents used nanomaterials. Then, the AuNP aptamer was bound to the QDs. The introduction of cocaine or benzoylecgonine 323 led to a conformational change of the aptamer, which brought the AuNPs and QDs close. Hence, 324 the extent of QD fluorescence quenching was observed when the target drug was added. aptamer that selectively coordinated with cocaine. In this case, the cocaine resulted in the release 339 of crystal violet, and the delivered crystal violet was detected by SERS upon cocaine adsorption 340 on the AuNTs, which displayed an efficient electromagnetic field enhancement and high colloidal 341 stability. We also designed a structure of Au@Ag core-shell nanoparticles for better sensitivity and 342 developed a new biosensor based on Au@Ag for the detection of methamphetamine with SERS [60]. 344 Electrochemical analytical techniques, as an essential branch of modern analytical chemistry, 346 play a key role in illicit drug analysis. Electrochemical aptasensors hold the advantage of 347 simplicity, rapid detection, high sensitivity, low cost and compatibility with use in different 348 settings and therefore are also widely applied in illicit drug determination [58, 87] To improve the analytical performance, electrodes were usually decorated with suitable 356 materials in the development of electrochemical aptasensors. In the last few years, various 357 modification protocols have been explored, ranging from biological receptors such as DNA or 358 proteins to synthetic metal ionophores. Recent reports have suggested that nanoparticle-modified electrodes can be very effective in electrochemical sensors. Therefore, we discuss this in relation 360 to electrodes modified by different materials, mainly including nanobiomolecule-modified 361 electrodes, noble metal nanoparticle-modified electrodes, carbon nanoparticle-modified electrodes, 362 and some other nanoparticle-modified electrodes. 363 The most common electrochemical aptasensor was to directly modify the corresponding An electrode surface can be modified with nanomaterials, such as AuNPs, which seems to add 391 complexity to the synthesis but improves the analytical performance of the aptasensors and (AuNPs/graphite electrode), which endowed the electrode with a larger surface area for 395 modification with thiolated single-stranded DNA (ssDNA). This could become a biocathode in a 396 microbial fuel cell system (seen in Fig. 3B ). This self-powered sensor was dependent on the 397 microbial fuel cell system and acted as an efficient tool for ketamine analysis in a complex matrix. Under optimal experimental conditions, the difference between the power densities of 399 the ssDNA-modified electrode with and without accumulated ketamine served as the 400 determination signal with a limit of detection of 0.54 nM. 401 In contrast to AuNPs, Su et al. [102] used gold nanoclusters (AuNCs) as a modification and 402 developed another electrochemical sensor for cocaine. Two-dimensional zirconium-based 403 metal-organic framework nanosheets embedded with AuNCs (AuNCs@MOF) were synthesized 404 by a one-pot method under moderate conditions. The optimized AuNCs@MOF nanosheets not 405 only had good electrochemical activity, high specific surface area, and physicochemical stability 406 but also held a strong bio-affinity towards biomolecules. Therefore, an adequate aptamer could be 407 decorated onto the substrate modified by AuNCs@MOF nanosheets, further forming a biosensing 408 platform that successfully monitored cocaine based on the specific binding between the aptamer 409 and cocaine. This novel method has a great opportunity for simple and convenient cocaine 410 analysis due to its simple operation and its merits of excellent stability, repeatability, and 411 Another noble metal nanomaterial, platinum nanoparticles, was usually applied to modify the 413 electrode for aptasensor construction [93]. Roushani and Shahdost-fard designed a voltammetric 414 and impedimetric determination method for cocaine by using a platinum nanoparticle-modified 415 glassy carbon electrode and rutin as a redox probe. As shown in Fig. 3C , the electrode was 416 cocaine-responsive by decorating with platinum nanoparticles and aptamers. The platinum 417 nanoparticles on the electrode could accelerate the electron transfer kinetics of the rutin reduction 418 and enhance sensitivity. Graphene oxide (GO), a typical carbon nanomaterial, was also often used as a modified triggered autonomous isothermal replication/nicking processes and the displacement of a acid as a readout signal for the analysis (seen in Fig. 4B ). The target-triggered isothermal 517 autonomous replication/nicking process on the template could form the Mg 2+ -dependent 518 DNAzyme tethered to a free strand consisting of the target sequence. This activated additional 519 template units for the nucleic acid self-replication process, which could then determine the target 520 nucleic acid sequence. According to the above concept, this amplification detection method for Fig. 4C , an aptamer was coated on AuNP-modified magnetic beads hybridized with a short 532 DNA sequence. When cocaine was added, the short DNA sequence was displaced from the 533 aptamer due to the specific binding between the cocaine and aptamer. Next, the short DNA glucoamylase-trapped aptamer-cross-linked hydrogel with a glucometer for portable detection of 570 cocaine. As shown in Fig. 5B , in the presence of cocaine, the hydrogel was dissolved and the 571 glucoamylase was released, which catalysed the hydrolysis of amylose to produce more glucose 572 for a quantitative readout using a glucometer. As shown in Fig. 5C, Mao et al. [120] also utilized a 573 DNA crosslinked hydrogel as a target-responsive unit and gold nanorods as a multicolour signal 574 readout circuit for visual detection of cocaine. The colour variation of the gold nanorod solution was correlated with the cocaine concentration. These simple platforms will have a wide range of 576 applications for visual determination of different illicit drugs because the aptamer cross linked 577 hydrogel can be targeted to any ligand depending on the corresponding aptamer. In addition to increased efforts being devoted to illicit drug aptasensor construction, many 591 attempts have been devoted to integrating existing analytical systems or miniaturized commercial 592 equipment with illicit drug aptasensors into field-portable devices. Many miniaturized or portable 593 devices, such as microfluidic chips, paper devices, and commercial devices, have been employed The characteristic of this microfluidic platform was that multiple targets could be recognized and 614 detected depending on the readout signal on a certain electrode by only one electrochemical probe. 615 [Ru(NH 3 ) 6 ] 3+ , as an electrochemical probe that produced a chronocoulometric signal in this 616 method, could quantitatively bind to surface-confined nucleic acids with electrostatic interactions; 617 additionally, AuNPs were applied for sensitivity improvement by amplifying the detection signal. 618 This Au-Ag dual metal electrochemical chip detector integrated with a microfluidic 619 electrochemical illicit drug aptasensors has the advantages of simplicity, sensitivity, and selectivity 620 and there is great potential for it to be further applied in multiplex illicit drug analysis with high 621 integration, high throughput, and high automation. 622 Paper materials hold the merits of abundance, low cost, simple fabrication, and portability and 624 act as an ideal supporting material for developing sensor devices with a wide range of applications, 625 particularly in the field of point of care diagnostics. Hashemian et al. developed a paper device for 626 codeine detection through an aptamer immobilized on cellulose paper for thin-film 627 microextraction of codeine from practical samples followed by electrospray ionization for ion 628 mobility spectrometry [129] . Immobilization was based on the covalent linking of an 629 amino-modified codeine aptamer to the aldehyde groups of the oxidized cellulose paper. The 630 comparison of the results between their method and high-performance liquid chromatography 631 validated the accuracy of this paper device, which could become a choice for simple and rapid 632 codeine analysis in a complex matrix. Fig. 6B also describes an aptamer based paper 633 microfluidic device coupled with AuNPs for a colorimetric determination of seized cocaine 634 samples [124] . The mechanism was a paper strip that produced a colour change due to the aggregation of AuNPs that was induced by salt in the presence of the drug. These methods based 636 on paper materials not only are easy to operate and have a rapid response without expensive 637 instrumentation but also all of the paper materials utilized in the device are safe and 638 environmentally friendly, which is a huge benefit in analysing illicit drugs in different samples. 639 Aptasensors are often integrated with some commercial miniaturized devices for on-site 641 analysis. Portable devices with characteristics of rapid detection, easy-to-use, and low cost have 642 been employed over a wide range of applications in daily life. A glucometer, as the most 643 successful portable device, is widely used due to its portable size, simple manipulation, low cost 644 and quantitative analysis. However, typically glucometers can only directly monitor blood glucose. 645 To use glucometers for the analysis of more targets, some experts successfully combined 646 glucometers with aptamers to analyse a variety of non-glucose targets, including illicit drug 647 analysis [119]. Fig. 6C shows a novel determination method using personal glucose meters and 648 aptamers to quantify cocaine [125] . However, only a few quantized portable devices have been 649 successful in commercialization, and many more commercial portable devices should be explored 650 for illicit drug analysis in the future. 651 Drug abuse is a global concern, and effective evaluation and control are urgently needed to fight 653 against drug abuse. WBE is a low-cost but effective approach to evaluate drug consumption in a 654 certain area in comparison with traditional population surveys. As an alternative and powerful 655 analytical tool, community sewage sensors have been recently proposed to monitor biomarkers in 656 sewage for evaluation of drug consumption and prediction of infectious disease in our group [15, 37] . Compared with the conventional method, the developed sensors have minimal sample 658 pretreatment, fast response time, and potential for on-site monitoring. A wide range of community 659 sewage sensors have been recently reported for various target analyses in wastewater, such as 660 illicit drugs [37], prevalence of cancer-associated prostate-specific antigens [37] [130] , and 661 population biomarkers [15, 131] . In particular, our group has developed a lateral flow device 662 through a combination of loop-mediated isothermal amplification methods as a portable tool to 663 determinate human nucleic-disease markers in sewage, which has allowed for on-site monitoring 664 of genetic pollution biomarkers for public health assessments [132, 133] . Aiming at the epidemic 665 situation of new coronavirus that is raging all over the world, we also put forward an important 666 innovative view that paper-based biosensor depending on WBE is used to screen potential new 667 coronavirus patients [39] . This technology overcomes the traditional detection method's inability 668 to quickly and large-scale screen potential new coronavirus patients, and the inability to detect 669 through PCR in areas with limited resources, which holds great significance for the timely 670 diagnosis and early warning of new coronavirus. These works demonstrate that community 671 sewage sensors hold great promise in playing a significant role in wastewater analysis, particularly 672 for field testing, to rapidly inform drug consumption patterns and epidemiology studies at the 673 community level. 674 As mentioned, aptasensors have attracted increasing interests in a variety of illicit drug 675 analyses, such as methamphetamine, cocaine, ketamine, and codeine [18, 60, 66, 70, 95] . 676 Aptasensor signals from the binding between an aptamer and drug can be output as colorimetric reporter probe nucleic acids for cocaine and methamphetamine, respectively. The magnetic beads 708 were conjugated with two capture probe nucleic acids that separately targeted cocaine and 709 methamphetamine. The respective reporter probes and capture probes were hybridized with each 710 illicit drug-binding aptamer and then formed a sandwich structure, which could be removed by an 711 external magnetic field. However, in the presence of illicit drugs, the special structure would be 712 disassembled due to the high affinity of the aptamers with the illicit drugs, which caused a colour 713 change of the supernatant. The biosensor showed the capability of duplex detection of 714 methamphetamine and cocaine after a non-negative matrix factorization algorithm process. 715 We demonstrated that the wastewater analysis ability of aptasensors can act as a potential 716 monitoring approach for the assessment of drug consumption in a certain area by WBE. Thus, the 717 use of biosensors instead of advanced analytical method such as LC-MS/MS has profound potential 718 in WBE. However, there are a few challenges in addition to the biosensor development itself. These include the uncertainty associated with the continuous monitoring, instead of using daily composite 720 samples or even grab samples. The uncertainty will increase because of more frequent data 721 monitoring. What's worse, low analyte concentrations in combination with the complexity and 722 unknown composition of the wastewater matrix might hamper not only the sensitive and accurate 723 quantification but also a sound identification. For the biosensor itself, how it can cope with the 724 complex components in wastewater is a big issue. The analytical tools for drug use trends utilize 725 mass spectrometry-based techniques due to their robustness, sensitivity and selectivity. Various 726 illicit drugs or their metabolites presented in wastewater can be quantified using mass spectrometry 727 system (Tab S1). However, troublesome sample purification, costly measurements and the 728 requirement for well-trained personnel may burden the assessment process. To this end, there is a 729 great need for novel analytical tools to perform a rapid and on-site analysis of wastewater with 730 minimal sample processing by un-skilled personnel. Sensors may have a shortage of sensitivity for 731 the lower concentration of drugs in wastewater as well as the stability of sensors for long-term 732 monitoring. Therefore, we need to improve the performance of the sensor by utilizing a range of 733 above-mentioned strategies. One can use signal amplification (e.g. nano-material and 734 nanotechnology and molecular amplification) to enhance the signal to meet the target analytes. 735 There is also an alternative to integrate a sample processing technique either with a simple 736 enrichment of illicit drug using microfluidic techniques, for example, using a paper-microfluidic 737 technique which does not need power and laborious facilities, to purify wastewater samples and 738 integrate with sensors for the rapid test. This will also provide a potential to perform the assay in the 739 field. Furthermore, the ultimate goal of WBE is to provide results in real time, which can't be 740 realized by large-scale LC-MS/MS instrument. This data can be achieved with biosensor due to its simplicity, rapidity and portability. There are, however, several issues that need to be addressed as 742 mentioned above, mainly including the relatively low sensitivity of biosensors, most probably low 743 selectivity and other drawbacks to overcome. Herein, much more studies should be carried out on 744 these aspects to make any biosensors useful in the wastewater environment. Due to fast 745 advancements in the field, we believe that wastewater biosensor will provide a novel avenue to 746 analyse the abuse of illicit drugs and even discover illicit drug manufacturing bases along sewage 747 pipe networks. 748 The challenge of drug abuse worldwide is that it takes both time and money to combat. An 750 aptamer is a good option for drug sensor construction due to its high specificity and highly Combinations of aptasensors and new integrated devices, such as paper microfluidic, and 760 commercial biosensors devices, are conducive to the miniaturization and portability of illicit drug 761 aptasensors, which can be easily used in complex environments. We also present several recent 762 publications about community sewage sensors for illicit drug analysis in wastewater. Therefore, we are hoping that most of those developed aptasensors can be implanted as an alternative method 764 to analyse wastewater for drug consumption evaluation for WBE. 765 However, commercial illicit drug aptasensors for wastewater have yet to be achieved. 766 Practically, the complex matrix in real wastewater samples has a significant effect on the 767 sensitivity and selectivity of aptasensors. The selectivity of the sensors in wastewater matrices has 768 not been widely evaluated but is often relatively poor given the concentrations of analogues in 769 natural waters. Apart from the interference found in wastewater samples, the aptasensor is not 770 sensitive enough to monitor illicit drugs at the low concentrations present in wastewaters. All of 771 these limitations hinder the further wide application of illicit drug aptasensors in WBE. Moreover, 772 apart from the selectivity and sensitivity, one also needs to take into consideration the stability, 773 reproducibility, robustness, ease-to-use, portability, and cost before they can be applied in practical 774 use or for commercialization potential. 775 Therefore, many more new methods and efforts are needed to address the major challenge of 776 screening illicit drug aptamers with excellent sensitivity and selectivity in wastewater. In the 777 future, we deem the following work needs to be done in the following areas: aptamers, 778 nanomaterials, new technologies and finally commercial applications. For example, for the 779 identification of the aptamer probes, it's essential to understand the intrinsic properties and 780 functions between the aptamer and illicit drugs, such as binding structure [138] and kinetic effects 781 [139], and these should be studied for further optimization. Then, according to the characteristics 782 of the aptamer, the selectivity of the aptasensor can be improved by optimizing the stem length 783 and different bases of aptamer [140] . Another avenue is that many more aptamers need to be 784 developed and implemented for more drug detection because the drug market is very large, and many new drugs appear every year. For the implementation with nanomaterials and 786 nanotechnology, the characteristics of existing materials should be further explored for better use 787 in drug aptasensors; on the other hand, depending on the needs of drug aptasensor design, we need 788 to synthesize new specific nanomaterials that hold wonderful optical, electrochemical, and other 789 necessary characteristics, which ultimately will contribute to improving the analytical 790 performance of sensors. The current progress in the field of nanotechnology shows great potential 791 in improving some aspects of biosensors and may open up new opportunities for improvement of 792 sensing performance. Although we reviewed some new technology, such as DNA technology and 793 device integration technology, used in the design of illicit drug aptasensors, we further recommend 794 incorporating other promising technologies, such as 3D printing technology, into aptasensor 795 designs to improve their miniaturization along with their portability, robustness, stability, 796 reusability, and/or reproducibility. 797 The last and most important consideration is a practical application of illicit drug aptasensors. 798 Although the reported aptasensors are highly selective and sensitive and have excellent 799 reproducibility and repeatability in a buffer, they may not work well when considering the 800 complex matrix and presence of multiple drugs for in situ detection in wastewater. Therefore, 801 more efficient and useful aptasensors for the on-site detection of illicit drugs in wastewater are an 802 urgent need in the future. Such demand for illicit drug aptasensors before the practical or 803 commercial application may be satisfied by paying special attention to the development of (i) a 804 single analytical device for detecting multiple illicit drugs, (ii) robust aptasensors that can be 805 applied in complex environments, (iii) highly selective aptasensors for illicit drugs, and (iv) 806 portable aptasensors for on-site monitoring. Because of the rapid progress of versatile nanomaterial syntheses and the rapid occurrence of new technology, we believe that the 808 integration of these new technologies and versatile nanomaterials into aptasensors will promote 809 tremendous progress in illicit drug analysis to be marketed and applied in WBE in the near future. aptamer folding-based sensory platform decorated with nanoparticles for simple cocaine testing. 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magnetic nanoparticles as the separation and 1008 amplification element Structure-Switching Signaling Aptamers Electron-transfer quenching of nucleic 1011 acid-functionalized CdSe/ZnS quantum dots by doxorubicin: A versatile system for the optical 1012 detection of DNA, aptamer-substrate complexes and telomerase activity Cocaine Detection in Blood Serum Using Aptamer Biosensor on 1015 Gold Nanoparticles and Progressive Dilution A novel platform for detection of 1020 protooncogene based on Au nanocluster enhanced fluorescence Simple and Sensitive Molecularly Imprinted Polymer -1023 Mn-Doped ZnS Quantum Dots Based Fluorescence Probe for Cocaine and Metabolites Determination 1024 in Urine Nanomaterials for the sensing of 1026 narcotics: Challenges and opportunities Fluorescent sensing of cocaine 1028 based on a structure switching aptamer, gold nanoparticles and graphene oxide Surface-Enhanced Raman Spectroscopy: A 1031 Facile and Rapid Method for the Chemical Component Study of Individual Atmospheric Aerosol Gold nanorods as SERS 1034 substrate for the ultratrace detection of cocaine in non-pretreated oral fluid samples Electrochemical Nucleic Acid-Based Biosensing of 1037 Drugs of Abuse and Pharmaceuticals Highly sensitive and specific on-site detection of serum 1039 cocaine by a low cost aptasensor A Novel PbS 1043 Nanparticle Based Electrochemical Codeine Sensor DNA nanostructure-decorated 1046 surfaces for enhanced aptamer-target binding and electrochemical cocaine sensors Shahdost-fard, A highly selective and sensitive cocaine aptasensor based on 1049 covalent attachment of the aptamer-functionalized AuNPs onto nanocomposite as the support 1050 platform An aptasensor for voltammetric and impedimetric determination 1052 of cocaine based on a glassy carbon electrode modified with platinum nanoparticles and using rutin as 1053 a redox probe Conformation switching of an aptamer based on cocaine 1055 enhancement on a surface of modified GCE Microbial fuel cell-based self-powered 1057 biosensing platform for determination of ketamine as an anesthesia drug in clinical serum samples Polypeptide Functional Surface for the Aptamer Immobilization: 1064 Electrochemical Cocaine Biosensing A label-free 1066 electrochemical biosensor based on a DNA aptamer against codeine An ultrasensitive aptamer biosensor for the 1068 detection of codeine based on a Au nanoparticle/polyamidoamine dendrimer-modified screen-printed 1069 carbon electrode Impedance Spectroscopic Sensing of Methamphetamine by a Specific Aptamer Engineering an aptamer-based recognition sensor for electrochemical 1074 opium alkaloid biosensing Two-Dimensional Zirconium-Based Metal-Organic Framework Nanosheet Composites Embedded with 1077 Au Nanoclusters: A Highly Sensitive Electrochemical Aptasensor toward Detecting Cocaine Highly Sensitive Detection for Cocaine Using 1080 Graphene Oxide-Aptamer Based Sensors in Combination with Tween 20 Fabrication of a novel 1083 aptasensor based on three-dimensional reduced graphene oxide/polyaniline/gold nanoparticle 1084 composite as a novel platform for high sensitive and specific cocaine detection Isothermal nucleic acid amplification technologies for point-of-care 1087 diagnostics: a critical review Isothermal Amplification of Nucleic Acids Isothermal exponential amplification techniques: From basic 1091 principles to applications in electrochemical biosensors Spotlighting of cocaine by an autonomous 1093 aptamer-based machine Fluorescence Aptameric 1095 Sensor for Strand Displacement Amplification Detection of Cocaine Proximity-dependent isothermal cycle amplification for 1097 small-molecule detection based on surface enhanced Raman scattering In situ amplification signaling-based 1100 autonomous aptameric machine for the sensitive fluorescence detection ofcocaine A novel label-free fluorescence 1106 aptamer-based sensor method for cocaine detection based on isothermal circular 1107 strand-displacement amplification and graphene oxide absorption Autonomous Replication of Nucleic Acids by 1109 Polymerization/Nicking Enzyme/DNAzyme Cascades for the Amplified Detection of DNA and the 1110 Cocaine detection via rolling circle amplification of short 1112 DNA strand separated by magnetic beads Electrochemical aptasensor for highly 1114 sensitive determination of cocaine using a supramolecular aptamer and rolling circle amplification Label-free and ultrasensitive fluorescence 1117 detection of cocaine based on a strategy that utilizes DNA-templated silver nanoclusters and the 1118 nicking endonuclease-assisted signal amplification method An 1120 aptamer cross-linked hydrogel as a colorimetric platform for visual detection Target-responsive "sweet" hydrogel with glucometer readout for portable and quantitative detection 1124 of non-glucose targets A portable visual 1126 detection method based on a target-responsive DNA hydrogel and color change of gold nanorods Label-Free Nanopore Biosensor for Rapid and Highly Sensitive 1129 Cocaine Detection in Complex Biological Fluids Single Nanochannel-Aptamer-Based Biosensor for 1131 Ultrasensitive and Selective Cocaine Detection Microfluidic Electrochemical Aptameric Assay 1133 A Potentially Convenient Sensing Platform for the Amplified and Multiplex 1134 Analysis of Small Molecules An aptamer-based paper microfluidic device for the 1136 colorimetric determination of cocaine Using personal glucose meters and functional DNA sensors to quantify a variety of 1138 analytical targets A microfluidic affinity sensor for the 1142 detection of cocaine Microfluidic Technology for Nucleic Acid Aptamer Evolution and Application Anticodeine aptamer immobilized on a Whatman 1147 cellulose paper for thin-film microextraction of codeine from urine followed by electrospray ionization 1148 ion mobility spectrometry Graphene nanocomposites modified electrochemical 1150 aptamer sensor for rapid and highly sensitive detection of prostate specific antigen Monitoring Genetic Population 1153 Biomarkers for Wastewater-Based Epidemiology Rapid Veterinary Diagnosis of Bovine Reproductive Infectious Diseases from Semen Using 1156 Paper-Origami DNA Microfluidics Paper-based microfluidics for DNA diagnostics of malaria in low resource underserved rural 1159 communities Latent Fingerprints and Identification of Cocaine Ultra-high frequency piezoelectric 1163 aptasensor for the label-free detection of cocaine A label-free immunosensor for ultrasensitive 1165 detection of ketamine based on quartz crystal microbalance Social, demographic, and economic correlates of food and chemical consumption measured 1168 by wastewater-based epidemiology Triplex-quadruplex structural scaffold: a new binding structure of aptamer. Sci Rep Single-Molecule Kinetic Investigation of 1172 Cocaine-Dependent Split-Aptamer Assembly Optimizing 1174 Stem Length To Improve Ligand Selectivity in a Structure-Switching Cocaine-Binding Aptamer • The concept of wastewater-based drug epidemiology (WBE) was outlined.• The recent advances of nanomaterial-based aptasensors for illicit drugs detection are described, focusing on optical and electrochemical strategies • The emerging DNA technology are discussed for drug detection as well as engineering of aptasensors for portable assay.• The feasibility of illicit drug aptasensors for WBE are discussed.• The future trends and our insights on aptasensors for illicit drug are provided. The authors declared no conflict of interest.