Application of aptamers in detection and chromatographic purification of antibiotics in different matrices

Accepted Manuscript Application of aptamers in detection and chromatographic purification of antibiotics in different matrices Yi Yang, Shuo Yin, Yongxin Li, Dan Lu, Jing Zhang, Chengjun Sun PII:

S0165-9936(17)30151-6

DOI:

10.1016/j.trac.2017.07.023

Reference:

TRAC 14971

To appear in:

Trends in Analytical Chemistry

Received Date: 1 May 2017 Revised Date:

24 July 2017

Accepted Date: 25 July 2017

Please cite this article as: Y. Yang, S. Yin, Y. Li, D. Lu, J. Zhang, C. Sun, Application of aptamers in detection and chromatographic purification of antibiotics in different matrices, Trends in Analytical Chemistry (2017), doi: 10.1016/j.trac.2017.07.023. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Application of aptamers in detection and chromatographic

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purification of antibiotics in different matrices

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Yi Yang a, Shuo Yin a, Yongxin Li a,b, Dan Lu c, Jing Zhang a, Chengjun Sun a,b,*

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West China School of Public Health, Sichuan University, Chengdu 610041, China

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Provincial Key Laboratory for Food Safety Monitoring and Risk Assessment of Sichuan, Chengdu 610041, China

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School of Public Health, Xi’an Jiaotong University Health Science Center, Xi’an 710061, China

* Corresponding author：[email protected]. Tel/Fax: +86-28-85501301

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antibiotic resistance has emerged as a serious issue globally in recent years. To detect antibiotics and their residues in the environment, foods, drugs and biological samples, numerous analytical techniques have been developed. Among them, aptamer-based methods are considered to be highly sensitive and selective and have been applied to the detection of various antibiotics in different samples. We present a systematical and critical review on the antibiotic-specific aptamers and their application in detection of antibiotics in different matrices, focusing on the recent advances in optical and electrochemical aptasensors, aptamer-affinity based sample purification, as well as the promising label-free and multiplex determination.

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Keywords: Aptamer; Antibiotic; SELEX; Sensor; Purification; Detection; Environment;

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Food; Drinking water; Biological sample

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Antibiotic contamination and abuse are universal phenomena, accordingly

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Contents

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1. Introduction ................................................................................................................................... 3 2. Antibiotic-specific aptamers ......................................................................................................... 4 2.1. Selected sequences of antibiotic aptamers ......................................................................... 4 2.2. Modification of antibiotic aptamers ................................................................................... 5 2.2.1. Methylation ............................................................................................................. 5 2.2.2. Structure adjustment................................................................................................ 5 2.2.3. Length alteration ..................................................................................................... 5 3. Aptasensors for antibiotics and general sensing mechanisms ....................................................... 5 4. Luminescent aptasensors............................................................................................................... 6 4.1. Photoluminescent aptasensors ............................................................................................ 6 4.1.1. Signal labeling......................................................................................................... 6 4.1.2. Fluorescence resonance energy transfer and fluorescence quenching .................... 7 4.2. Chemiluminescent aptasensors ........................................................................................ 10 5. Colorimetric aptasensors ............................................................................................................. 10 5.1. Gold/silver nanoparticles-based colorimetric aptasensors ............................................... 11 5.1.1. Salt-induced AuNPs/AgNPs aggregation.............................................................. 11 5.1.2. Cationic conjugated polymer-induced AuNPs aggregation .................................. 12 5.1.3. Modified AuNPs ................................................................................................... 12 5.1.4. Peroxidase-like activity of AuNPs ........................................................................ 13 5.2. Enzyme-linked aptamer assay (ELAA)............................................................................ 13 5.3. Antibody-assisted catalytic colorimetric aptasensors....................................................... 14 6. Electrochemical aptasensors ....................................................................................................... 14 6.1. Modification of electrodes ............................................................................................... 15 6.1.1. Gold nanoparticles (AuNPs) ................................................................................. 15 6.1.2. Conducting polymers ............................................................................................ 15 6.1.3. Graphene ............................................................................................................... 16 6.1.4. Multi-walled carbon nanotubes (MWCNTs) ........................................................ 16 6.1.5. Magnetic nanoparticles (MNPs)............................................................................ 17 6.2. Competitive electrochemical aptasensors ........................................................................ 17 6.3. Folding-based aptasensors ............................................................................................... 17 6.4. Signaling-probe displacement aptasensors....................................................................... 18 6.5. Electrochemiluminescent aptasensors .............................................................................. 18 6.6 Photoelectrochemical aptasensors ..................................................................................... 19 7. Other aptasensors ........................................................................................................................ 19 8. Some sensing strategies for antibiotic aptasensors ..................................................................... 20 8.1. Microfluidic biosensors.................................................................................................... 20 8.2. Enzyme-recycled amplification aptasensors .................................................................... 20 8.3. Multiplex detection .......................................................................................................... 21 9. Aptamer affinity-based chromatographic purification ................................................................ 21 10. Sample detection ....................................................................................................................... 22 11. Conclusion and perspectives ..................................................................................................... 23

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Antibiotics are a large category of chemicals used in the treatment of bacterial infections and promotion of livestock growth. There are many classes of antibiotics, such as β-lactam antibiotics, tetracyclines, aminoglycosides, streptogramins, macrolides, lincosamides, quinolones, nitroimidazoles, etc[1]. Antibiotics can be accumulated in human body[2] because of their residues in food and water, which may be direct toxic and result in allergic reactions[3], arrhythmias[4], hearing loss[5], nausea, vomiting and acute pancreatitis, etc[6]. Furthermore, antibiotics abuse can lead to antimicrobial resistance and make infection treatment become more difficult[7]. Many countries, such as the United States and the European Union, have set maximum residue limits (MRLs) in different food commodities, which are in low microgram per kilogram level[8, 9]. Take the Code of Federal Regulations[10] as an example, it established acceptable daily intake (ADI) and tolerances for residues of antibiotics. The ADI for total residues of tetracyclines and neomycin are 25 mg/kg/day and 6 mg/kg/day, respectively. The tolerances are established as 2 ppm in animal muscles and 0.3 ppm in milk for tetracyclines, 1.2 ppm in animal muscles and 0.15 ppm in milk for neomycin, 0.1 ppm in tissues and 0.01 ppm in milk for sulfadimethoxine, 0.01 ppm in milk and cattle for ampicillin, 0.5 ppm in animal tissues for streptomycin, respectively. The currently available detection methods for antibiotics in different matrices are mainly high performance liquid chromatography (HPLC)[11], gas chromatography-mass spectrometry (GC-MS)[12] and liquid chromatography-tandem mass spectrometry (LC-MS/MS)[13]. Other techniques such as capillary electrophoresis (CE)[14], enzyme-linked immunosorbent assay (ELISA)[15], surface plasmon resonance (SPR)[16] and the microbiological multi-residue system[17] have been reported too. Aptamers are oligonucleotides (DNA and RNA) that can bind to specific targets with high affinity and high specificity[18, 19]. They are usually identified by the process “Systematic Evolution of Ligands by Exponential Enrichment (SELEX)”[20]. While many people believed that only antibodies could bind to targets with high affinity and specificity, it had to be recognized that aptamers’ oligonucleotide structures could do these jobs as well, sometimes even better.[21] The main advantages of aptamers over antibodies involve their simple synthesis with high purity, stable reproducibility, and relatively low cost; wide target ranges; easily chemical modification; and high stability under different circumstances and in target-binding reaction[22, 23]. Due to these advantages aforementioned, over the past decades, aptamers have been extensively used in drug delivery, in vivo therapeutics, molecular sensor, selective chromatography and biological imaging. In the recent years, aptamers have received particular attention in the field of analytical chemistry to realize the recognition of a wide range of analytes including small molecules, peptides, proteins and even macromolecular ribosomal and viral particles. Up to now, several commercial aptamer-based kits such as Myco-Sense System (NeoVentures, Canada) for detection of ochratoxin A and aflatoxins, Apta-Beacon Demonstration Kit (GQ-EXPAR, TMB) for theophylline versus caffeine detection, AptoPrepTM Protein Isolation Kit (Aptsci, Korea) and Aptoprecipitation (AP)/Co-aptoprecipitation (Co-AP) Kits (AMSBIO, UK) for protein isolation have been on the market. Commercial columns such as Ultra-Fast Affinity Columns (NeoVentures, Canada) for HPLC clean-up of ochratoxin A and aflatoxin are available as well. A few authors have summarized the application of aptamers in analysis of antibiotic residues[24-27], mainly focused on kanamycin (KANA) detection[25] and electrochemical

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1. Introduction

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ACCEPTED MANUSCRIPT aptasensors fabrication[26, 27], but a systematical and comprehensive overview of the application of aptamers in detection of antibiotics in different matrices is still needed. Consequently, we have made an in-depth summary of the history and the application of antibiotic-specific aptamers in detection and chromatographic purification of antibiotics in different matrices, and also discussed their future trends and perspectives.

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2. Antibiotic-specific aptamers In the 1990s, RNA molecules were supposed to fold and form binding pockets to accommodate small molecules. Subsequently, antibiotics were recognized as RNA-binding compounds. Attempts to dissect how the antibiotics bind to RNA have resulted in the development of SELEX of low molecular antibiotics-binding RNA aptamers, which were utilized as the investigation model since the natural binding ribosomal RNAs and ribozymes were too large for the small antibiotics molecules. The interaction mechanism has been reviewed elsewhere[28]. Due to the limitation of RNA aptamers (e.g. instability) in designation of bioassays[29], DNA aptamers that specifically binding to antibiotics were isolated[30].

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2.1. Selected sequences of antibiotic aptamers The frequently utilized sequences of antibiotics-binding aptamers and their dissociation constants (Kd) are listed in Table 1. The target-binding affinity is inversely proportional to its Kd value. Ever since Wang et al.[31] isolated aptamers X1 and J6 against tobramycin and their distinctive stem-loops containing aptamers X1SL and J6SL in 1995, these aptamers have been extensively employed in tobramycin recognition[32-34]. Though these aptamers could bind to KANA and gentamicin[34] as well, they bound to tobramycin with much higher affinity[31, 35]. In 2008, Gu’s team[36] selected single-stranded DNA (ssDNA) aptamers specific to oxytetracycline (OTC) for the first time. Among the selected aptamers, No. 4 had the strongest affinity (Kd= 9.61 nM), while No. 20 showed the highest selectivity followed by No. 5 and 4 with no or insignificant binding to tetracycline (TET) and doxycycline (DOX). As No. 5 showed both strong affinity and selectivity towards OTC, this team[37] studied its utility in electrochemical sensing of OTC with discrimination of TET and DOX. Thus in the following studies, researchers usually used aptamer No. 5 to bind to OTC. Subsequently, Gu’s team[38] isolated seven ssDNA aptamers specifically binding to more than one type of tetracyclines, i.e. TET, OTC and DOX. Among these aptamers, T20, T24 and T23 showed stronger affinities. T20 bound with much higher affinity to TET (Kd[TET]= 63.6 nM) than OTC or DOX, while the rest six aptamers showed higher affinity toward OTC than TET and DOX. Accordingly, for the recognition of TET, T20 was most employed in later research. In the meantime, T24 was also used for binding to OTC as it had both high affinity and selectivity towards OTC. This team[39] built a T24-based aptasensor that showed a dynamic range from 25 nM to 1 µM. In comparison, an antibody-based ELISA showed a narrow range from 0.9 nM to 2.3 nM with low specificity. In 2011, Mehta et. al.[30] selected two aptamers namely Aptamers 7 and 16 towards chloramphenicol (CAP). Then Miao et al.[40] reported a colorimetric aptasensor based on Aptamer 7 and achieved higher sensitivity (LOD= 0.0003 ng/mL) than ELISA as a sample (0.12 ng/mL) spiked at 0.10 ng/mL was detected by this aptasensor but undetected by ELISA. Song et al.[41] isolated an aptamer against KANA, KANA B and tobramycin in 2011, and an AMP17 aptamer against ampicillin in 2012[42]. A streptomycin (STR) binding aptamer was selected by Zhou et al.[43] with high affinity and selectivity in 2013. These sequences have been widely adopted to recognize their

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ACCEPTED MANUSCRIPT specific targets thereafter. In addition to aptamers against these frequently analyzed antibiotics, other aptamers have been isolated and utilized as well for detection of antibiotics like penicillin G[44], sulfadimethoxine (SDM)[45] and ofloxacin[46]. Among these antibiotic-binding aptamers, some are highly selective, while some bind to more than one target[34, 41, 47, 48].

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2.2. Modification of antibiotic aptamers To improve the stability of an antibiotic-specific aptamer and enhance its affinity or improve its selectivity, it can be modified by methylation and altering its structure or length. 2.2.1. Methylation Numerous small molecules like antibiotics preferred to interact with structured RNA over DNA in animal and human body via disturbing the synthesis of the encoded-protein. Consequently, the isolated antibiotics binding aptamers are mainly RNAs. However, due to their intrinsic lower endonuclease stability, RNAs were precluded in application in biological fluids.[49] To make RNA aptamers nuclease-resistant in antibiotics sensing, 2’-O-methylated RNA aptamer was found able to improve the stability without loss of selectivity[49]. The binding properties of aptamers in different methylation degree were studied by modifying a selected tobramycin-binding aptamer[31] into a partially or a fully O-methylated aptamer[50]. The two modified aptamers showed similar affinity and reproducibility in aqueous solution. Other investigations[51, 52] indicated that the modification could not significantly increase the affinity of the RNA aptamers toward the target antibiotics but could confer biological stability to the aptamers against nucleases, which demonstrated the viability of designing endonuclease-resistant aptamers rationally without compromising the analytical characteristics. Thereupon, 2’-O-methylated RNA aptamers have promisingly been applied to sense their specific antibiotics in biological samples[53].

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2.2.2. Structure adjustment Structure adjustment can improve the aptamers' sensitivity. Taking the selected tobramycin aptamer[31] which exhibited no target-induced conformation change as a parent, Schoukroun-Barnes et al.[54] deleted the parent aptamer’s 5'-terminal bases GGGA and 3'-terminal bases UCCC to produce a mutant. This mutant destabilized the parent’s stem-loop to allow a large conformation change upon target binding. Since bases participating in target-binding interaction were conserved, the sensitivity and affinity of the generated mutant aptamer were improved from 16± 3 µM of the patent aptamer to 0.22± 0.05 µM without compromising the original specificity and selectivity.

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2.2.3. Length alteration The modification approaches can enhance the binding affinity and selectivity of aptamers by altering the sequence length. An 8-mer aptamer[55] was truncated from its parent OTC-specific 76-mer aptamers[36]. Though conserved only the original stacking pocket and 6 additional specific bases, this 8-mer aptamer yielded higher binding affinity and broader selectivity (Kd[OTC]= 1.104 nM, Kd[TET]= 1.067 nM), and has been successfully applied to analyze tetracyclines in milk and rat serum samples[56].

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3. Aptasensors for antibiotics and general sensing mechanisms Aptamer-based sensors for antibiotics include luminescent aptasensors, colorimetric aptasensors, electrochemical aptasensors etc. All the aptasensors share general sensing mechanisms, including direct/indirect competition, signal displacement, folding-based detection

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ACCEPTED MANUSCRIPT and labeled/label-free strategies. For the reason that sandwich assay scheme is not suitable because of antibiotics’ small size, detection approaches based on competitive formats including direct and indirect competition are preferred[57]. For direct competition, free targets in samples competitively bind to immobilized aptamers with standard targets or signal probes. As for indirect competition, free targets in samples compete with immobilized standard targets for aptamers. When aptamers preferentially bind to targets and are released from signal probes, signal displacement happens. Folding based detection is based on the inner structural change of aptamers to bring in signal changes. Labeling is the earliest utilized signal tagging method, with multiplex labels simultaneous detection could be realized, and sometimes labeling can even generate a stronger signal. For example, González-Fernández et al.[58] employed a biotin-streptavidin multivalent labeling system to induce a bigger detection signal triggered to the electrochemical aptasensor for tobramycin detection with LOD of 5 µM. Subsequently, they established a fluorescein isothiocyanate (FITC)-antiFITC-enzyme-Fab monovalent labeling system to analyze tobramycin[53], which greatly improved the sensitivity with LOD improved from 5 µM to 0.1 µM in aqueous solution compared with the biotin-streptavidin multivalent system[58]. This enhancement was ascribed to the use of the monovalent labeling system to give an exclusion of multiple binding of enzymatic conjugate to several recognition events that hindered the detection of low concentrations. Though labeling can provide a detectable signal and even enhance the signal, an alteration of the affinity of the aptamer after labeling may sacrifice its selectivity, which is ascribed to a lower affinity of the labeled aptamer for its cognate ligand and an increased affinity for structurally related compounds[58]. Meanwhile, the labeling procedure greatly increases the consumption of laboratory resources and analysis time. Accordingly, label-free strategy is preferred.

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4. Luminescent aptasensors Luminescent signaling combined to fluorescent detection offers the platform for analysis of antibiotics with high sensitivity. According to the mechanism of luminescence, luminescent aptasensors can be categorized into photoluminescent, chemiluminescent and electrochemiluminescent (see 6.5) aptasensors. In photoluminescent aptasensors, fluorescence quenching or recovery are measured, which are achieved by typical means of signal labeling, materials-induced quenching, inner structural change of the aptamer and so on. Table 2 lists the luminescent aptasensors and their performances for detection of antibiotics in various samples.

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4.1. Photoluminescent aptasensors 4.1.1. Signal labeling

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For sensing a detectable signal, photoluminescent dyes are commonly used as markers to evaluate the performance of aptamers via adding or labeling dyes such as OliGreen[59], FAM[46, 48] and SYBR Gold[60] on the antibiotic target-aptamer mixture. For example, a STR sensing aptasensor[60] utilized SYBR Gold which possessed specificity for nucleic acids as signal label and exploited Exonuclease III (Exo III, a double-stranded DNA (dsDNA) digestion enzyme) to assist the determination. Upon target binding, the induced aptamer-STR conjugate and released complementary DNA (cDNA) were protected from the degradation of Exo III. After staining by SYBR Gold, the fluorescence intensity increased. Seminaphthorhodafluor (SNARF) is a representative red-emitting fluorophore that shows pH dependent dual-emission property and has been employed in a ratiometric fluorescence sensing system to show the ratiometric fluorescence

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ACCEPTED MANUSCRIPT spectra change in response to TET and STR[61]. Dye-displacement assay has also been employed using dyes like thiazole orange (TO)[62] to competitively bind to aptamers against the analytes. New materials that require no ultraviolet (UV) or visible light induced photobleaching have been used as signaling probes. For example, upconversion nanoparticles (UCNPs) are excellent emitters which possess high penetration depth in biosamples. They can effectively amplify luminescent signal as well as entirely avoid auto-luminescence originating from biomolecules possibly contained in samples. Two aptasensors have utilized UCNPs as signaling probes for the detection of CAP[63] and OTC[64]. As shown in Fig. 1 A, in this system, aptamers were immobilized onto the surface of magnetic nanoparticles (MNPs) to capture and concentrate CAP. In the absence of target, MNPs-aptamer hybridized to the UCNPs-modified cDNA to form a duplex structure, giving a maximum fluorescent signal. Exploiting the CAP-induced conformational change from the duplex complex of MNPs-aptamer/UCNPs-cDNA to MNPs-aptamer/CAP complex, the hybridized UCNPs-cDNA was released from the MNPs-aptamer, leading to substantially decrease of fluorescence intensity. Liu et al.[65] combined the double recognition of aptamer- molecularly imprinted polymers (MIPs) with signal transduction of UCNPs to analyze enrofloxacin with high sensitivity (LOD=0.04 ng/mL).

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Labeling a donor fluorophore on an aptamer, it would be quenched when the aptamer adsorbs on some materials serving as quenchers. But a false positive signal may be generated due to some ligands or solvents’ interference with quenching. To obviate this problem, fluorescence resonance energy transfer (FRET) is utilized. FRET is a physical phenomenon describing energy transfer from a donor molecule to an acceptor molecule, and the energy transfer efficiency is inversely proportional to the distance between donor and acceptor[66]. After FRET, the fluorescence of the donor reduced and that of the acceptor enhanced. Aptamer-based antibiotics detection can be realized by fluorescence quenching effects or FRET, employing different materials (especially nanomaterials) as energy acceptors, donors, quenchers and assistant carriers and linkers.

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4.1.2.1. Materials-induced FRET and fluorescence quenching Among the materials studied as quenchers, gold nanoparticles (AuNPs) can specifically combine with aptamers and have extensive applications, due to their high surface-to-volume ratio, easy modification and the ability to adsorb ssDNA. Using AuNPs as both DNA/RNA nanocarrier and quencher, the fluorescence of the FAM-labeled aptamers can be quenched by their adsorption onto the surface of AuNPs, and recovered by their releasing from AuNPs upon binding to the target antibiotic[42, 67-69]. Likewise, coordination polymer nanobelts (CPNBs)[45] and graphene oxide (GO)[70-73] have been used as quenchers to adsorb target-free dye-labeled single strand aptamers directly or indirectly via π-π stacking interaction. Fig. 1 B shows the process of fluorescence quenching induced by AuNPs, CPNBs and GO. For instance, Zhao et al.[70] adsorbed fluorescein-labeled 76-mer long-chain OTC aptamers onto GO sheet with quenched fluorescence and then added OTC which changed the conformation of the aptamer to restore the fluorescence. But as a long-chain aptamer, the existing secondary structure caused an indistinctive structural transformation after binding OTC, which hindered its effective desorption from the graphene sheet, so hindered the fluorescence restoration. Instead of labeling the fluorescein directly on the long-chain aptamer, Yuan et al.[71] applied an indirect fluorescein-labeled OTC aptasensor by inducing a FAM-labeled short-chain ssDNA (S1) to hybridize with the 5’-end of the

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GO adsorbed long-chain aptamer. Upon OTC binding, S1 was released from the aptamer and then brought away from GO by its complementary short-chain ssDNA (C1) to restore its fluorescence. Consequently, this aptasensor avoided the negative effects from the intrinsic secondary structure of the long-chain aptamer. A target-induced florescence-off model was developed by Zhang et al.[72]. They found that the fluorescence of cyanine dye (Cy5)-labeled ssDNA aptamer was just partially quenched when adsorbed on low concentration of GO, while in the presence of aminoglycoside antibiotics, the antibiotic-aptamer duplex’s binding efficiency toward GO surface increased through amide coupling, resulting in fluorescence attenuation. These aforementioned GO sheets were in two-dimensional (2D) since ssDNA was flatly laying on the surface of GO sheet. According to Tan et al.[73], three-dimensional (3D) macrostructural GO hydrogels could be readily prepared by physically mixing GO solution with adenosine. In this hydrogel, adenosine and aptamer served together as cross-linkers between the GO sheets to form the 3D macrostructures. Compared to the wrinkled surface of 2D GO sheets, hydrogel showed a well-defined and interconnected 3D network, with pore sizes ranging from submicrometer to several micrometers. Recently, materials showing photoluminescence such as UCNPs and quantum dots (QDs) were also used as energy donors. A KANA detection aptasensor applied UCNPs as the energy donor and graphene as the energy acceptor[74]. The KANA aptamer was tagged to UCNPs, then the π-π stacking interaction between the aptamer and graphene brought UCNPs and graphene in close proximity, resulting in the quenching of UCNPs fluorescence. The addition of KANA changed the conformation of the aptamer into a hairpin structure, hence blocked the energy transfer and recovered the fluorescence. Many researchers have employed QDs as donors with different acceptors to fabricate aptasensors for analyzing antibiotics. For example, Lin et al.[75] developed an aptamer against daunorubicin (DNR) and combined it with NIR CuInS2 QDs. They found that the analyte DNR could intercalate into the double-stranded CG sequence of the developed aptamer to form a DNR-aptamer-QDs complex, by which CuInS2 QDs photoluminescence emission was quenched through photo induced electron transfer process. Another aptasensor for CAP detection was fabricated based on self-quenching of QDs with the assistance of a dsDNA-antibody (antibody that specifically bound to dsDNA) covalently bound on the surface of CdSe QDs[76]. The aptamer-cDNA formed dsDNA acted as a bridge for the probes to contact with each other, resulting in the fluorescence quenching. This dsDNA-antibody-aptamer/cDNA system was adopted to analyze CAP by Miao et al.[77] who employed magnetic SiO2@AuNPs probe as acceptor for QDs. In the CAP aptasensor proposed by Alibolandi et al.[78], GO was served as the quencher for the CdTe QDs donor. Assistant materials have also been applied to antibiotics sensing. Similar to the dsDNA-antibody, as a linkage connecting aptamers and signal tracers, ssDNA binding protein (SSB)[79-81] was employed to competitively bind to aptamers with targets. Based on SSB's specific affinity towards aptamer, the SSB-linked aptamer probe was formed. Because the aptamer had a higher affinity towards the target over SSB, in the presence of target, the aptamer preferentially bound to the target and released from the SSB-linked probe. Liposomes (Lip)[79] and QDs[80, 81] were induced to fabricate aptamer-based sensors. Liposomes are spherical lipid vesicles in magnitude of nanometer with a double layer membrane structure consisting of amphiphilic lipid molecules, possessing many attractive features[79], such as the good hydrophily, hydrophobic inner membrane and the vesicle cavity. These features enable them to be

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ACCEPTED MANUSCRIPT immobilized with many hydrophobic fluorescence probes in their inner hydrophobic membrane and encapsulate numerous hydrophobic molecules in their nanometer cavity. By choosing different precursors for preparing liposome, grafting ligands such as -COOH and -NH2 can be fabricated on their outer surface to immobilize proteins. Therefore, to enhance signals, fluorescence vesicle tracers can be fabricated employing liposomes to encapsulate a large number of fluorescence probes and label biological proteins like SSB. A magnetic aptamer-liposome vesicle probe-based fluorescence aptasensor was developed to sense CAP by Miao et al.[79]. The vesicle signal tracer was built by coimmobilizing SSB and DIL (1,1’-dioctadecyl-3,3,3’,3’-tetramethyl-indocarbocyanineper-chlorate) on liposomes (SSB/DIL-Lip). Aptamers were labeled on functionalized magnetic beads (MBs) as capture adsorbent (MB-Apt). By the recognition of SSB towards aptamer on vesicle signal tracer, the composite vesicle probe was formed between SSB/DIL-Lip and MB-Apt. Upon the vesicle probe reacted with CAP, the aptamer on the MBs preferentially bound to CAP and released SSB/DIL-Lip vesicle signal tracer from MB-Apt to emit fluorescence. Combining AuNPs, SSB and QDs together, Wang et al.[80] fabricated an aptasensor to analyze CAP. The FRET switch was synthesized by connecting the donor SSB labeled QDs (QDs-SSB) with the acceptor aptamer labeled AuNPs (AuNPs-Apt). The interaction between core-shell QDs-SSB and AuNPs-Apt led to a dramatic quenching. After addition of CAP, AuNPs-Apt bound to the target specifically and separate QDs-SSB from AuNPs-Apt-target, so the QDs’ fluorescence was restored. In addition, the SSB-QDs/AuNPs-Apt based FRET system was conceived by Miao et al.[81] to sense CAP, involving liposomes to form a vesicle composite probe. In this probe, a nanotracer was prepared through labeling SSB on liposome-CdSe/ZnS QDs (SSB/Lip-QD) complexes. Despite of the similar SSB-QDs/AuNPs-Apt based FRET switch scheme described above, the use of liposomes encapsulated with a number of QDs and SSB allowed excellent signal amplification by about 15 folds compared with that only QDs were used.

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4.1.2.2. Structural change of aptamers-induced FRET and fluorescence quenching The inner structural change of an aptamer could be employed to realize a signal-switch amplification. An aptasensor was reported by Leung et al.[82] for the detection of KANA in aqueous solution, basing on the speculation that the used KANA aptamer could vary from a random-coiled structure into a hairpin-like structure upon the addition of KANA. As shown in Fig. 1 C, this structural change facilitated the intercalation of a luminescent platinum (II) complex into the bound aptamer segments and enhanced the luminescence signal. The use of relatively inexpensive unmodified DNA aptamers and platinum (II) complexes is economically advantageous over those requiring nanoparticles, immunological reagents, or functionalized oligonucleotides. However, most aptamers have no changeable inner conformation. Their basic structural change could be realized through the formation or dissociation of the duplex dsDNA by the ssDNA aptamer and its cDNA. With the aptamer-cDNA duplex structure, a SYBR Green I dye which could insert into the duplex structure of dsDNA was introduced to an OTC aptasensor[83]. By the insertion of this dye into the dsDNA formed by UCNPs-linked aptamer and its cDNA, the energy donor and acceptor were brought in close proximity, yielding a luminescence transfer from UCNPs to SYBR Green I. The aptamer was prone to bind to OTC, therefore, the dehybridization of aptamer-cDNA resulted in the liberation of SYBR Green I and restoration of the UCNPs' luminescence.

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ACCEPTED MANUSCRIPT Jalalian et al.[84] presented a triple-helix molecular switch (THMS) system (Fig. 1 D). The system was composed of a label-free TET specific aptamer sequence with two arm segments, a fluorophore and a quencher dual-labeled oligonucleotide signal transduction probe (STP). The two arm segments of the aptamer bound to the loop sequence of the STP to form a THMS complex. Upon TET-aptamer complex formation, the THMS was disassembled and the STP was released to form a stem loop structure, so the fluorescence was quenched. G-quadruplex (G4) structures have recently drawn much attention. The structures are higher-order DNA and RNA structures formed from G-rich sequences built around tetrads of hydrogen-bonded guanine bases and stabilized by stacked G-G-G-G tetrads in monovalent cation-containing solution[29]. A G4-DNA aptamer-based fluorescent intercalator displacement assay for the analysis of KANA has been developed by Xing et al.[29]. The authors verified that KANA-binding DNA aptamer could form stable G4 structures by themselves. TO emits strong fluorescence when it binds to quadruplex DNA. Once KANA existed, it replaced TO from the G4-DNA-TO conjugate, so the fluorescent signal decreased. In addition, the hemin contained G4 structure (hemin/G4) could also be used as a signal indicator to catalyze hydrogen peroxide to produce reactive oxygen species, which could enhance luminol emission intensity. A magnetic aptamer probe was prepared to detect CAP residue in food by Miao et al.[85]. As shown in Fig. 1 E, the probe was fabricated through immuno-reactions between the capture beads (dsDNA-antibody labeled on magnetic dynabeads, MNP-ds-Ab) and nanotracer (nano-Pt-luminol labeled with double-strand aptamer-cDNA and hemin/G4) to form hemin/G4-labeled aptamer-Pt-luminol nanocomposites. Upon mixing with CAP, the aptamer preferentially reacted with CAP to decompose the double-strand aptamer to ssDNA, which could not be recognized by the dsDNA-antibody on the capture probes. Hence, after magnetic separation, the nanotracer was released into solution. As the hemin/G4 and PtNPs in nanotracer both catalyzed luminol-H2O2 system to emit fluorescence, a dual-amplified “switch-on” signal appeared.

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4.2. Chemiluminescent aptasensors Chemiluminescent enzyme immunoassay (CLEIA) has been applied by Ni et al.[86] to detect enrofloxacin in milk, using biotin modified aptamer that was immobilized on streptavidin-coated microplate to recognize enrofloxacin. With the direct competition of enrofloxacin-spacehorseradish peroxidase (HRP) and enrofloxacin in samples, quantification of enrofloxacin was realized. Hao et al.[87] applied a chemiluminescent (CL) aptasensor based on N-(4-aminobutyl)-N-ethylisoluminol(ABEI)-functionalized gold nanostructures and magnetic nanoparticles to analyze CAP in milk. In the method, biotinylated aptamer was immobilized directly on avidin-modified MNPs as capture probes. The flower-like gold nanostructure (AuNFs) with larger specific surface area over AuNPs was employed to allow a greater amount of CL reagents to be adsorbed on the surface. The signal probes were the thiolated cDNA conjugated on the surface of ABEI-functionlized AuNFs. In the presence of CAP, it competed with signal probe to bind to the capture probe, forming a competitive format in which the CL intensity was negatively correlated with the concentration of CAP. Additionally, piodophenol (PIP) was added to the immobilized ABEI-H2O2 CL system as a CL enhancer and stabilizer, significantly prolonged the detection time.

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5. Colorimetric aptasensors Colorimetric aptasensors are convenient for rapid on-site analysis, and they are commonly

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ACCEPTED MANUSCRIPT associated with AuNPs, silver nanoparticles (AgNPs), enzymes and antibodies (Table 3).

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5.1. Gold/silver nanoparticles-based colorimetric aptasensors Gold nanoparticles were superior nanomaterials in developing colorimetric sensors for their good biocompatibility, large surface area, high absorption efficiency, well-established synthesis and surface modification protocols. Due to AuNPs’ remarkable SPR properties, they can serve as indicators to give visual output of the sensing events by color change, indicating the presence or absence of targets. A colorimetric sandwich aptasensor was fabricated by Abnous et al.[88] for CAP detection based on an indirect competitive enzyme-free assay using AuNPs, biotin and streptavidin. In the absence of CAP, AuNPs remained on the plate, yielding a sharp red color. In the presence of target, functionalized AuNPs could not bind to the plates, resulting in a faint red color. Besides, the SPR shift caused by AuNPs aggregation has been well exploited by colorimetric aptasensors for antibiotics detection.

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5.1.1. Salt-induced AuNPs/AgNPs aggregation As shown in Fig. 2, AuNPs have a tendency to aggregate non-specifically in the presence of salt and some other molecules, but the unfolded ssDNA aptamers are strongly adsorbed on unmodified AuNPs (umAuNPs) to stop them from salt-induced aggregation via electrostatic repulsion that prevented the strong van der Waals attraction. Pang et al.[89] have pointed out that only if the aptamer-target interaction was stronger than the AuNPs-aptamer and the AuNPs-target, the conformation of aptamers would be transmuted to folded state upon aptamer-target interaction. The folded aptamers are not easily adsorbed on the umAuNPs, thus salt-induced aggregation of AuNPs happens. The colorimetric method based on the SPR shift of AuNPs by salt-induced aggregation is advantageous over the other conventional methods for its simple signal generation and detection ability with the naked eye, which can be applied to on-site and label-free detection. Based on this mechanism, AuNPs have been applied in fabricating aptasensors for detection of OTC[39], KANA[41], ampicillin[42], STR[43] and sulfadimethoxine(SDM)[90]. In contrast to ssDNA’s protection, owing to the rigid structure of duplex dsDNA, it could not protect AuNPs against salt-induced aggregation. Emrani et al.[67] designed a colorimetric aptasensor for detection of STR using aqueous AuNPs and dsDNA. In the absence of STR, aptamer-cDNA formed dsDNA was stable, resulting in the aggregation of AuNPs by salt and an obvious color change from red to blue. In the presence of STR, aptamer bound to its target and the cDNA adsorbed on the surface of AuNPs to protect the well-dispersed AuNPs against salt-induced aggregation with a wine-red color. To fabricate an aptasensor of variable detection sensitivity for antibiotics in different samples, Kim et al.[91] adjusted the affinity ratio of the aptamers toward targets and umAuNPs via changing the capping ligands (aptamers with different affinities). Similar to AuNPs, AgNPs would be aggregated by salt inducement. Notwithstanding, this aggregation could be prevented by KANA due to its inherent amino-groups that could adsorb on the surface of unmodified AgNPs via Ag-N and maintain the stability of AgNPs against salt-induced aggregation. According to this analyte-protected AgNPs and aptamer-selective sensing mechanism, Xu et al.[92] designed an aptasensor to detect KANA. Salt concentration, ionic strength and pH value influence the aggregation of AuNPs. Chen et al.[90] observed that when the pH value was up to 9.0, the sensitivity decreased and the AuNPs aggregations took place even without SDM. This was in consistence with a former research[93]. Ascribed to this phenomenon, there was a lack of aptamer to protect AuNPs from the easily

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ACCEPTED MANUSCRIPT occurred salt-induced aggregation even with very low salt concentration or no SDM targets. This observation was verified by Liu et al.[94] who used SDM and thrombin as model analytes to study the effects of remnant non-AuNPs components on the AuNPs-based aptasensor, proving that the low pH value caused by the remnant non-AuNPs components increased the adsorption of aptamer to surface of AuNPs. These non-AuNPs components were the remnant sodium citrate and the reaction products citric acid, HCl and ketoglutaric acid that remained as impurity during the synthesis of AuNPs. They demonstrated that these components played counteractive roles in AuNPs based fluorescence quenching and colorimetric aptasensor, and found that water resuspended AuNPs could remove these remnant non-AuNPs components and improve the sensitivity by at least 10-fold. Seo et al.[95] reported a reflectance-based colorimetric system that could generate more stable and 25-fold sensitive signals (LOD=1 nM) in the determination of OTC compared to the absorbance-based colorimetric aptasensor (LOD=25 nM)[39]. They directly measured the peak shift of the localized surface plasmon resonance, as the unmodified AuNPs aggregated in the solution and the reflectance signals increased at high AuNPs concentrations.

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5.1.2. Cationic conjugated polymer-induced AuNPs aggregation AuNPs are dispersed by the absorbed negative charge (like aptamer) because of electrostatic stabilization, but aggregated at high salt concentration. As a positively charged material, poly(diallyldimethylammonium) (PDDA) can disturb the charge surface of AuNPs. In a colorimetric aptasensor for detecting TET developed by He et al.[96], PDDA was used to control the aggregation of the AuNPs and the hybridization of the aptamer, as it could compete with targets to bind to aptamers and aggregate the aptamer-free AuNPs. In the absence of TET, PDDA bound to the negatively charged phosphate backbone of the aptamer to form a complex structure, while the DNA still maintained its original conformation. So AuNPs did not aggregate and their color showed no change. On adding TET, aptamer lost the ability to protect AuNPs under PDDA, so the color change from wine red to blue could be detected. In this method, calcium cation need to be removed to avoid salt-induced AuNPs aggregation interference.

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5.1.3. Modified AuNPs The aggregation of AuNPs can be triggered by factors such as the presence of a high concentration of salt and the binding of modified ligands on the surface of AuNPs with targets. The hybridization between complementary ssDNAs is similar to the interaction between ligands and targets. Therefore it can be utilized to induce the aggregation of AuNPs by modification on the AuNPs. Zhou et al.[97] established a colorimetric method for detection of KANA based on a KANA-specific aptamer and AuNPs. They synthesized two types of functionalized AuNPs which were complementary to the 5’ terminal and 3’ terminal sequences of the KANA aptamer. The AuNPs aggregated due to the hybridization of the aptamer with the complementary ssDNAs on the surface of AuNPs. However, when KANA was present, it bound to the aptamer specifically and competitively, liberating the AuNPs. Luo et al.[98] employed a modified (+)AuNPs in colorimetric aptasensor for the detection of TET. The aptasensor was easy to use as it avoided the procedure of salt solution-induced AuNPs aggregations. Cysteamine was capped onto the surfaces of the AuNPs through strong Au-S bonds to be reporters (CS-AuNPs), imparting positive charges from the -NH3+ terminus of cysteamine to the AuNPs. Due to the positive capping agent’s electrostatic repulsion between AuNPs, the

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ACCEPTED MANUSCRIPT CS-AuNPs were resistant to aggregation. When treated with TET, the distribution of CS-AuNPs almost exhibited no change hence remained the color of the solution. When adding the negatively-charged aptamer, the electrostatic attraction between the CS-AuNPs and the aptamers resulted in the aggregation of CS-AuNPs and a rapid color change. In the presence of TET, aptamers preferentially bound to TET and CS-AuNPs was released from aggregation. The absorbance change of CS-AuNPs was linearly proportional with TET concentration in the range of 0.20- 2.0 µg/mL with the LOD of 0.039 µg/mL.

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5.1.4. Peroxidase-like activity of AuNPs Particularly, AuNPs have the ability to oxidize peroxidase substrates in the presence of H2O2 to form colored products. Sharma et al.[99] exploited this peroxidase-like activity of AuNPs to detect KANA which gave a rapid visual readout within 3-8 min with high selectivity. By blocking the surface of AuNPs through the adsorption of ssDNA aptamer molecules, the peroxidase-like activity of AuNPs could be turned-off. In the presence of the target analyte, the aptamer preferentially bound to the target and be dissociated from the AuNPs surface, subsequently permitting the peroxidase-like activity of AuNPs be turned-on again. Yuan et al.[100] fabricated a synergistic 3D graphene-supported bimetallic Fe3O4, AuNPs (3D graphene/Fe3O4-AuNPs) enzyme mimetic catalyst to detect OTC, which exhibited flexibly switchable peroxidase-like activity via a typical color reaction in the presence of H2O2. The catalytic activity was flexibly regulated by the adsorption and desorption of ssDNA aptamers via target-binding. The traditional 2D graphene-based monometallic composite might suffer from agglomeration, which shielded abundant active sites on the surface to a significant drop of the catalytic activity. On the contrary, the glutamic acid induced 3D structure and bimetallic anchoring approach markedly improved the catalytic activity (about 2 folds of graphene/Fe3O4NPs and 5 folds of graphene/AuNPs), as well as the catalysis velocity (about 6.1 and 1.1 times higher than those of HRP with tetramethylbenzidine (TMB) and H2O2 as substrates) and its affinity for substrate (with TMB and H2O2 as substrates, about 1.1 and 23 times stronger than those of HRP; with H2O2 as substrate, about 20, 3.6 and 3 times stronger than those of graphene oxide, graphene/Fe3O4 and graphene/Au, respectively.). What’s more, this aptasensor simplified the analysis process including labeling and modification in other methods.

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5.2. Enzyme-linked aptamer assay (ELAA) Owing to aptamers’ remarkable advantages over antibodies, they were used to replace antibodies in ELISA as the binder, and maintained ELISA’s high-throughput screening ability. A direct competitive enzyme-linked aptamer assay (dc-ELAA) utilizing both DNA and RNA aptamers as recognition elements was first established by Jeong et al.[101] to determine TET residues in milk. The biotinylated aptamer was immobilized onto microplate via avidin-biotin interaction. TET-HRP was used to compete with TET in samples and offered color change by reaction with TMB. But this method was not superior to the ELISA method in terms of specificity, detection limit (LODs in buffer were 32.7 nM for DNA aptamer-based ELAA, 21.0 nM for RNA aptamer-based ELAA and 0.174 nM for ELISA, respectively), and dynamic range. Changing the biotin-avidin interaction into biotin-streptavidin interaction to immobilize the biotinylated-aptamer, Wang et al.[57] built another dc-ELAA colorimetric aptasensor and successfully applied it to detect TET in honey. Using the same DNA aptamer, they also established an aptasensor[102] for TET detection in honey based on an indirect competitive enzyme-linked aptamer assay (ic-ELAA).

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ACCEPTED MANUSCRIPT They introduced a biotin-streptavidin mediated system to improve target detection efficiency. After competition between immobilized TET-bovine serum albumin (BSA) and free TET analyte for the biotinylated aptamer, the solution was incubated with the streptavidin-HRP conjugate, and followed by enzyme label detection. With the highly specific biotin-streptavidin combination, streptavidin-HRP brought a signal amplification. Similarly, Kim et al.[103] established an ic-ELAA for detection of OTC in milk. However, all these methods required a long-time (16 h or overnight) well/plate coating procedure. If these coated wells/plates could not be reused (which was unmentioned by all the researchers), they were quite time-consuming. An analytical method for the detection of OTC in chicken, milk and honey was proposed by Lu et al.[104] based on dc-ELAA and magnetic separation technique. In their method, OTC aptamer was immobilized on magnetic beads and the application of magnetic separation and the concentration effect of magnetic nanoparticles significantly improved the method’s efficiency (the detection time was 100 min, without considering of preparation time of functionalized-MNPs).

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5.3. Antibody-assisted catalytic colorimetric aptasensors The HRP-catalysis of TMB-H2O2 system to form a blue product (measured at 650 nm) is widely applied in colorimetric aptasensor. Three similar aptasensors have been reported for CAP determination as exemplified in Fig. 3. Their capture probe was fabricated using Fe3O4@Au magnetic nanoparticles (AuMNPs) as the core, immobilizing dsDNA[105] or cDNA[106] or dsDNA-antibody[40] on the core to bind to the tracer. Correspondingly, signal tracers were constructed by co-immobilizing HRP and dsDNA-antibody[105] or ssDNA-antibody[106] or dsDNA[40] on the core AuNPs[105, 106] or platinum nanoparticles (PtNPs)[40]. Upon target-aptamer binding and magnetic separation, signal tracers were substituted into supernatant to catalyze TMB-H2O2, so the target could be colorimetrically measured.

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6. Electrochemical aptasensors Electrochemical aptasensor has drawn increasing attention in recent years. Compared to other aptasensors, it is highly sensitive over the dynamic range of fM- mM for antibiotics. For instance, using the same peroxidase-like activity of AuNPs and target-induced replacement of aptamer for KANA detection, electrochemical aptasensor achieved much higher sensitivity (LOD=0.06 nM) than colorimetric aptasensor (LOD=2.28 nM)[107]. Meanwhile, its simple instrumentation, cheapness and miniaturation allow it for on-site detection. Various detection methods have been utilized in electrochemical aptasensors for antibiotics. Among these methods, electrochemical impedance spectroscopy (EIS) monitors the change of electron-transfer resistance to show impedance signal, square wave voltammetry (SWV), differential pulse voltammetry (DPV), cyclic voltammetry (CV) and linear sweep voltammetry (LSV) all observe faradaic current. Electroactive reporters such as methylene blue, hemin, hydroquinone, prussian blue, H2O2, ferrocene, thionine, ferri/ferrocyanide ([Fe(CN)6]3-/4-), metal ions and quantum dots have been used for signal transduction conjugated to the aptamer or its cDNA. The basic schemes of electrochemical aptasensors for antibiotics detection are shown in Fig. 4. Electrodes are modified by functional groups or nanomaterials to immobilize aptamers or standard targets. Analytes are directly bound to aptamers (Fig. 4 A) or compete with immobilized standard targets for aptamers (Fig. 4 B). Generally, sensing mechanisms include direct target-aptamer binding (Fig. 4 A-a), target-induced aptamer folding (Fig. 4 A-b), label intercalation into self-hybridized aptamer and target-induced label liberation (Fig. 4 A-c), and signal probe displacement (Fig. 4 A-d). Table 4 lists the

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ACCEPTED MANUSCRIPT electrochemical aptasensors used for antibiotics detection.

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6.1. Modification of electrodes Numerous electrochemical aptasensors have been fabricated for antibiotics detection using aptamers immobilized on various types of electrodes modified with different functional groups. As shown in Fig. 4 A-a, Pilehvar et al.[108] reported an EIS detection method using thiolated aptamers immobilized on a gold electrode (GE) for analysis of CAP. Song et al.[109] detected KANA by DPV immobilizing thiolated aptamer on a GE. Kim et al.[37] detected OTC at 1-100 nM by CV and SWV using thiolated OTC-aptamers immobilized on gold interdigitated array (IDA) electrode chip. Later, they[110] changed the gold IDA electrode to a streptavidin-modified screen-printed gold electrode (SPGE) and functionalized it with biotinylated TET-aptamers for detection of TET with broader detection range (10 nM- 10 µM). Zhang et al.[111] modified a glassy carbon electrode (GCE) with amino group and functionalized it with aptamer for detecting TET and OTC with LOD down to 1 ng/mL. Paniel et al.[44] analyzed penicillin G by EIS with aptamers and amine modified on SPCE. Wang et al.[112] detected ampicillin using DPV on indium tin oxide electrode. Instead of just being modified by functional groups, electrodes that modified by AuNPs, conducting polymers, graphene and multi-walled carbon nanotubes (MWCNTs) offered platforms with larger loading capacity for aptamer immobilization and accelerated electron transfer. When modified by MNPs, experimental processes were simplified with the help of external magnets.

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6.1.1. Gold nanoparticles (AuNPs) AuNPs, as good conductors, can accelerate the electron transfer of the electrode. Due to their good biocompatibility, AuNPs can be used as a versatile platform to provide biocompatible surface area for the immobilization of abundant aptamers. Accordingly, they were frequently used in electrochemical aptasensors. By fixing prussian blue-chitosan-glutaraldehyde (PB-CS-GA) onto the surface of GCE, AuNPs could be adsorbed on the electrode, facilitating the electron transfer and immobilizing aptamers to improve the sensitivity of the electrochemical aptasensor for TET detection[113]. This AuNPs/PB-CS-GA/GCE system showed good conductibility, biocompatibility, as well as higher sensitivity (LOD= 3.2×10-10 M, much lower than HPLC, an MIPs-based sensor etc.) and stability. Another AuNPs modified electrochemical aptasensor was constructed for determination of KANA[114], using molybdenum disulfide (MoS2) formed single or few-layer nanosheets to modify a GCE, then conjugated AuNPs on the MoS2 layer to increase electric conductivity and finally added hemin in as a synergetic catalyst towards the electroreduction of H2O2. An electrochemical aptasensor was developed by Bagheri et al.[115] for the determination of CAP based on the binding of thiolated CAP aptamer and AuNPs, and 1,4-diazabicyclo[2.2.2]octane (DABCO)-supported mesoporous silica SBA-15 (SBA-15@DABCO) modified SPGE. Owing to the high surface to area ratio, porosity, uniform pore size distributions and thermal stability of SBA-15, large amount of AuNPs deposited on SBA-15@DABCO and formed a dendritic Au nanostructure.

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6.1.2. Conducting polymers Conducting polymers can be utilized to modify electrodes as well, since their functional groups can ensure stable immobilization of biomolecules on the electrode and increase the surface area of aptamer binding sites. For instance, Dapra et al.[116]prepared an all-polymer microfluidic biosensor using TopasR as substrate and a conductive polymer bilayer as electrode material for

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ACCEPTED MANUSCRIPT detecting ampicillin and KANA A. Conductive bilayer consisted of tosylate doped poly(3,4-ethylenedioxythiophene) (PEDOT: TsO) and the hydroxymethyl derivative PEDOT-OH: TsO, which was covalently functionalized with two aptamers to ampicillin and KANA A, respectively. Another electrochemical aptasensor[117] was built for CAP detection, using aptamers to immobilize on the poly-(4-amino-3-hydroxynapthalene sulfonic acid) (p-AHNSA) modified edge plane pyrolytic graphite (EPPG) electrode. The polymer electrodes were much cheaper than the metallic electrodes, but their low conductivity limited their use in electrochemical sensors. Chandra et al.[118] improved the conductivity by combining the high conductive AuNPs with the conducting polymer polyTTBA layer to establish an electrochemical aptasensor for daunomycin detection. Compared with the sensor modified with electrodeposited AuNPs and poly-2,5-di-(2-thienyl)- 1H-pyrrole-1(p-benzoic acid) (poly-DPB), a self-assembling of conducting polymer on AuNPs scheme further improved the sensitivity of the sensor about five times[119]. The sensor was fabricated by covalently immobilizing a ssDNA KANA aptamer onto an AuNPs-comprised conducting polymer. The polymer was constructed by self-assembling of DPB on AuNPs that prepared on a screen-printed electrode. CV and LSV were applied to detect KANA.

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6.1.3. Graphene A novel composite film consisting of a magnetic graphene nanocomposite (GR-Fe3O4NPs) and a PEDOT-AuNPs composite was used to modify a bar GCE for an electrochemical aptasensor of penicillin detection[120]. OTC was analyzed via the antibody-OTC-aptamer sandwich-type electrochemical aptasensor[121], employing graphene-three dimensional nanostructure gold nanocomposite (GR-3D Au) to modify the GCE, and aptamer-AuNPs-HRP was used to capture targets. The use of the AuNPs as capture probes could load a large number of aptamers, yielding an amplification effect. A simple synthesized porous carbon nanorod (PCNR) fabricated with porous carbon nanosphere and multifunctional graphene composite (GR-Fe3O4-AuNPs) exhibited high electrocatalytic activity, large specific pore volume and high specific surface area. It has been utilized to modify GCE for detecting STR[122]. Thionine functionalized graphene (GR-TH) and hierarchical nanoporous (HNP) PtCu alloy were employed to construct an electrochemical aptasensor for determining KANA in pork meat and chicken liver[123]. Here, GR-TH composite acted as a charge-transfer bridge to facilitate the electrode transfer rate, while TH performed as an electron transfer mediator whose electroactivity was greatly enhanced in combination with GR or HNP-PtCu. A competitive aptasensor for OTC detection was reported by Xu et al.[124], who immobilized the GO-conducting polymer polyaniline (PANI) film on the surface of the GCE and then electrodeposited AuNPs on the electrode surface. The incorporation of PANI in GO could promote the composite in its higher electrochemical capacitance and charge-discharge cyclic stability. The researchers[125] have also constructed an antibody-KANA-aptamer sandwich-type electrochemical aptasensor by introducing polyamidoamine (PAMAM) dendrimers, a KANA-antibody, assembled GR-PANI and PAMAM-AuNPs nanocomposites on the surfaces of a GCE.

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6.1.4. Multi-walled carbon nanotubes (MWCNTs) MWCNTs were applied to modify electrodes as well to immobilize the TET aptamer and to construct an electrochemical aptasensors[126]. Guo et al.[127] utilized a composite film consisted of MWCNTs, ionic liquid of 1-hexyl-3-methylimidazolium hexafluorophosphate (HMIMPF6), and

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ACCEPTED MANUSCRIPT nanoporous PtTi (NP-PtTi) alloy to fabricate an electrochemical aptasensor for KANA detection. Sun et al.[128] developed an electrochemical aptasensor for the detection of kanamycin with improved response time compared to the aptasensor without nanomaterials based on the synergistic contributions of chitosan-gold nanoparticles (CS-AuNPs), graphene-gold nanoparticles (GR-AuNPs) and MWCNTs-cobalt phthalocyanine (MWCNTs-CoPc) nanocomposites.

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6.1.5. Magnetic nanoparticles (MNPs) Electrochemical detection of TET in tablet, milk, honey and blood serum was achieved using aptasensors based on both a modified oleic acid (OA)-carbon paste electrode (CPE) and a Fe3O4 MNPs-OA-magnetic bar carbon paste electrode (MBCPE)[129]. In the structure of CPE, OA was used to modify the surface of the electrode to provide a free carboxyl group which offered a suitable base for attaching the TET aptamer. To fabricate an MBCPE/Fe3O4MNPs@OA/aptamer aptasensor, MBCPE was constructed, then Fe3O4MNPs was used to modify the electrode surface followed by OA and TET aptamer. The result showed that the MBCPE-Fe3O4MNPs-OA aptasensor (LODs= 3.8 fM by EIS and 0.31 pM by DPV) showed higher sensitivity than the CPE-OA aptasensor (LODs= 0.3 pM by EIS and 29 pM by DPV). The use of MNPs leaded a better immobilization process at the electrode surface and allowed the placement of a magnet on the electrode surface which leaded to the physical attraction of iron oxide, resulted in high selectivity, sensitivity, reproducibility and stability. As shown in Table 4, taking KANA as the model target, probably due to their excellent electron transfer ability, complex aptasensors[125, 127] with many components usually showed higher sensitivities. But it was not the higher complexity the aptasensor possessed, the higher sensitivity it achieved[128]. Aptasensors with simpler fabrication and better performance are recommended.

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6.2. Competitive electrochemical aptasensors Using immobilized standard targets to compete with free targets in samples for aptamers, two competitive electrochemical aptasensors were built using EIS as a transducer to detect neomycin B[49] and tobramycin[50]. As shown in Fig. 4 B, standard analytes were immobilized on gold electrodes, binding specific aptamers to their surface. In the presence of targets, the competitive displacement of surface-bound aptamer occurred to form a complex with the target analytes in solution and release from the surface, leading to a drop in the electron-transfer resistance. A competition between MPs-immobilized tobramycin and free tobramycin for the limited amount of the biotinylated-aptamer was employed to detect tobramycin by Fernández et al. too[58]. Also, a competitive aptasensor for OTC detection was explored, using the complete antigen OTC-BSA to fix on an AuNPs/GO-PANI modified GCE, and OTC in sample to compete with OTC-BSA to bind to HRP-aptamer[124].

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6.3. Folding-based aptasensors Compared with the modification-based electrochemical means, folding-based electrochemical sensing based on the inner structural change of aptamers is simple, time-saving and convenient. As shown in Fig. 4 A-b, Pilehvar et al.[130] developed an electrochemical aptasensor to detect CAP using a thiolated ssDNA aptamer which could change into a hairpin structure in response to CAP to fix onto a GE surface. The hairpin structure brought the target molecules close to the surface of the electrode and triggered electron transfer. Schoukroun-Barnes et al.[33] developed a mixed ratio sensor surface using a combination of a mutant aptamer[54] and

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ACCEPTED MANUSCRIPT its parent aptamer[31] of different binding abilities to tobramycin. By varying the ratio of these two aptamers on a single sensor surface, the affinity, dynamic range, and sensitivity of the resulting sensors could be tuned and predicted by a bi-Langmuir-type isotherm. The properties of particular aptamer-based sensors could be tuned by the intercalation of a DNA dye to stabilize the long bases ssDNA aptamer containing self-hybridized segments. Pilehvar et al.[131] used proflavine as this specific intercalator for the self-hybridized dsDNA segments of an 80 bases CAP ssDNA aptamer which was immobilized on gold electrodes. As shown in Fig. 4 A-c, upon addition of the target CAP, the intercalator was released.

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6.4. Signaling-probe displacement aptasensors The dependence of aptamers’ conformational change-introduced signal transduction requires well-designed aptamers, which limits their general applications. Liu et al.[132] constructed a general signaling-probe displacement electrochemical aptamer-based sensor, using KANA A as model, a short cDNA as the capture probe and a ferrocene-labeled aptamer as the signal probe (Fig. 4 A-d). As a result, signal transduction was independent of the conformational state of the aptamer. Since the short cDNA and the aptamer used as capture and signal probes could be exchanged, the aptasensor using short cDNAs as the signaling probes was found to be 500-fold more sensitive than that using aptamers as the signaling probes.

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6.5. Electrochemiluminescent aptasensors Electrochemiluminescent (ECL) aptasensors possess the advantages such as wide dynamic range, high specificity and sensitivity. A label-free ECL aptasensor for determination of KANA was developed by Zhao et al.[133] based on an “on-off-on” switch system. As shown in Fig. 5, the first “switch on” state was obtained by the tri-layer composite films modified glassy carbon electrode towards the S2O82--O2 system. The AuNPs decorated C60 nanoparticles (Au@nano-C60) was as the inner-layer. The poly-L-histidine (PLH) as a coreactant of S2O82--O2 system was adsorbed on the Au@nano-C60 modified electrode as the inter-layer. A self-assembling layer of colloidal AuNPs was the outer-layer. Successively, KANA aptamers were anchored on the tri-layer composite films modified electrode. In the presence of hemin, the DNA hybridization reaction of assistant probes (guanine-rich nucleic acid) with aptamers generated a large amount of hemin/G4 DNAzymes (DNA oligonucleotides that are capable of performing a specific chemical reaction, here they performed catalytic ability). Accordingly, the “switch off” state was made by the quenching effect of hemin/G4 DNAzymes which electro-catalyzed the reduction of dissolved O2 of S2O82--O2 system to reduce the background signal for further improving sensitivity. Attributing to the formation of target KANA-aptamer complex, the hemin/G4 DNAzymes was released from the sensing interface, in response the ECL signal recovered to exhibit the second “switch on” state. This aptasensor had a low LOD of 45 pM. To eliminate the possible false positive signals of ECL aptasensors[134], Feng et al.[135] designed a screen-printed carbon electrode (SPCE) array composed of two spatial-resolved working electrodes (WE1 and WE2) as the platform for R-ECL detection of CAP based on the ratio of working signal to internal reference signal. The ratio of ECL signal from working tags (luminol-AuNPs, L-AuNPs) compared with the internal standard tags (CdS QDs) provided a correction to eliminate the environmental interferences. The two ECL signal tags were separately immobilized on two separate working electrodes, hence the working signal and internal standard signal were respectively generated at different electrode, which eliminated the potential crosstalk

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ACCEPTED MANUSCRIPT and cross-reactivity, hence the stability was improved. The authors have further employed this R-ECL to fabricate a “dual-potential” ECL aptasensor array[136] for simultaneous detection of malachite green (MG) and CAP in one single assay. With CdS QDs and L-AuNPs labels respectively modified on WE1 and WE2, the distinct ECL signal potentials not only avoided the potential crosstalk and cross-reactivity as much as possible, but also reflected the identity of the targets MG and CAP and enabled the multiplexed capability of the aptasensor array.

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6.6 Photoelectrochemical aptasensors Photoelectrochemical (PEC) sensors combine the advantages of optical methods and electrochemical sensors, having great potential in analytical applications. In PEC sensors, semiconductors especially visible light-active materials with narrow band-gap may absorb visible light more efficiently, and also avoid high background arising from the direct UV photolysis of analytes[137]. Using water-dispersible graphite-like carbon nitride (w-g-C3N4) as visible light-active material, doped by metal-free GO, a PEC aptasensor for the detection of KANA was reported by Li et al.[138]. Liu et al.[137] constructed a PEC sensing platform for CAP detection using synthesized nitrogen-doped graphene quantum dots (N-GQDs) as visible light-responsive materials. Yan et al.[139] employed a p-type semiconductor BiOI together with graphene to fabricate a BiOI-graphene nanocomposites based cathodic “signal-off” PEC aptasensor for OTC detection.

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7. Other aptasensors Other aptasensors including SPR, scattering spectra and cantilever aptasensors have been used to detect antibiotics combining with aptamers (Table 5). SPR is a surface-sensitive label-free technique and has been applied in aptasensors to analyze neomycin B[51] and tobramycin[140]. To miniaturize the tobramycin aptasensor[140], Cappi et al. built a portable, palm-sized transmission-localized surface plasmon resonance (T-LSPR) setup. With this T-LSPR setup, real-time label-free direct determination of tobramycin was realized in buffer and in filtered undiluted blood serum. Kim et al.[141] developed a so-called “light scattering particle immunoagglutination assay” (LSPIA) which could provide real-time detection of OTC through agglutination assay of ssDNA aptamer conjugated polystyrene (PS) latex microspheres by proximity optical fibers. This application offered a versatile platform for the detection of various antibiotics in one-step and needed no sample pretreatment. Luo et al.[142] proposed a resonance scattering (RS) aptasensor for detecting TET in milk, exploiting AuNPs’ strong catalytic activity on Fehling reaction, which could reduce the Cu2+-glucose system to yield Cu2O cubic thus enhance the RS intensity along with the increase of non-aggregated AuNPs’ concentration. Surface-enhanced Raman scattering (SERS) can provide a non-destructive approach for identifying molecular species by the unique fingerprint spectrum, which effectively avoids the interference of background signal and improves the specificity of detection. Based on these features, a SERS sensing platform was established by Yan et al.[143] to analyze CAP through embedding Cy5-labeled aptamer-cDNA between the SERS-active Au core-Ag shell nanostructures (Au@AgNSs). The presented CAP competitively bound to the aptamer, dissociate the Cy5-labeled aptamer from AuNPs surface, hence greatly decreases the SERS signals of Au@AgNSs. Cantilever sensors can be applied for the label-free detection as a real-time and highly sensitive approach to detect biomolecules. An aptamer-based cantilever array sensor for OTC

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ACCEPTED MANUSCRIPT detection was developed by Hou et al.[144]. The sensing cantilevers in the array were functionalized with self-assembled monolayers (SAMs) of OTC aptamer, which acted as a recognition molecule for OTC. Once OTC interacted with the aptamer, a surface stress changed, which caused the sensing cantilevers to bend downward, resulting in a deflection of the cantilever instantaneously. Reference cantilevers were utilized to eliminate the influence of environmental disturbances, such as temperature and nonspecific adsorption. Similarly, an aptamer-based cantilever sensor was fabricated to quantify KANA by this team[145]. Their KANA detection cantilever method was highly selective, but its detection limit (50 µM) was not satisfactory compared with other techniques.

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8. Some sensing strategies for antibiotic aptasensors 8.1. Microfluidic biosensors Microfluidic systems fabricated on silicon, glass or other polymers have attracted significant attention due to their miniaturization, integration, and automation. For instance, to achieve real-time detection of OTC, Kim et al.[141] conducted a LSPIA inside a Y-channel PDMS microfluidic device. Dapra et al.[116] fabricated an all-polymer electrochemical microfluidic biosensor as well to detect ampicillin and KANA A. The polymer-based microfluidic system met the requirements of disposable devices for low sample consumption, cost efficiency, reliability, and fast response time, which made the systems ideal for rapid analysis. Additionally, a biosensor platform of microfluidic electrochemical detector for in vivo continuous monitoring (MEDIC) was constructed by Ferguson et al.[146] to detect doxorubicin and KANA in real-time. This device required no exogenous reagents, operated at room temperature, and could be reconfigured to measure different target molecules by exchanging aptamer probes in a modular manner. Paper based microfluidic device, has been used by Zhang et al.[72] to detect neomycin.

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8.2. Enzyme-recycled amplification aptasensors When analytes or signal indicators were substituted into solution containing DNA polymerase or endonuclease, a target recycling system was triggered. For instance, as shown in Fig. 3, utilizing exonuclease I (Exo I) which could digest ssDNA, Miao et al.[40] established a colorimetric aptasensor for CAP sensing. When CAP was captured by the aptamer on the signal probes, the aptamer-cDNA duplex departed. As the single-stranded aptamer and cDNA could not be recognized by the dsDNA-antibody on MNPs core capture probes, after magnetic separation the CAP-signal tracer complex and cDNA were released into the supernatant. With the assistance of Exo I in the supernatant, further digestion of the bound aptamer-CAP happened and CAP was released again to further participate in a new cycling process to react with the probes. In a like manner, they detected CAP using a CdSe QDs-SiO2@Au FRET based aptasensor with Exo I amplified signal[77]. Ramezani et al.[147] employed Exo III to digest the KANA-aptamer and its FAM-labeled cDNA formed dsDNA to recycle into strong fluorescence emission. The present target KANA competitively bound to the aptamer and left FAM-cDNA to be quenched on AuNPs surface. Since no dsDNA formed, Exo III could not show any activity. In the absence of KANA, aptamer formed dsDNA with FAM-cDNA to bring FAM-cDNA away from AuNPs and recovered the fluorescence. The formed dsDNA had unique characteristic 3’-overhang end at aptamer and 3’-blunt end at the cDNA. So upon the addition of Exo III, only the FAM-labeled cDNA was digested and leaded to the release of aptamer. The released aptamer interacted with the FAM-labeled cDNA on the surface of AuNPs again and the cycle went on as mentioned above.

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ACCEPTED MANUSCRIPT Thus, the catalytic recycling of aptamer by Exo III amplified the fluorescence with minimum consumption of aptamer. Wang et al.[148] proposed a cascade enzymatic amplification approach for electrochemical detection of ampicillin. By coupling the target-triggered structural change of programmable hairpin probes with polymerase and nicking endonuclease-assisted target recycling, the target ampicillin achieved a high quadratic amplified signal. Likewise, several electrochemical aptasensors were established based on enzyme-recycled amplification[112, 149-154]. These enzyme-amplification allowed reaching detection limits as low as 0.0002 ng/mL[77], signal improvements of 10- 15 [40, 77, 152, 153] orders of magnitude over detection strategies that no enzyme-recycled amplifications were used. These approaches showed good reproducibility with relative standard deviations (RSDs) less than 8 % and were successfully used with real samples such as milk[40, 112, 147-154], serum [147, 151, 154] and seafood [77].

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8.3. Multiplex detection To save analysis time and increase efficiency, one of the key aims for developing the detection methods is to easily and rapidly measure multiplex antibiotics in one course. A salt-induced AuNPs aggregation-based colorimetric aptasensor was reported by Niu et al.[155] for multiplex detection of SDM, KANA and adenosine using three aptamers with the same sensitivity as a corresponding single-target aptasensor. The use of an universal buffer allowed the omission of salt, which simplified the sensing procedure. Nevertheless, the multiplex aptasensor could not distinguish the targets. With multi-metal ions encoded nanospherical brushes as nanotracers, an electrochemical aptasensor was established by Yan et al.[156] to simultaneously detect CAP and polychlorinated biphenyl-72 (PCB72). The aptamers of CAP and PCB72 were labeled on magnetic gold nanoparticles as capture probes (aptamer-MGPs), and their cDNA encoded with Cd2+ and Pb2+ respectively were immobilized on nanospherical branched polyethylene imine brushes as tracers (cDNA-MSPEIs). The Cd2+ and Pb2+ tags could be simultaneously detected referring to the concentrations of CAP and PCB72, respectively. Another Cd2+ and Pb2+ signal tags utilized electrochemical aptasensor was reported by Chen et al.[157] for multiplex determination of KANA and CAP. In this aptasensor, amine-functionalized nanoscale metal organic framework (NMOF) was employed as the carrier of the metal ions (M-NMOF) due to its large internal surface areas, ultrahigh porosity and abundant amine groups in the pores. Consisted with this strategy, they achieved simultaneous detection of OTC and KANA based on the Cd2+ and Pb2+ labeled M-NMOFs signal tracers, while introduced RecJf exonuclease (a kind of ssDNA specific exonuclease)-catalyzed targets recycling amplification[152]. They also used QDs as signal tags to simultaneously determine CAP and PCB72 basing on the interaction between Fe3O4@AuNPs-aptamers and their QDs-tagged cDNAs (cDNA1-CdS or cDNA2-PbS)[158]. Moreover, simultaneous detection of STR, CAP and TET residues was achieved by Xue et al.[159] using the specific aptamers as recognition elements and PbS, CdS, ZnS QDs as tags. However, presumably ascribed to the degradation of the oligonucleotides in milk matrix, less QDs were immobilized onto the electrode surface, in turn, a slight signal suppression existed in milk samples compared with in buffers.

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9. Aptamer affinity-based chromatographic purification The antibiotic-aptamer binding reaction was applied in affinity purification early in 1999 as an assistant technique to isolate proteins that interacted with modified functional RNA[160]. STR was coupled to sepharose to form the affinity stationary phase of the purification column. The

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ACCEPTED MANUSCRIPT STR-specific aptamer sequences were combined to a protein-binding RNA as the tag. After incubation of the tagged RNA with crude extract, the binding of the in RNA-protein complex to STR-Sepharose was mediated by the aptamer. Subsequent elution with free STR allowed the recovery of the specifically bound proteins. Nevertheless, the antibiotic-aptamer binding based purification column had not been employed in antibiotic determination until 2013 by Aslipashaki et al.[161]. In their study, a combination of solid-phase extraction using amino modified TET aptamer to mix with CNBr-activated sepharose as sorbent and electrospray ionization-ion mobility spectrometry (ESI-IMS) was adopted for the determination of TET in human urine and plasma. This method achieved LODs of 0.019 µg/mL in urine and 0.037 µg/mL in plasma with recoveries of 84 %- 91 % in urine and 77 %- 89 % in plasma. An aptamer-based organic-silica hybrid monolithic capillary column was synthesized by Jiang et al.[162] to separate the enantiomers of doxorubicin and epirubicin followed by ultraviolet detection. This prepared monolithic column had good stability and permeability, large specific surface, and showed excellent selectivity towards the enantiomers of doxorubicin and epirubicin. When applied in biological samples, this method showed LOD of 0.3 µg/mL and recoveries of 90.1 %- 97.8 % in both serum and urine. Hence, such a combination of aptamer-based purification and instrumental detection exhibits potential use in enantiomers separation and low-level analytes detection in complicated matrix.

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10. Sample detection Antibiotic residues are widely found in foods, environments and biological samples. But up to now, most established aptasensors were just applied to buffers or simple samples like water. In fact, the usability of aptasensors in samples should be well investigated. Firstly, the antibiotic-specific aptasensor may suffer from cross-reaction even it is sensitive and precise[101]. Secondly, signal suppression usually exists in the detection of real samples compared with those in buffers[56, 151, 159]. Thirdly, for different matrix such as milk and serum the performance of a same method may differ as well[56]. To our knowledge, samples involved in the reported methods included water[46, 84, 132], milk[104, 129], honey[43, 104, 129], urine[163], serum[129, 163], plasma, drug[117, 129, 139] and animal tissues such as chicken[104, 123], fish[79, 105, 106, 135, 136, 158], duck [106], pork[123] and in vitro cells supernatant[117]. The contained protein, fat, carbohydrate, ions and high viscosity for serum and honey may affect the performance of aptasensors. Ionic strength is a critical factor for the recognition event[50] and some metal ions can contribute to the stability and structure of DNA[57, 102], but cations such as Ca2+ and Mg2+ which can form chelation complexes with various antibiotics[101] have negative effect on the combination of antibiotics and aptamer[57, 102]. Proteins are tending to induce nonspecific adsorption of the enzymatic conjugate which may bring false-positive signals[53], so they should be removed. Dilutions were usually utilized to maintain constant the ionic strength of the sample solution[53], preclude the nonspecific absorption[42, 53], liberate the protein associated antibiotics to free state and pass through the filter in some degree[53] and reduce the high viscosity[43]. Some materials-assisted aptasensors require mild conditions as the extreme conditions (acids, alkalis and high temperature) can decrease the performance of composite probes[158]. Thereupon, samples were usually pretreated by dilution, filtration, degreasing, deproteinization and extraction. Despite the fact that analysis of antibiotics in samples is challenging, several researchers have done good jobs in various complex matrix with detailed performance evaluation. For instance, Lu

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ACCEPTED MANUSCRIPT et al.[104] applied an ELAA-based aptasensor for detection of OTC in chicken, milk, and honey with recoveries ranging from 71.0 % to 91.2 % with RSDs of 3.17%-11.94%. In their study, 40 food samples from the laboratory stock were comparatively analyzed using both the proposed method and HPLC to assess the accuracy and applicability. Qin et al.[123] conducted GR-TH/HNP-PtCu based electrochemical detection of KANA in pork and chicken liver. Employing the established CPE-OA based aptasensor and MBCPE-Fe3O4MNPs-OA based aptasensor, Jahanbani et al.[129] detected TET in tablet, milk, honey and blood serum. Zheng et al.[163] established an electrochemical aptasensor and directly detected OTC in unpurified mouse serum and urine. They obtained repeatable and nearly identical results compared with an HPLC method.

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11. Conclusion and perspectives Aptamers have been utilized in antibiotics detection, following the basic principles, involving signal-tagging, conformation switch, mass change, materials and enzymes association. To meet the requirement of trace determination of antibiotics in foods, drugs and environmental samples, efforts have been made to achieve signal enhancement by material assistance, conformation change enlargement and enzyme-recycled amplification. Several materials have been employed to indicate the signal change, offer platforms for aptamer immobilization, accelerate electron transfer and provide signal amplification. Particularly, QDs and UCNPs can offer highly sensitive luminescence to allow the detection and even multiplex detection. AuNPs have widely been used owing to their superior colorimetric and electrochemical properties. AuNPs need to react with thiol or amino modified aptamers when used as the carrier, while CPNB, GO and MWCNTs can adsorb unmodified aptamers via π−π stacking with large loading capacity, though π−π interaction is not so stable. Conductive polymers provide a platform of high loading capacity for aptamers immobilization. The application of MBs or MNPs as bioreceptor or solid support greatly simplifies the experimental processes with the help of external magnets. The combination of aptamers with enzyme-cycled amplification enhances detection signal dramatically. Most of the developed aptasensors are for a single use, while some are for multiple tests[50, 114, 157]. As shown in the tables, their reproducibilities are satisfying. Compared with the standard HPLC or ELISA methods, the response time, sensitivity, selectivity and repeatability of many aptasensors are acceptable. Due to the limited number of aptamers available at present, current aptamer-based analytical methods are mainly focused on antibiotics like KANA, TET and OTC. Aptamers for more antibiotics with high affinity and specificity are still required. With regard to the analysis of antibiotics in complicate sample matrices, many studies showed certain degree of signal suppression. Furthermore, quite a large amount of antibiotic-specific aptamers have not applied in real samples. So the development of aptamer-based methods for detection of antibiotics in different matrix should be emphasized in the future. For commercial use, though ELISA is a quite mature technique, aptamers have many advantages over antibodies to make aptamer-based analysis kits promising. Despite the fact that another molecular recognition unit MIPs already has some antibiotic-specific commercial products for sample preparation, yet no antibiotics-specific aptamer-based kits and columns for antibiotics appeared. Various aptasensors and aptamer affinity-based chromatographic purification methods have been established. Among these methods, luminescent aptasensors with fluorescent detection show

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high sensitivity and efficiency, though signal probes labeling are usually required which may influence the target-binding affinity of aptamer. New fluorescent probes like UCNPs bring the possibility for robust sample analysis with less interference; Colorimetric aptasensors exhibit great convenience in read-out naked-eye analysis and on-site detection, they can be applied for multi-channel detection but not multiplex detection; Electrochemical aptasensors have advantages in high sensitivity, simple instrumentation, the ability to be readily miniaturized and low cost, yet they need electrode modification; SPR, scattering spectra and cantilever sensors are label-free, but their sensitivity and reproducibility are still an impediment. Aptamer-based columns and sorbents need relatively large amounts of aptamers, but they can be used for more than one time. Considering the need of multiplex detection and label-free strategy, the aptamer affinity-based purification combining to instrumental detection is a potential approach to achieve high-throughput analysis with higher efficiency and lower cost. Further focus can be put beyond aptamer affinity-based column separation which needs tedious fabrication. As for therapy monitoring, real-time, small-size and easy-to-use devices are expected. This can be achieved by microfludic devices, while their applications in real samples are still required to exclude the possible interference.

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ACCEPTED MANUSCRIPT oxytetracycline using light scattering agglutination assay with aptasensor. Electrophoresis 31 (2010) 3115-3120. [142] Y. Luo, L. He, S. Zhan, Y. Wu, L. Liu, W. Zhi, and P. Zhou, Ultrasensitive resonance scattering (RS) spectral detection for trace tetracycline in milk using aptamer-coated nanogold (ACNG) as a catalyst. J. Agri. Food Chem. 62 (2014) 1032-1037. [143] W. Yan, L. Yang, H. Zhuang, H. Wu, and J. Zhang, Engineered “hot” core–shell nanostructures for patterned detection of chloramphenicol. Biosens. Bioelectron. 78 (2016) 67-72. [144] H. Hou, X. Bai, C. Xing, N. Gu, B. Zhang, and J. Tang, Aptamer-based cantilever array sensors for

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oxytetracycline detection. Anal. Chem. 85 (2013) 2010-2014.

[145] X. Bai, H. Hou, B. Zhang, and J. Tang, Label-free detection of kanamycin using aptamer-based cantilever array sensor. Biosens. Bioelectron. 56 (2014) 112-116.

[146] B.S. Ferguson, D.A. Hoggarth, D. Maliniak, K. Ploense, R.J. White, N. Woodward, K. Hsieh, A.J. Bonham, M. Eisenstein, T.E. Kippin, K.W. Plaxco, and H.T. Soh, Real-time, aptamer-based tracking of circulating therapeutic

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agents in living animals. Sci. Transl. Med. 5 (2013) 213ra165.

[147] M. Ramezani, N.M. Danesh, P. Lavaee, K. Abnous, and S.M. Taghdisi, A selective and sensitive fluorescent aptasensor for detection of kanamycin based on catalytic recycling activity of exonuclease III and gold nanoparticles. Sens. Actuators B Chem. 222 (2016) 1-7.

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[148] H. Wang, Y. Wang, S. Liu, J. Yu, W. Xu, Y. Guo, and J. Huang, Target-aptamer binding triggered quadratic recycling amplification for highly specific and ultrasensitive detection of antibiotics at the attomole level. Chem. Commun. (Camb) 51 (2015) 8377-8380.

[149] H. Wang, Y. Wang, S. Liu, J. Yu, Y. Guo, Y. Xu, and J. Huang, Signal-on electrochemical detection of antibiotics at zeptomole level based on target-aptamer binding triggered multiple recycling amplification. Biosens. Bioelectron. 80 (2016) 471-476.

[150] H. Wang, Y. Wang, S. Liu, J. Yu, Y. Guo, Y. Xu, and J. Huang, Signal-on electrochemical detection of

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antibiotics based on exonuclease III-assisted autocatalytic DNA biosensing platform. RSC Adv. 6 (2016) 43501-43508.

[151] S.M. Taghdisi, N.M. Danesh, M. Ramezani, and K. Abnous, A novel M-shape electrochemical aptasensor for ultrasensitive detection of tetracyclines. Biosens. Bioelectron. 85 (2016) 509-514. [152] M. Chen, N. Gan, Y. Zhou, T. Li, Q. Xu, Y. Cao, and Y. Chen, An electrochemical aptasensor for multiplex

EP

antibiotics detection based on metal ions doped nanoscale MOFs as signal tracers and RecJf exonuclease-assisted targets recycling amplification. Talanta 161 (2016) 867-874. [153] Z. Yan, N. Gan, T. Li, Y. Cao, and Y. Chen, A sensitive electrochemical aptasensor for multiplex antibiotics detection based on high-capacity magnetic hollow porous nanotracers coupling exonuclease-assisted cascade target

AC C

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recycling. Biosens. Bioelectron. 78 (2016) 51-57. [154] N. Mohammad Danesh, M. Ramezani, A. Sarreshtehdar Emrani, K. Abnous, and S.M. Taghdisi, A novel electrochemical aptasensor based on arch-shape structure of aptamer-complimentary strand conjugate and exonuclease I for sensitive detection of streptomycin. Biosens. Bioelectron. 75 (2016) 123-128. [155] S. Niu, Z. Lv, J. Liu, W. Bai, S. Yang, and A. Chen, Colorimetric aptasensor using unmodified gold nanoparticles for homogeneous multiplex detection. PLOS ONE 9 (2014) e109263. [156] Z. Yan, N. Gan, D. Wang, Y. Cao, M. Chen, T. Li, and Y. Chen, A "signal-on'' aptasensor for simultaneous detection of chloramphenicol and polychlorinated biphenyls using multi-metal ions encoded nanospherical brushes as tracers. Biosens. Bioelectron. 74 (2015) 718-724. [157] M. Chen, N. Gan, Y. Zhou, T. Li, Q. Xu, Y. Cao, and Y. Chen, A novel aptamer- metal ions- nanoscale MOF based electrochemical biocodes for multiple antibiotics detection and signal amplification. Sens. Actuators B Chem. 242 (2017) 1201-1209.

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ACCEPTED MANUSCRIPT 1366 1367 1368 1369 1370 1371 1372 1373 1374 1375 1376 1377 1378 1379 1380 1381

[158] M. Chen, N. Gan, H. Zhang, Z. Yan, T. Li, Y. Chen, Q. Xu, and Q. Jiang, Electrochemical simultaneous assay

1382 1383 1384 1385 1386 1387 1388 1389 1390 1391 1392 1393 1394 1395 1396 1397 1398 1399 1400 1401 1402 1403 1404 1405

Figure captions:

of chloramphenicol and PCB72 using magnetic and aptamer-modified quantum dot-encoded dendritic nanotracers for signal amplification. Microchim. Acta 183 (2016) 1099-1106. [159] J. Xue, J. Liu, C. Wang, Y. Tian, and N. Zhou, Simultaneous electrochemical detection of multiple antibiotic residues in milk based on aptamers and quantum dots. Anal. Methods 8 (2016) 1981-1988. [160] R.S.A.U. M. Bachler, StreptoTag: a novel method for the isolation of RNA-binding proteins. RNA 5 (1999) 1509-1516.

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[161] S.N. Aslipashaki, T. Khayamian, and Z. Hashemian, Aptamer based extraction followed by electrospray ionization-ion mobility spectrometry for analysis of tetracycline in biological fluids. J. Chromatogr. B 925 (2013) 26-32.

[162] H.P. Jiang, J.X. Zhu, C. Peng, J. Gao, F. Zheng, Y.X. Xiao, Y.Q. Feng, and B.F. Yuan, Facile one-pot synthesis of an aptamer-based organic-silica hybrid monolithic capillary column by "thiol-ene" click chemistry for

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detection of enantiomers of chemotherapeutic anthracyclines. Analyst 139 (2014) 4940-4946.

[163] D. Zheng, X. Zhu, X. Zhu, B. Bo, Y. Yin, and G. Li, An electrochemical biosensor for the direct detection of

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oxytetracycline in mouse blood serum and urine. Analyst 138 (2013) 1886-1890.

Fig. 1 Luminescent aptasensors for detection of antibiotics.

A: The employment of UCNPs as signaling probes (adapted from [63] with permission). B: Quenching effect of AuNPs, CPNBs and graphene. C: Inner structural change of a kanamycin aptamer (adapted from [82] with permission). D: THMS system (adapted from [84] with permission). E: The application of hemin/G4 as the

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indicator (adapted from [85] with permission). The abbreviations and more details are given in the text.

Fig. 2 Colorimetric aptasensor for antibiotics detection based on salt-induced AuNPs aggregation (reproduced from [41] with permission).

Fig. 3 The scheme of a triple-amplification colorimetric assay for CAP based on magnetic aptamer-enzyme

permission).

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co-immobilized platinum nanoprobes and exonuclease-assisted target recycling (reproduced from [40] with

AC C

Fig. 4 Basic schemes of antibiotics electrochemical aptasensors. Electrodes are without or within modifications (here supposing within), possessing functional groups or materials to immobilize aptamers or standard target. Schemes A-a, b, c, d represent direct target-aptamer binding, target-induced aptamer folding, label intercalation into self-hybridized aptamer and target-induced label liberation, and signal probe displacement, respectively. Scheme B describes the competition between immobilized standard targets and free targets.

Fig. 5 The “on-off-on” switch system of an electrochemiluminesence aptasensor for KANA (reproduced from [133] with permission).

33

ACCEPTED MANUSCRIPT

Targets CAP

a

Aptamer sequences

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Table 1 Sequences of the frequently used aptamers for detection of antibiotics Dissociation constants (Kd)

Aptamer 7: 5'-AGC AGC ACA GAG GTC AGA TG -ACT TCA GTG AGT TGT CCC ACG

Aptamer 7: Kd[CAP]= 0.766 µM

GTC GGC GAG TCG GTG GTA G- CCT ATG CGT GCT ACC GTG AA-3’

Aptamer 16: Kd[CAP]= 1.16 µM

Refs [30]

SC

Aptamer 16: 5'-AGC AGC ACA GAG GTC AGA TG -ACT GAG GGC ACG GAC AGG AGG GGG AGA GAT GGC GTG AGG T- CCT ATG CGT GCT ACC GTG AA-3’ Tobramycin

X1: GGG ACU UGG UUU AGG UAA UGA GUC CC

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X1SL: GGG UGA CUU GGU UUA GGU AAU GAG UCA CCC J6: AGG UUU AGC UAC ACU

No. 4: 5’-CGA CGC GCG TTG GTG GTG GAT GGT GTG TTA CAC GTG TTG T-3’

No. 4: Kd[OTC]= 9.61 nM

No. 5: 5’-ACG TTG ACG CTG GTG CCC GGT TGT GGT GCG AGT GTT GTG T-3’

No. 5: Kd[OTC]= 12.08 nM

No. 20: 5’-CGA GTT GAG CCG GGC GCG GTA CGG GTA CTG GTA TGT GTG G-3’

No. 20: Kd[OTC]= 56.84 nM

T7: 5’-GGG CAG CGG TGG TGT GGC GGG ATC TGG GGT TGT GCG GTG T-3’

T7: Kd[TET]= 357.8 nM

T15: 5’-GGA GGA ACG GGT TCC AGT GTG GGG TCT ATC GGG GCG TGC G-3’

T15: Kd[TET]= 197 nM

T19: 5’-CGG GAG GGC GGG GTG TGG TAT GTA TTG AGC GTG GTC CGT G-3’

T19: Kd[TET]= 424.8 nM

T20: 5’-CCC CCG GCA GGC CAC GGC TTG GGT TGG TCC CAC TGC GCG T-3’

T20: Kd[TET]= 63.6 nM

T22: 5’-GGG CGG ACG CTA GGT GGT GAT GCT GTG CTA CAC GTG TTG T-3’

T22: Kd[TET]= 483.5 nM

T23: 5’-GGG GGC ACA CAT GTA GGT GCT GTC CAG GTG TGG TTG TGG T-3’

T23: Kd[TET]= 100.6 nM

T24: 5’-GGG CGG GGG TGC TGG GGG AAT GGA GTG CTG CGT GCT GCG G-3’

T24: Kd[TET]= 70.7 nM

Ky2: 5’-TGG GGG TTG AGG CTA AGC CGA-3’

Kd [KANA]= 78.8 nM

AC C

KANA, KANA B and tobramycin

J6: Kd[Tobramycin]= 2±1 nM J6SL: Kd[Tobramycin]= 9±3 nM

EP

Tetracyclines

[31]

X1SL: Kd[Tobramycin]= 12±5 nM

J6SL: GGG ACG AGG UUU AGC UAC ACU CGU CCC

TE D

OTC

X1: Kd[Tobramycin]= 3±1 nM

[36]

[38]

[41]

Kd[KANA B]= 84.5 nM Kd [Tobramycin]= 103 nM

Ampicillin

AMP17: 5’-GCG GGC GGT TGT ATA GCG G-3’

Kd[Ampicillin]= 13.4 nM

[42]

STR

STR1: 5’- TAG GGA ATT CGT CGA CGG ATC C-GGG GTC TGG TGT TCT GCT TTG TTC

Kd[STR]= 199.1 nM

[43]

ACCEPTED MANUSCRIPT

TGT CGG GTC GT-CTG CAG GTC GAC GCA TGC GCC G-3’ Su13: 5’-GAG GGC AAC GAG TGT TTA TAG A-3’

Kd[SDM]= 84 nM

Ofloxacin

Q1: 5’-ATA CCA GCT TAT TCA ATT CGA TGG TAA GTG AGG TTC GTC CCT TTA ATA

Q1: Kd[Ofloxacin]= 6.9 nM (±11.3)

AAC TCG ATT AGG ATC TCG TGA GGT GTG CTC TAC AAT CGT AAT CAG TTA G-3’

Q2: Kd[Ofloxacin]= 0.11 nM (±0.06)

Q2: 5’-ATA CCA GCT TAT TCA ATT GCA GGG TAT CTG AGG CTT GAT CTA CTA AAT

Q3: Kd[Ofloxacin]= 0.20 nM (±0.09)

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SDM

SC

GTC GTG GGG CAT TGC TAT TGG CGT TGA TAC GTA CAA TCG TAA TCA GTT AG-3’ Q8: 5’-ATA CCA GCT TAT TCA ATT AGT TGT GTA TTG AGG TTT GAT CTA GGC ATA

M AN U

GTC AAC AGA GCA CGA TCG ATC TGG CTT GTT CTA CAA TCG TAA TCA GTT AG-3’

AC C

EP

TE D

a: All abbreviations in Tables 1- 5 are explained in the text.

[45] [46]

ACCEPTED MANUSCRIPT

Table 2 Luminescent aptasensors for detection of antibiotics preparation

Method performances a

Detection techniques and employed materials

RI PT

Sample matrix and

Analytes Ampicillin

Milk

Fluorescent and colorimetric detection

Extracted

AuNPs, FAM-ssDNA aptamer

CAP

Milk

Fluorescent detection

Centrifuged, diluted, filtered

UCNPs-cDNA, MNPs- biotin-ssDNA aptamer

Milk

Fluorescent probe

Diluted, filtered

QDs, dsDNA antibody, cDNA, thiol-ssDNA aptamer

Seafood

Fluorescent detection

Extracted

QDs, SiO2@AuNPs, Exo I, dsDNA antibody, cDNA,

CAP

Filtered

QDs, GO, amino-ssDNA aptamer

CAP

CAP

SC

[76]

3.6 %- 4.5 % (n= 6)

Range= 0.001- 10 ng/mL; LOD= 0.0002 ng/mL; Recoveries=

[77]

87.3 %- 99.5 %; Reproducibility: RSD= 2.15% (n= 5)

LOD= 0.2 ng/mL in milk; Reproducibility: RSD= 4.73 % (n= 10)

[78]

[79]

Fish

Fluorescent detection

Range= 0.003- 10 nM; LOD= 1 pM; Recoveries= 90.00 %-

SSB/DIL-Lip, thiol-ssDNA aptamer-MB

105.56 %; Reproducibility: RSD= 2.05 %- 4.97 % (n= 5)

Milk

Fluorescent detection

Range= 0.005- 100 ng/mL; LOD= 3 pg/mL; Recoveries= 88.3 %-

Centrifuged, deproteinized

QDs-SSB, AuNPs-thiol-ssDNA aptamer

93.6 %; Precision: RSD= 3.1 %- 4.8 % (n= 6)

and diluted

[63]

101.41 %; Precision: RSD= 2.84 % (n= 7)

Extracted

TE D

CAP

Florescent detection

EP

CAP

Milk

Milk

Fluorescent detection

LOD=0.3 pM; Range=0.001 nM- 10 nM; Recoveries=

Diluted

SSB/Lip-QD, thiol-ssDNA aptamer

90 %-103 %; Reproducibility: RSD= 2.73 % (n=5)

Milk

Fluorescent detection

Range= 0.001-100 ng/mL; LOD= 0.0005 ng/mL; Recoveries=

-

Hemin/G4, nano-Pt–luminol, thiol-ssDNA aptamer,

86.6 %- 93.5 %; Reproducibility: RSD= 1.42 % (n= 3)

AC C

CAP

Range= 0.01- 1 ng/mL; LOD= 0.01 ng/mL; Recoveries= 93.67 %-

Range= 0.05 -100 ng/ mL; LOD= 0.002 ng/mL; Precision: RSDs=

thiol-ssDNA aptamer

[42]

10 ng/mL by colorimetric detection in both milk and distilled water

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CAP

Range= 0.5- 500 ng/mL; LODs= 2 ng/mL by fluorescent detection,

Refs

[80]

[81]

[85]

thiol-cDNA, dsDNA antibody-MB, H2O2, TMB CAP

Milk

Chemiluminescent detection

Range= 0.01-0.2 ng/mL; LOD= 0.01 ng/mL; Recoveries=

Centrifuged and diluted

N-(4-aminobutyl)-N-ethylisoluminol-functionalized

94.8 %-103.1%; Precision: RSD= 2.7- 6.4 % (n= 3)

[87]

ACCEPTED MANUSCRIPT

flower-like gold nanostructures and MNPs, biotin-ssDNA

Daunomycin and

-

doxorubicin Daunorubicin

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aptamer Fluorescent detection OliGreen, ssDNA aptamer Prostate cancer cells

Fluorescent detection

KANA

KANA

KANA

Fluorescent detection

Extracted

MIPs-ssDNA aptamer-UCNPs

Bovine milk

Chemiluminescent enzyme immunoassay (CLEIA),

Centrifuged and diluted

ssDNA aptamer

Milk

Fluorescent detection

Deproteinized, centrifuged

Thiazole orange, G4 structural ssDNA aptamer

[75]

[65]

3.16 %- 4.83% (n= 6)

Range= 6.43- 89.99 ng/mL; LOD= 2.26 ng/mL; Recoveries=

[86]

89.7 %- 108.6 %; Precision: coefficient of variation= 14.0- 20.7 % Range= 0.1- 20 µM; LOD= 59 nM; Recoveries= 80.1 %- 98.0 %

[29]

Ranges= 0.01- 3 nM in buffer, 0.03- 3 nM in human serum; LODs=

[74]

Human serum

Fluorescent detection

Diluted

UCNPs, graphene, amino-ssDNA aptamer

9 pM in buffer, 18 pM in human serum

Fish

Florescent detection

Range= 0.2- 150 µM

Extracted, evaporated and

Platinum(II) complex, ssDNA aptamer

redissolved

LOD= 143 nM

[82]

Fluorescent detection

Deproteinized

Exo III, AuNPs, ssDNA, FAM-cDNA

Waste water

Fluorescent detection

Filtered

FAM-ssDNA aptamer, streptavidin-MB-cDNA-biotin

KANA A

Milk

Fluorescent detection

Solid phase extraction

AuNPs, FAM-ssDNA aptamer

105.1 %

Neomycin

Water

Florescent detection, paper-based microfluidic device

Range= 0- 2 µM; LOD= 153 nM; Recoveries= 95 %- 101 %

[72]

Range= 0.1- 10 µM in milk; LOD= 0.01 µM in milk

[69]

KANA A

EP

Rat serum and milk

AC C

KANA

Range= 33- 88 nM; LOD= 19 nM

Range= 0.5- 10 ng/mL; LOD= 0.04 ng/mL; Precision: RSD=

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Enrofloxacin

Fish

TE D

Enrofloxacin

[59]

SC

QDs, ssDNA aptamer, amino-cDNA

LOD= 8.4 ng/mL

Range= 0.5- 20 nM; LOD= 321 pM (0.185 µg/L)

[147]

Range= 0- 50 µM; LOD= 5 µM

[48]

Range= 0.8 nM- 350 nM; LOD= 0.3 nM; Recoveries= 99.2 %-

[68]

GO, Cy5-ssDNA aptamer Neomycin B

Milk

Fluorescent detection

ACCEPTED MANUSCRIPT

AuNPs, RNA aptamer that splitted into two shorter half

ultracentrifugation

fragments, one fragment was labeled with FAM

OTC

Milk

Fluorescent detection

Centrifuged and diluted

Amino-ssDNA aptamer-MNPs, cDNA-UCNPs

OTC

Tap water: no pretreatment;

Fluorescent detection

Milk: denatured, centrifuged

Graphene sheet, FAM-cDNA(S1), cDNA(C1), ssDNA

and filtered

aptamer

Tap water: no pretreatment;

Fluorescent detection

River water: filtered

Graphene oxide hydrogel, FAM-ssDNA aptamer

Milk

Florescent detection

Centrifugation and dilution

UCNPs, SYBR Green I, cDNA, amino-ssDNA aptamer

2.5 % (n= 11)

Milk

Fluorescent detection

Range= 10- 500 ng/mL in distilled water and milk; LOD= 10

Liquid liquid extraction

CPNBs, ssDNA aptamer

Milk and rat serum

Fluorescent assay

No pretreatment

Exo III, SYBR Gold, cDNA, ssDNA aptamer

STR

TET

-: unmentioned.

SC

Range= 0.01- 0.2 µM; LOQ= 0.01 µM

[73]

Range= 0.1- 10 ng/mL; LOD= 0.054 ng/mL; Precision: RSD=

[83]

[45]

ng/mL in distilled water and milk Ranges= 60- 1000 nM in rat serum, 60- 2000 nM in milk; LODs=

[60]

54.5 nM in buffer, 71 nM (51.73 µg/kg) in rat serum and 76.05 nM (0.054 µg/mL) in milk

Fluorescent determination

Range= 0- 300 nM; LODs= 8.48 nM in rat serum, 7.3 nM in tap

Rat serum: mixed with

THMS system: STP (fluorophore and quencher

water

buffer

dual-labeled oligonucleotide) and ssDNA aptamer

AC C

[71]

Range= 25- 1000 ng/mL; LOD= 25 ng/mL

Tap water: unmentioned

a: Method performances refer to the performances in buffer if not specially stated.

[64]

3.71 % (n= 10)

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SDM

Range= 0.05- 100 ng/mL; LOD= 0.036 ng/mL; Precision: RSD=

TE D

OTC

EP

OTC

RI PT

Diluted and

[84]

ACCEPTED MANUSCRIPT

Table 3 Colorimetric aptasensors for detection of antibiotics

CAP

Detection techniques and employed materials

Method performances

Milk

Colorimetric and fluorescent detection

Range= 0.5- 500 ng/mL; LODs= 2 ng/mL by fluorescent detection, 10

Extracted

AuNPs, FAM-ssDNA aptamer

ng/mL by colorimetric detection in both milk and distilled water

Milk and serum

Colorimetric sandwich assay

Range= 1- 120 nM; LODs= 451 pM in buffer, 697 pM for milk and

-

AuNPs, biotin-ssDNA aptamer, streptavidin

601 pM for serum; Recoveries

Refs [42]

[88]

98.57 %- 96.77 %; Precision: RSD=

SC

Ampicillin

Sample matrix and preparation

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Analytes

7.1 %

Fish

Colorimetric detection

-

AuMNPs-cDNA-ssDNA aptamer, dsDNA

Range= 0.05- 100 ng/mL; LOD= 0.015 ng/mL; Recovery= 85.95 %-

[105]

95.86 %; Reproducibility: RSD= 2.36 % (n= 5)

M AN U

CAP

a

antibody/ Envision reagents -AuNPs-HRP CAP

Duck and fish

Colorimetric assay

Range= 0.01- 100 ng/mL; LOD= 3 pg/mL; Recoveries= 92.3 %-

Extraction

AuMNPs-cDNA, ssDNA aptamer, ssDNA

[106]

110.0 %; Reproducibility: variation coefficient= 3.85- 4.76 %

b

antibody/HRP/PowerVision -AuNPs Milk

Colorimetric assay

Extraction

AuMNP-dsDNA antibody, thiol-ssDNA

TE D

CAP

Range= 0.001- 10 ng/mL; LOD= 0.0003 ng /mL; Recovery =90.0 %-

[40]

100.8 %; Reproducibility: RSD= 2.4 % (n= 5)

aptamer-cDNA–HRP– PtNPs, Exo I

DOX and TET

Colorimetric detection

Range= 0.05- 0.6 µg/mL; LOD= 2.6 ng/mL

[92]

Diluted

AgNPs, ssDNA aptamer

Milk

Colorimetric detection

Range= 1- 500 ng/mL in buffer and milk; LOD= 1 ng/mL in buffer and

[97]

Deproteinized and defatted

Two thiol-cDNA-AuNPs, ssDNA aptamer

milk

Milk and rat serum

Colorimetric detection

Range= 0.3-10 nM; LODs= 266 pM (0.127 ng/mL) in buffer, 347

Deproteinized using acetonitrile, and

THMS (STP+8-mer ssDNA aptamer),

(0.166 mg/L) in milk and 393 pM (0.189 mg/L) in rat serum

centrifuged

AuNPs

OTC

Tap water

OTC

Milk

No pretreatment

EP

KANA

Milk

AC C

KANA

Reflectance-based colorimetric detection

[56]

Range= 1 nM- 1 µM; LOD= 1 nM

[95]

LODs= 10.01 ng/mL in buffer, 12.3 ng/mL in milk

[103]

AuNPs, ssDNA aptamer Ic-ELAA colorimetric detection

Denature, defatted, deproteinized, extracted

Biotin-ssDNA aptamer,

and filtered

streptavidin-HRP-TMB, immobilized BSA-TET

Chicken: extracted, centrifuged and

Dc-ELAA colorimetric detection

Range= 0.5- 100 ng/mL; LOD= 0.88 ng/mL; Recoveries= 71.0 %-

diluted;

Amino-Fe3O4 MNPs, avidin, biotin-ssDNA

91.2 %; Precision: RSD= 3.17- 11.94 %

Milk: shaken, defatted and deproteinized

aptamer

SC

OTC

by centrifugation, diluted; Honey: mixed with PBS and ultrasonicated

STR

Honey

Colorimetric detection

Diluted

AuNPs, ssDNA aptamer

Milk and rat serum Diluted

[104]

Range= 0.2- 1.2 µM; Precision: RSD= 1.48- 1.82 %

[43]

Colorimetric and fluorescent detection

Ranges= nanomolar for colorimetry, ~2000 nM for fluorescent assay;

[67]

AuNPs, FAM-cDNA, ssDNA aptamer

LODs= 47.6 nM (34.7 µg/kg) and 73.1 nM (53.3 µg/kg) for

M AN U

STR

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ACCEPTED MANUSCRIPT

fluorescent assay and colorimetry in buffer; 58.2 nM (0.041 µg/mL)

in milk Range= 0.01- 2 µM; LODs= 1 µM for the naked eyes, 45.8 nM for

Acid added and centrifuged

AuNPs, PDDA, ssDNA aptamer

machine; Recoveries= 80.8 %- 112.1 %

Milk

Colorimetric detection

Range= 0.20- 2.0 µg/mL; LOD= 0.039 µg/mL; Precision: RSD=

Diluted, deproteinized, sonicated and

CS-AuNPs, ssDNA aptamer

2.33 % (n= 10)

Dc-ELAA colorimetric detection

Ranges= 0.1- 1000 ng/mL in buffer and honey; LODs= 0.0659 in

centrifuged TET

(72.19 µg/kg) for fluorescence quenching and colorimetric aptasensors

Colorimetric detection

Honey

Metal ions and protein were moved with solvents

EP

TET

colorimetric assay in serum; 56.2 nM (40.94 µg/kg) and 108.7 nM

Milk

AC C

TET

TE D

and 102.4 nM (0.072 µg/mL) for fluorescence quenching and

TET-HRP, biotin-ssDNA aptamer

buffer, 0.0978 ng/mL in honey; Recoveries= 92.09 %- 109.7 %; Precision: RSD< 7 %

[96]

[98]

[57]

ACCEPTED MANUSCRIPT

Ranges= 0.01- 100 ng/mL in buffer and honey; LODs= 7.8 ×10-3

Ic-ELAA colorimetric detection

RI PT

Tetracyclines

Honey Mental ions were removed, deproteinized

Biotin-ssDNA aptamer, streptavidin-HRP,

and extracted

immobilized TET-BSA

108.07 %

Milk

Dc-ELAA colorimetric detection

DNA aptamer: Ranges= 1.00× 10-4 - 1.99× 10-7 M in buffer, 3.16× 10-4

Defatted, deproteinized and diluted

ng/mL in buffer, 9.6×10 ng/mL in honey; Recoveries= 85.38 %-

Avidin-modified microplate, Biotin-ssDNA aptamer or biotin- RNA aptamer

-7

- 3.16× 10 M in milk

LODs= 15.7 µg/mL in buffer, 45.7 µg/mL in

milk;

RNA aptamer: Ranges= 3.16× 10-8 - 3.16× 10-4 M in buffer, 1.00×

M AN U

10-4- 1.00× 10-7 M; LODs= 10.1 µg/mL in buffer, 16.8 µg/mL in milk

a: Envision reagent contains about 100 HRP and some anti-IgG antibody molecules.

EP

TE D

b: PowerVision is a polymeric conjugate of horseradish peroxidase and antibody with a high enzyme-to-antibody ratio.

AC C

[102]

-3

SC

TET

[101]

ACCEPTED MANUSCRIPT

Table 4 Electrochemical aptasensors for detection of antibiotics Ampicillin

Sample matrix and preparation

Detection techniques and employed materials

Method performances

RI PT

Analytes

Milk

DPV

Range= 5 pM- 10 nM; LOD = 1.09 pM (or 21.8 amol);

Dilution

GE, programmable hairpin probe (a ssDNA aptamer, a

Reproducibility: RSDs= 3.09- 3.23 % (n= 4)

specific recognition sequence of nicking endonuclease

SC

and a nucleic acid segment complementary to the

Refs [148]

5’-end of the aptamer), methylene blue-labelled probe, DNA polymerase, nicking endonuclease Milk

DPV

Range= 0.02- 40 nM; LOD= 4.0 pM; Recoveries= 96 %- 104 %;

-

Indium tin oxide electrode, ssDNA aptamer contained

M AN U

Ampicillin

[112]

Reproducibility: RSDs= 3.2- 4.6 % (n= 6)

hairpin probe-methylene blue, T7 exonuclease CAP

Fish

Ratiometric electrochemiluminescent (R-ECL)

Range= 0.1 nM- 120 nM; LOD= 0.03 nM; Recoveries= 93.2 %-

Extracted and filtered

detection

102.9 %; Reproducibility: RSD= 4.86 %

[135]

Amino-ssDNA aptamer, luminol-AuNPs, CdS QDs Fish

Electrochemiluminescent detection

Range= 0.2- 150 nM for CAP; LOD= 0.07 nM for CAP;

malachite

Extracted

CdS QDs, luminol-gold nanoparticles, COOH-ssDNA

Recoveries= 94.7- 101.5 % for CAP; Reproducibility: RSD= 5.63 %

[136]

CAP aptamer, COOH-RNA malachite green aptamer,

green Serum

DPV

-

Dendritic gold nanostructures, SPGE, SBA-15, hemin,

EP

CAP

TE D

CAP,

Recovery= 90.0 %- 93.3 %; LOD= 4.0 nM; Reproducibility:

[115]

RSDs=1.20 % (n= 8)

thiol-ssDNA aptamer In vitro detection of CAP: in the CAP treated H. influenza cells supernatant (CAP-s) Tablets and capsules: Urine: diluted CAP

Milk

CV and SWV

Range=0.1- 2500 nM; LOD= 0.02 nM; Recovery > 96.4 %;

Amino-ssDNA aptamer, p-AHNSA modified EPPG

Reproducibility: RSDs< 3.5 % (n= 4)

Folding-based SWV detection

LOD= 1.6× 10−9 mol/L; Range=1.6× 10−9 - 4.2× 10−7 mol/L

AC C

CAP

[117]

[130]

ACCEPTED MANUSCRIPT

Thiol-ssDNA aptamer-GE Photoelectrochemical detection

Range=10- 250 nM; LOD= 3.1 nM; Reproducibility: RSD= 2.6 %

-

N-GQDs, ssDNA aptamer

(n= 5)

CAP and

Milk

SWV, multiplex

Range= 0.0005- 50 ng/mL; LODs= 0.15 ng/mL for CAP, 0.10 ng/mL

OTC

Unmentioned

GCE, metal ions (Pb2+, Cd2+) encoded magnetic

for OTC; Reproducibility: RSDs= 7.1- 8.4 % for CAP and 4.9-

hollow porous nanoparticles, Exo I, cDNAs, ssDNA

5.7 % for OTC (n= 4)

aptamers Fish

SWV, multiplex

Range= 0.001-100 ng/mL for CAP; LOD= 0.3 pg/mL for CAP;

PCB-72

-

GCE, thoil-ssDNA aptamers-magnetic gold 2+

[137]

[153]

[156]

Recoveries= 92 %- 110 % for CAP; Reproducibility: RSDs= 5.9-

M AN U

CAP and

RI PT

Eye drops

SC

CAP

2+

nanoparticles, cDNA-metal ions (Cd and Pb ) on

6.7 % for CAP (n= 3)

nanospherical branched polyethylene imine brushes as tracers (MSPEIs) CAP and

Fish

PCB-72

-

Range= 0.001-100 ng/mL; LOD= 0.33 pg/mL for CAP; Recoveries=

SWV, multiplex

GCE, thiol-ssDNA aptamers-Fe3O4@Au, PbS/CdS

[158]

92.0 % - 102.1 %; Reproducibility: RSDs= 3.5 % (n= 5)

Daunomycin

TE D

QDs, cDNAs-QDs- dendritic polymerase Human urine

DPV

Filtered

GCE, AuNPs, polyTTBA conducting polymer,

Range= 0.1- 60.0 nM; LOD= 52.3± 2.1 pM; Recoveries= 94 %-

[118]

98 %

phosphatidylserine, amino-ssDNA aptamer Live rats: see ref

SWV, MEDIC microfluidic

Range= 0.1- 10 µM for doxorubicin in buffer; LOD= 10 nM for

and KANA

Human whole blood: Pass through a

GE, methylene blue, thiolated-ssDNA aptamer

doxorubicin in buffer (KANA is unmentioned)

KANA

Milk Centrifuged and diluted

KANA

Milk -

AC C

cell strainer

EP

Doxorubicin

Electrochemiluminesence detection

Range= 0.15 nM- 170 mM; LOD= 45 pM; Recoveries= 99.5 %-

AuNPs@nano-C60, thiol-ssDNA

107.1 %; Reproducibility: RSD= 2.6 % (n= 5)

DPV

Range= 0.010- 150 ng/mL; LOD= 0.005 ng/mL (8.6 pM);

GE, AuNPs, HRP-AuNP-cDNA, hydroquinone and

Recoveries= 99.6 %- 103.4 %; Reproducibility: RSD= 3.2 % (n=5)

H2O2, thiol-ssDNA aptamer

[146]

[133]

[109]

ACCEPTED MANUSCRIPT

KANA

Milk

DPV

Linear: 1.0- 1.0×105 ng/L; LOD= 0.8 ng/L; Recoveries= 98.8 %-

Diluted

GCE, cDNA-Au-glucose oxidase, glucose solution,

104.0 %; Reproducibility: RSD= 2.3 % (n=5) with one aptasensor

MoS2-Au-hemin, thiol-ssDNA aptamer

and 3.4 % (n= 4) with four electrodes

Milk

CV and LSV

Range= 0.05 µM- 9.0 µM; LODs= 9.4± 0.4 nM in buffer and 10.8±

Filtered and diluted

Screen-printed electrode, DPB, AuNPs, amine-ssDNA

0.6 nM in milk; Reproducibility: RSD= 4.7 % (n=5)

aptamer

SC

KANA

Range= 5× 10-7- 5× 10-2 µg/mL; LOD= 0.42 pg/mL; Recoveries=

Pork meat and chicken liver

DPV

Deproteinated, extracted and diluted

GCE, GR-TH, HNP-PtCu, ssDNA aptamer

Milk

DPV, sandwich-type

Diluted

GCE, GR-PANI, PAMAM-Au, anti-KANA antibody,

[114]

[119]

[123]

97.4 %- 106.1 %; Reproducibility: RSD= 4.0 % (n=5)

Range= 5× 10-6- 4× 10-2 µg/mL; LOD= 4.6× 10-6 µg/mL;

M AN U

KANA

RI PT

KANA

[125]

Recoveries= 91 %- 103 %; Reproducibility: RSD= 4.5 % (n=5)

BSA, streptavidin-HRP, H2O2, biotin-ssDNA aptamer KANA

Milk

DPV

Range= 0.05- 100 ng/mL; LOD= 3.7 pg/mL; Recoveries= 94.66 %-

Diluted

GCE, MWCNTs-HMIMPF6, nanoporous PtTi alloy, amino-ssDNA aptamer

108.95 %; Reproducibility: RSD= 3.1 % (n=5)

Milk

DPV, double aptamer sandwich scheme

Range= 1× 10-8 M- 1.5× 10-7 M LOD= 5.8 nM; Recoveries=

Filtered and diluted

GE, biotin- and amino-ssDNA aptamers, CS-AuNPs,

97.18 %- 103.1%; Reproducibility: RSD= 3.8 % (n=6)

TE D

KANA

[127]

[128]

GR-AuNPs, MWCNTs-CoPc, hydroquinone-H2O2

KANA

DPV and colorimetric detection

Electrochemical detection: Ranges= 0.1- 60 nM in buffer, 1- 60 nM

Diluted

Peroxidase-like activity of AuNPs, thionine, ssDNA

in honey; LODs= 0.06 nM in buffer, 0.73 nM in honey;

aptamer

Reproducibility: RSD= 2.79 % (n=5)

Milk Diluted

EP

Honey

AC C

KANA

Colorimetric detection in buffer: Range= 5- 100 nM; LOD= 2.28 nM.

DPV

Range= 5 fM- 100 pM; LOD= 1.3 fM (26 zmol); Reproducibility:

GE, methylene blue, programmed hairpin probes,

RSD= 4.06- 4.47 % (n=4)

polymerase, nicking endonucleasw, Exo III, ssDNA aptamer

[107]

[149]

ACCEPTED MANUSCRIPT

Milk

DPV

Range= 1 pM- 10 nM; LOD= 0.74 pM; Reproducibility: RSDs=

Diluted

GE, methylene blue, programmed hairpin probes,

4.06- 4.47 % (n= 4)

RI PT

KANA

Exo III, ssDNA aptamer SWV

-

GE, ferrocene-ssDNA aptamer, thiol-short cDNA

KANA A

Low fat milk

EIS, microfluidic

and

Diluted and ultra-high temperature

Conductive polymer (PEDOT: TsO and PEDOT-OH:

Ampicillin

treated

TsO), TopasR, amino-modified ssDNA aptamer

KANA and

Milk

SWV, multiplex

CAP

Extraction

GCE, NMOF-cDNAs, amino-ssDNA

pM for CAP; Reproducibility: RSDs= 4.7% and 5.2% for KANA (10

aptamers-magnetic beads

nM) and CAP (10 nM)

Neomycin B

Milk Dilution and ultracentrifugation

[132]

Ranges= 10 nM- 1mM for KANA A; 100 pM- 1µM for ampicillin

[116]

Range= 0.002 nM- 100 nM; LODs= 0.16 pM for KANA and 0.19

Competitive EIS detection

Neomycin-immobilized GE, [Fe(CN)6] 2’-O-methylated RNA aptamer

Range= 0.75- 500 µM in buffer, 25- 2500 µM in milk; Recoveries=

3-/4-

, fully

[157]

[49]

102 %- 109 %

Honey

Sandwich-type DPV detection

Range= 5× 10-10- 2× 10-3 mg/mL; LOD= 4.98× 10-10 mg/mL;

Diluted 10-fold

GCE, GR-3D Au, thiol-ssDNA aptamer-AuNPs-HRP,

Recoveries= 93.7 %- 102.7 %; Reproducibility: RSD= 3.8 % (n= 5)

TE D

OTC

Range= 1 nM- 10 mM; LOD= 1 nM

SC

Lake water

M AN U

KANA A

[150]

[121]

hydroquinone-H2O2 Honey

CV

Diluted and centrifuged

AuNPs/GO-PANI modified GCE, streptavidin-HRP,

EP

OTC

Range= 4.0× 10-6 - 1.0 µg/mL; LOD= 2.3× 10-6 µg/mL; Recoveries=

[124]

97.2 %- 101 %; Reproducibility: RSD= 4 % (n= 5)

biotin-ssDNA aptamer, OTC-BSA,

OTC

Mouse blood serum: centrifuged Urine : no pretreatment

OTC

Commercial tablet -

OTC and

Milk

AC C

hydroquinone-H2O2

Folding-based SWV sensing

Range= 10- 600 ng/mL; LOD= 9.8 ng/mL

[163]

Photoelectrochemical detection

Range= 0.4- 150 nM; LOD= 0.9 nM; Reproducibility: RSD= 4.5 %

[139]

BiOI-graphene, amino-ssDNA aptamer

(n= 6)

SWV, multiplex

Range= 0.5 pM- 50 nM; LOD= 0.18 pM for OTC, 0.15 pM for

GE, ferrocene-ssDNA aptamer, cDNA

[152]

ACCEPTED MANUSCRIPT

TET

GCE, NMOF, magnetic bead, RecJf exonuclease,

KANA; Precision: RSDs< 7.3% for OTC and < 5.5 % for KANA

ssDNA-antibody, amino-ssDNA aptamer

(n= 5)

Milk

DPV

Ranges= 1×10−9 M- 1×10−5 M and 10−5- 10−2 M; LOD= 3.2× 10−10

Diluted and ultracentrifugated

GCE, PB-CS-GA, AuNPs, amino-ssDNA aptamer

M; Recoveries= 92 %- 106 %; Reproducibility: RSD= 5.8 % (n= 5)

Milk

DPV

Range= 1× 10-8 M- 5× 10-5 M; LOD= 5× 10-9 M; Reproducibility:

Diluted and ultracentrifugated

GCE, MWCNBs, [Fe(CN)6]

3-/4-

, amino-ssDNA

aptamer Tablet: -

EIS and DPV

CPE-OA by EIS

Milk: diluted, extracted and

CPE-OA, MBCPE-Fe3O4MNPs-OA, [Fe(CN)6]

centrifuged

amino-ssDNA aptamer

[113]

[126]

RSD= 1 % (n= 6)

3-/4-

,

LOD= 3.0× 10

M AN U

TET

RI PT

TET

Extraction

SC

KANA

-13

Range= 1.0× 10-12- 1.0× 10-7 M;

[129] -10

-7

M. CPE-OA by DPV: Range= 1.0× 10 - 1.0× 10

M; LOD= 2.9× 10-11 M.

Honey: ethanol added, sonicated,

MBCPE-Fe3O4NPs-OA by EIS

Range= 1.0× 10-14- 1.0× 10-6 M;

LOD= 3.8× 10-15 M. MBCPE-Fe3O4NPs-OA by DPV: Range= 1.0×

diluted and filtered

10-12- 1.0× 10-6 M; LOD= 3.1× 10-13 M.

Blood serum: diluted

TET

TE D

Recoveries= 95 %- 104 % in serum, honey and milk.

Milk and serum

DPV

Unmentioned

SPGE, M-shape structure: ssDNA aptamer and

Milk

OTC

No pretreatment

Penicillin

Milk Diluted

CV

Amino-GCE, [Fe(CN)6]

Bar GCE, GR-Fe3O4NPs, PEDOT-AuNPs,

Milk Isopropanol treated, filtrated

3-/4-

and 6.8 % (n=3) for CPE-OA Ranges= 1.5 nM- 3.5 µM; LODs= 0.45 nM (0.216 µg/L) in buffer,

[151]

0.74 nM in milk and 0.71 nM in serum; Recoveries= 93.17 %- 103.8 %; Reproducibility: RSD= 7.9 % (n= 3) Range= 0.1- 100 ng/mL; LOD= 1 ng/mL

[111]

Range= 0.1- 200 ng/mL; LOD= 0.057 ng/mL; Recoveries=95.96 %-

[120]

, ssDNA aptamer

DPV

[Fe(CN)6]

Penicillin G

3-/4-

AC C

TET and

EP

thiol-cDNA, Exo I, [Fe(CN)6]

3-/4-

Reproducibility: RSDs= 6.2 % (n= 3) for MBCPE-Fe3O4NPs-OA

105.40 %; Reproducibility: RSD= 4.1 % (n= 5)

, amino-ssDNA aptamer Range= 0.4- 1000 ng/mL; LOD= 0.17 ng/mL; Recoveries= 83 %-

EIS

Amine-SPCE, [Fe(CN)6]

3-/4-

, ssDNA aptamer

100 %

[44]

ACCEPTED MANUSCRIPT

Milk Diluted

DPV

Range= 0.05- 200 ng/mL; LOD= 0.028 ng/mL; Reproducibility:

GCE, PCNR, GR-Fe3O4-AuNPs, [Fe(CN)6]

3-/4-

,

thiol-ssDNA aptamer STR

Milk and rat serum No pretreatment

DPV

Ranges= 30- 1500 nM in buffer and milk, 40- 1500 nM in serum;

SPGE, [Fe(CN)6]

3-/4-

[122]

RSD= 3.1 % (n= 10)

RI PT

STR

, Exo I, arch-shape structure of

LODs= 11.4 nM (8.3 µg/kg) in buffer, 14.1 nM (10.2 µg/kg) in milk and 15.3 nM (0.011 µg/mL) in serum; Recoveries= 95.4 %- 98.2 %

SC

thiol-ssDNA-cDNA

[154]

in serum; Reproducibility: RSD= 6.5 % (n= 4)

Milk

SWV, multiplex

Ranges= 50- 1000 nM for STR, 40- 1000 nM for CAP and 20- 2000

and TET

Precipitated, centrifuged and filtered

GE, Capture DNA, cDNA1s, PbS/CdS/znS QDs-cDNA2s, ssDNA aptamers

Tobramycin

Human serum Diluted

Competitive EIS detection

Tobramycin-immobilized GE, [Fe(CN)6]

[159]

nM for TET; LODs= 10 nM for STR, 5 nM for CAP and 20 nM for

M AN U

STR, CAP

TET

Range= 3- 72.1 µM in serum; LOD= 1.8 µM in serum;

3-/4-

, fully and

[50]

Reproducibility: RSD= 6 % (n= 3)

partially 2’-O-methylated RNA aptamer DPV, monovalent labeling

Diluted and ultracentrifugated

SPCE, Magnetic microparticles-immobilized

TE D

Human serum

tobramycin, antiFITC-alkaline phosphatase Fab,

EP

FITC-partially 2’-O-methylated RNA aptamer

AC C

Tobramycin

Ranges= 0.1- 1000 µM in buffer, 1- 200 µM in serum; LODs= 0.1 µM in buffer, 1 µM in serum; Reproducibility: RSD= 4.4 % (n= 3)

[53]

ACCEPTED MANUSCRIPT

Table 5 Other aptasensors and aptamer-based chromatographic purification methods for antibiotics

TET

TET

Method performances Range= 0- 1000 pg/mL; LOD= 0.19 pg/mL;

Surface-enhanced Raman scattering

-

SERS-active Au@AgNSs, Cy5-ssDNA aptamer

Recoveries= 96.6 %- 110.2% Range= 10- 80 µM; LOD= 3.4 µM

[140]

[142]

Undiluted fetal bovine serum

Transmission- localized surface plasmon resonance

Filtered

Thiol-ssDNA aptamer

Milk

Resonance scattering spectral detection

LOD= 11.6 nM; Reproducibility: RSDs= 1.9- 4.2 %

Deproteinized, centrifuged and filtered

ssDNA aptamer

(n= 5)

Drug-free and drug-taken human urine and plasma

Aptamer-based SPE coupled to ESI-IMS

Ranges= 0.05- 5.00 µg/mL in urine, 0.10- 5.00

Plasma: precipitated, diluted, filtered and SPE extracted

Amino-ssDNA aptamer

µg/mL in plasma; LODs= 0.019 µg/mL in urine,

TE D

Urine: diluted, filtered and SPE extracted

and 82.8 % (RSD= 6.3 %) for plasma spiked at 2 µg/mL; Recoveries= 84 %- 91 % in urine, 77 %89 % in plasma.

Aptamer-based hybrid affinity monolithic capillary

In serum and urine: Range= 5-1000 µg/mL; LOD=

and

Filtered

purification-liquid chromatography-UV detection

0.3 µg/mL; LOQ= 0.8 µg/mL; Recoveries= 90.1 %-

Thiol-ssDNA aptamer

97.8 %; Reproducibility: RSD< 3.7 % (n= 3)

EP

[161]

86.5 % (RSD= 5.9 %) for urine spiked at 1 µg/mL,

Serum and urine

AC C

[143]

0.037 µg/mL in plasma; Extraction efficiency=

Doxorubicin

epirubicin

Refs

Milk

SC

Tobramycin

Detection techniques and employed materials

M AN U

CAP

Sample matrix and preparation

RI PT

Analytes

[162]

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT Highlights ● Recent advances in aptamers-based detection methods of antibiotics in different matrices are reviewed. ● Different aptasensors and aptamer-based purification methods are presented.

RI PT

● Current sensing strategies of antibiotics-detecting aptasensors are summarized.

AC C

EP

TE D

M AN U

SC

● Future trends of the application of aptamers in antibiotics detection are discussed.