Aptamer-based environmental biosensors for small molecule contaminants

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ScienceDirect Aptamer-based environmental biosensors for small molecule contaminants Van-Thuan Nguyen*, Young Seop Kwon* and Man Bock Gu Aptasensors are promising biosensors, which take advantage of using aptamers as a recognition element. The combination of the excellent characteristics of aptamers and the leading detection platform techniques, such as optical, electrochemical with nanomaterial-integrated, or masssensitive techniques with high sensitivity and specificity draws a promising view for the application of the aptasensors for the detection of harmful small toxic chemicals and real-time monitoring in the environments. In spite of attraction of aptasensors, application of them is limited to the complex environment due to the facts that how the immobilization of aptamers onto the surface affects the functions of aptamers and their structures for the detection of environmental contaminants are not clearly known. This review examines the most recent update on the selection of aptamers for small molecules, the development and application of aptasensors in the detection of small molecule contaminants in environment. Additionally, their applications to the real samples as environmental monitoring reported in the publications also are reviewed. Address Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Anam-dong, Seongbuk-gu, Seoul 136713, South Korea Corresponding author: Gu, Man Bock ([email protected]) Both authors contributed equally to this work.

*

Current Opinion in Biotechnology 2017, 45:15–23 This review comes from a themed issue on Environmental biotechnology Edited by Jan Roelof Van Der Meer and Man Bock Gu

http://dx.doi.org/10.1016/j.copbio.2016.11.020 0958-1669/Published by Elsevier Ltd.

Introduction Nowadays, monitoring requirements of environmental contaminants are ever-increasing, since the chances of exposure by toxic chemicals that can affect human and animal life are huge. Some contaminants cause mild effects even after long term exposure, however, some others can have deadly effects and lead to global disasters. Although there have been a lot of efforts in developing techniques for monitoring various environmental toxic www.sciencedirect.com

molecules, there is still a great need in particular for portable, field deployable, and highly robust technologies. The pollutants that need to be monitored in the environment can be broadly divided into four classes: toxins, pesticides, environmentally polluting hormones and persistent organic toxic chemicals (POTC), and pharmaceuticals and personal care products (PPCPs). Since most of those are small molecules less than 1000 Da, they are nonimmunogenic and antibodies are inappropriate as recognizing receptors. In other cases, it is too tedious and complicated to develop the antibodies by binding the small targets to a certain protein. Moreover, due to expensive production costs for the synthesis of antibodies and their instability upon exposure to environmental conditions, alternatives are needed. Aptamers as recognizing receptors into environmental monitoring systems have attracted attention and their applications have been continuously studied. Aptamer-based environmental biosensors, so called aptasensors, have been developed especially for the detection of harmful toxic agents in the environment. Aptamers are short single-stranded nucleic acids obtained in a process, which is called SELEX (for Systematic Evolution of Ligands by EXponential enrichment). Applying aptamers to biosensors can have many advantages. They show high flexibility and stability, are cheap to produce, and easy to modify. These are interesting properties for real sample applications. Aptamers can be developed by in vitro screening of the various types of targets, ranging from small molecules to whole cells, thus making it possible to develop a wide range of aptasensors [1–4]. However, there are a few drawbacks in the conventional SELEX procedure using solid supporting materials (SSMTSELEX): 1) upon immobilization of the target molecule, around one of third of target surface is lost or inaccessible; 2) immobilization steps become very complicated or unsuitable for small molecular targets, in particular when molecules do not have any functional groups or are difficult to conjugate; 3) Some properties of the targets may change upon modification and thus affect the affinity of the aptamer to bind the original free target molecule. To overcome these issues, new SELEX procedures have been developed recently, such as structure-switching SELEX and Capture-SELEX [5,6]. The newer methods, however, still may lose some of the high affinity existing aptamers in the procedure, resulting in an enrichment of selected aptamers with low affinities. In addition, aptamer refolding relies mostly on their random region, especially for small molecule targets, which also results in selection Current Opinion in Biotechnology 2017, 45:15–23

16 Environmental biotechnology

of aptamers with low affinities. In response to this, Gu and co-workers have developed an immobilization-free aptamer screening method based on graphene oxide (GO) assistance (GO-SELEX) [7]. In this procedure, instead of immobilizing the targets on a rigid surface, the random DNA pool is adsorbed on a GO sheet via the p–p stacking interactions with the surface. The key advantage of GO-SELEX is that, especially, small molecular targets are placed without modification. In addition, aptamer selection is independent of the target’s size and molecular weight, because of target-induced aptamer detachment from the GO surface [8–11]. A recent demonstration included Multi-GO-SELEX to screen for flexible highaffinity aptamers for multiple pesticides [12].

Aptamer-based environmental biosensors for small molecule contaminants Among many different signal transducing platforms available for aptasensors, some limitations exist for implementing every platform technology, mainly due to the size of the target molecules. In other words, some of the platform technologies based on the mass of the targets or the multiple binding capabilities of the targets are hardly applied to the environmental monitoring. So, in this review, it is highlighted how these limitations caused from the intrinsic nature of the environmental contaminants, such as its small molecular weight or nonmodifiable targets, are appropriately dealt or overcome by introducing nanotechnology or complicated multiple steps, even though some of the methodologies are not feasible in field. Optical aptasensors

Fluorescence detection is widely employed for aptamer– ligand interactions, due to its convenience of labeling the aptamers with fluorescent dyes, the choice of many different fluorophores and quenchers, and the inherent capability for real-time detection. Several main strategies have been developed for converting aptamer–ligand binding into fluorescent signals, such as molecular beacons, duplex structure with complementary sequences, and competitive laser-based flow assays (Figure 1). The most famous and widely used fluorescence method to detect ligand-binding is by molecular structure-switching [13–20]. Turn-off style lateral flow assays (LFA) for aptasensors have been developed particularly for small molecular targets, which are based on the target-induced displacement of aptamers [21]. As an example, aflatoxinB binding aptamers were modified by biotin and their complementary DNA sequences were labeled with Cy5 as a signal probe. Another principle uses salt-dependent aggregation of gold nanoparticles (AuNPs), of a size of 10–50 nm, leading from a color change of wine red to purple and size-dependent surface plasmon absorbance peak shifts. The aptamers are placed on the surface of AuNPs Current Opinion in Biotechnology 2017, 45:15–23

and prevent aggregation via electrostatic repulsion. In presence of the ligand, the aptamers detach out from the AuNP surface to bind their own targets, and this induces the nanoparticle aggregation [22]. Based on this principle, a number of colorimetric aptasensors were developed for different environmental toxic chemicals, such as for Bisphenol A [23], Ochratoxin A [24], and pesticides [10,12]. Since the AuNP-based colorimetric aptasensors have relatively low compound detection sensitivities, horseradish peroxidase (HRP) mimicking DNAzyme has been introduced, which enhances the detection of the aptasensor [25]. As example, a competitive assay using an aflatoxin B1 (AFB1)-ovalbumin coated 96 well plate was developed with aptamers specific for AFB1, which generated chemi-luminescence from a hemin/ G-quadruplex horseradish peroxidase-mimicking DNAzyme (HRP-DNAzyme) that was linked to the AFB1specific aptamer. The advantage of using a DNAzyme forming a hemin-G-quadruplex complex, is that it shows higher catalytic activity compared to hemin itself. Electrochemical aptasensors

Electrochemical detection of small molecule binding to aptasensors has recently been developed on different platforms (Figure 2). Among them, label-free electrochemical impedance spectroscopy (EIS) has appeared as a promising strategy. EIS is not only a powerful method to characterize biomolecule-functionalized substrates, but also a sensitive technique to monitor aptamer–ligand binding occurring on the electrode surface. More importantly, EIS is nondestructive, which makes it highly attractive for aptamer-based small molecule detection [26,27]. Demonstrating one of the first electrochemical aptasensors, Kim et al. reported detection of 17b-estradiol [28]. The 17b-estradiol was detectable up to 0.1 nM, and the linear range of this biosensor was from 0.01 to 1 nM. Another label-free competitive electrochemical aptasensor was described for Brevetoxins (BTXs) [29], which was based on the competition between the BTX-beads and BTX-HRP conjugate. The limit of detection in this aptasensor for BTXs was calculated at 106 pg/ml. Elshafey et al. reported a newly selected aptamer embedded in an EIS aptasensor for the detection of anatoxin-a (ATX), the smallest potent neurotoxin [30]. This aptasensor exhibited an excellent detection limit (0.5 nM), and a linear range from 1 nM to 100 nM. More recently, electrochemical aptasensors integrated with nanomaterials have shown great potential for detection of small molecules. Fan et al. reported an ultrasensitive photoelectrochemical (PEC) sensing platform for detection of 17b-estradiol (E2) based on TiO2 nanotube arrays modified with CdSe quantum dot nanoparticles [31]. The CdSe nanoparticles were electrodeposited on the inner and outer surface of the TiO2 nanotubes, and the E2 aptamer was immobilized onto the CdSe nanoparticles attached to the TiO2 nanotubes (Figure 3a). www.sciencedirect.com

Aptasensors for small molecule contaminants Nguyen, Kwon and Gu 17

Figure 1

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3,3′, 5,5′-Tetramethylbenzidine (TMB) Current Opinion in Biotechnology

Illustration of detection strategies of optical aptasensors. (a) Molecular Beacon-based aptasensor, (b) complementary sequence-based aptasensor, (c) graphene/CNT-based aptasensor, (d) lateral flow assay (LFA) aptasensor, (e) gold-nano-particle (AuNP) aggregation-based aptasensor, (f) DNAzyme-based aptasensor, (g) surface-enhanced Raman scattering (SERS)-based aptasensor, and (h) localized surface plasmon resonance (LSPR) shift-based aptasensor.

Upon binding of the aptamer to E2, the aptamer complex increased steric hindrance, which blocked the diffusion of ascorbic acid and resulting in a decreased signal. The developed PEC aptasensor exhibited a wide linear range www.sciencedirect.com

from 0.05 to 15 pM E2 with a limit of detection of 33 fM. The Wang group described a gold nanoparticle bound reduced graphene oxide (AuNPs–rGO) as a signal amplification material for detection of Ochratoxin A (OTA) Current Opinion in Biotechnology 2017, 45:15–23

18 Environmental biotechnology

Figure 2

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Current Opinion in Biotechnology

Illustration of different electrochemical aptasensors. (a) Labeled aptamer-based electrochemical aptasensors, (b) labeled/unlabeled split aptamerbased electrochemical aptasensors, (c) enzyme-based electrochemical aptasensors, (d) label free aptamer-based electrochemical aptasensors, (e) nanomaterial-based electrochemical aptasensors.

[32] (Figure 3b). The capture DNA1 was immobilized on the gold electrode, the reporter DNA was conjugated with AuNPs–rGO whereas AuNPs–rGO-DNA was used as a carrier of reporter DNA. This impedimetric aptasensor could detect OTA around 0.74 pM. In another study [33], an electrochemical aptasensor was developed based on a sandwich model (Figure 3c). AuNPs-conjugated guanine-rich DNAs, which enable binding with methylene blue, were utilized as a signal amplification nanocarrier material. Upon addition of OTA, the AuNPsDNAs were replaced, resulting in a decrease of the signal. Alternative configurations of electrochemical aptasensors include polyamidoamine (PAMAM) dendrimers as immobilization platform [34], AuNPs integrated with Current Opinion in Biotechnology 2017, 45:15–23

enzyme-based platforms [35], or integrated reduced graphene oxide and magnetite (Fe3O4) nanoparticles [36]. To date, there are a few direct mass-based aptasensors reported for the detection of small molecules. Exceptions are the surface plasmon resonance aptasensors for OTA and the antibiotic tobramycin (467 Da) [37]. Further development of mass-based aptasensors will depend on new unique nanomaterials and/or other amplification signal techniques, in order to detect small ligands with high sensitivity and selectivity. Some studies reported an AuNP-based surface plasmon resonance aptasensor for detection of adenosine, with a detection limit in the pM or fM range [38]. www.sciencedirect.com

Aptasensors for small molecule contaminants Nguyen, Kwon and Gu 19

Figure 3

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Nanomaterial-based electrochemical aptasensors. (a) Photoelectrochemical (PEC) CdSe NPs sensing platform (working electrode (WE), reference electrode (RE) and counter electrode (CE)), (b) r-GO nanosheet-based aptasensor, (c) AuNP electrochemical aptasensor.

Applications of aptasensors to real environmental samples

Although most aptasensors have been applied only on ligands spiked to distilled water or buffer solutions, more www.sciencedirect.com

applications to real samples have appeared in recent years (Table 1). Examples include detection of toxins, hormones, pollutants, drugs, and pesticides in food, soil, and water samples. Some representative examples are discussed below. Current Opinion in Biotechnology 2017, 45:15–23

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Table 1 Real field samples Class Toxins Toxins Toxins Toxins Toxins Toxins Toxins Toxins Toxins Toxins Toxins Toxins Toxins Toxins Toxins Toxins Toxins Toxins Hormones Hormones Hormones Pollutants Pollutants Pollutants Pollutants Pollutants Drugs Drugs Drugs Drugs Drugs Pesticides Pesticides Pesticides Pesticides Pesticides

Target

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Ochratoxin A T-2 Toxin Ochratoxin A Ochratoxin A Ochratoxin A Ochratoxin A Aflatoxin B1 Aflatoxin B1 Ochratoxin A Fumonisin B Ochratoxin A Ochratoxin A Aflatoxin B1 Aflatoxin B1 Anatoxin A Aflatoxin B1 Aflatoxin M1 Brevetoxin-2 17b-estradiol 17b-estradiol 17b-estradiol Bisphenol-A Bisphenol-A Bisphenol-A Polychlorinated biphenyls (PCB) Polychlorinated biphenyls (PCB) Tetracycline Tetracycline Kanamycin Streptomycin Streptomycin Organophosphorus pesticides Acetamiprid Acetamiprid Acetamiprid Acetamiprid

Beer Beer Wine Wine Wine Wine Corn Corn Corn Corn Wheat Peanut oil, wine Peanut Hay Drinking water Milk Milk Shellfish Urine Water Water Water Milk Milk Water Blood Milk Honey Milk Milk Milk Cabbage, Rice Soil Water Cucumber Cabbage

1 ng/ml 0.5 mM 0.74 pM 0.3 pM 0.75 pM 10 fM 0.3 ng/g 0.11 ng/mL 1 nM 2 pM 2 pg/mL 0.005 ng/mL 0.40  0.03 nM 25 fg/mL 0.5 nM 0.05 nM 1.15 ng/L 106 pg/ml 0.2 nM 33 fM nd 1.86 nM 5 nM 0.012 ng/mL 0.01 mg/L 10 nM 347 pM 9.6  10 3 ng/mL 59 nM 56.2 nM 14.1 nM 5 mg/L, 38 nM 5 nM 17 fM 0.18 nM 7.29 nM

[16] [20] [32] [39] [33] [40] [21] [25] [41] [42] [43] [44] [34] [45] [30] [46] [47] [29] [48] [31] [49] [14] [50] [23] [54] [55] [56] [57] [58] [59] [60] [10,15] [61] [62] [63] [64]

Toxins

Pollutants

Mycotoxins are fungal toxins that contaminate grains like wheat and grain-derived products such as beer or baby food. They can also be found in milk, wine or coffee. Aptasensors have been used to detect mycotoxins including OTA, AFB, AFM, Anatoxin-a Fumonisin B1, T2-toxin and Brevetoxin-2, in beer [16,20] wine [32,33,39,40], corn [21,25,41,42], wheat [43], peanut [34,44], hay [45], drinking water [30], milk [46,47], and shellfish [29] with a detection limits ranged from nanomolar to femtomolar (see LOD in Table 1).

Contamination by industrial origin is leading to severe environmental problems. Aptasensors have been developed to detect, for example, bisphenol-A in water [14] and milk [23,50]. Many aptamers have been developed as well for the detection of polychlorinated biphenyls [51– 53], however only few studies describe real sample application of polychlorinated biphenyl detection in tap water [54], or blood [55].

Hormones

It is well known that the female sexual hormones progesterone and 17b-estradiol disrupt the endocrine system aquatic animals. Since only limited amounts can actually be absorbed by the human body, the rest is released into the environment, notably in aquatic systems. Several different aptasensor systems have been used to detect estradiol in urine [48], tap water, lake water, and waste water [31,49]. Current Opinion in Biotechnology 2017, 45:15–23

Drugs

Because of the large scale usage of antibiotics in industrial livestock farming and agriculture, they widely contaminate the environment, water, and food. Aptasensors have been reported to detect oxytetracycline in spiked tap water [56], tetracycline in honey [57], kanamycin in milk [58], or streptomycin in milk [59,60]. Pesticides

Pesticides are still heavily used in agriculture, and they contaminate the water, soil and environment. Since so www.sciencedirect.com

Aptasensors for small molecule contaminants Nguyen, Kwon and Gu 21

many different pesticides are in use, few studies have attempted to develop an aptamer for multitarget pesticide detection. Zhang et al. described the detection of four organophosphate pesticides phorate, profenofos, isocarbophos, and omethoate, in Chinese cabbage by using a single aptamer [15]. Other aptasensors were developed for the detection of, for example, acetamiprid in soil [61], water [62], cucumber [63], and cabbage [64]. Colorimetric aptasensors were described for the detection of both edifenphos and iprobenfos in rice [10].

2016R1E1A2020541), the National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (No. 2016R1A2B3011422), the Nano Material Technology Development Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (No. 2016M3A7B4910555) and the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI16C0220). In addition, Mr. Young Seop Kwon was financially supported for visiting research at the University of Illinois at UrbanaChampaign during 6 months by BK21 PLUS, Korea University.

References and recommended reading There are still many problems that need to be overcome for real field application of aptasensors. As we mentioned above, most aptasensors for real samples are applied to aqueous samples, such as wine, beer, juice, milk or honey. This is because solid samples need pre-analytical procedures, such as extraction or sample treatment by specific buffers. In addition, most aptasensors have been demonstrated on spiked samples, not real unknown samples. As an exception, the group of Fan et al. reported aptasensor detection of 17b-estradiol (E2) in medical waste water, tap water and lake water. It is noteworthy that very low E2 concentrations were detected by the aptasensor in tap water, but not detected by HPLC [31]. Another study reported successful detection of OTA by a fluorescent aptasensor in corn and oat samples [65].

Papers of particular interest, published within the period of review, have been highlighted as:

Concluding remarks The number of reported aptasensors for detection of toxic chemicals have rapidly increased. However, there are limited studies that demonstrate successful aptasensor ligand detection in environmental samples. Environmental samples frequently contain a large variety of other molecules, which may cause non-specific interactions with the aptamers or false binding to the target molecules. It was shown that the small molecules often dock in the binding cavity of the aptamer, causing probably little or no binding for the interaction with a secondary aptamer [66]. Therefore, unlike large-sized biomolecules, small molecules are normally detected using the single binding mode [67]. Moreover, many studies have focused to develop aptasensors with or without splitting previously well-characterized aptamers, such as for ATP, cocaine, or OTA [32,38]. In addition to the splitting of aptamers, molecule-switching or trip-helix are potential techniques which can improve the problem of poor binding sites of aptamers for small molecules. Many research groups have combined aptamers with novel nanomaterials to improve sensitivity and selectivity of the aptasensors. The future will definitely see the development of integrated aptasensors, which are not only sensitive, robust, automatic, and miniaturized but also allow usage with various kinds of real samples (solution, air, soil).

 of special interest  of outstanding interest 1.

Clark SL, Remcho VT: Aptamers as analytical reagents. Electrophoresis 2002, 23:1335-1340.

2.

Willner I, Zayats M: Electronic aptamer-based sensors. Angew Chem Int Ed 2007, 46:6408-6418.

3.

Mairal T, Cengiz O¨zalp V, Lozano Sa´nchez P, Mir M, Katakis I, O’sullivan CK: Aptamers: molecular tools for analytical applications. Anal Bioanal Chem 2007, 390:989-1007.

4.

Tombelli S, Minunni M, Mascini M: Analytical applications of aptamers. Biosens Bioelectron 2005, 20:2424-2434.

5.

Handy SM, Yakes BJ, DeGrasse JA, Campbell K, Elliott CT, Kanyuck KM, DeGrasse SL: First report of the use of a saxitoxin–protein conjugate to develop a DNA aptamer to a small molecule toxin. Toxicon 2013, 61:30-37.

6.

Elshafey R, Siaj M, Zourob M: In vitro selection, characterization, and biosensing application of high-affinity cylindrospermopsin-targeting aptamers. Anal Chem 2014, 86:9196-9203.

7. 

Park JW, Tatavarty R, Kim DW, Jung HT, Gu MB: Immobilizationfree screening of aptamers assisted by graphene oxide. Chem Commun 2012, 48:2071-2073. The paper developed a new SELEX method for obtaining aptamers using graphene oxide as assisted nanomaterial. The random DNA pool was freely immobilized on graphene oxide surface and then specific aptamers were selected by the targets. 8.

Raston N, Gu M: Highly amplified detection of visceral adipose tissue-derived serpin (vaspin) using a cognate aptamer duo. Biosens Bioelectron 2015, 70:261-267.

9.

Nguyen V-T, Seo HB, Kim BC, Kim SK, Song C-S, Gu MB: Highly sensitive sandwich-type SPR based detection of whole H5Nx viruses using a pair of aptamers. Biosens Bioelectron 2016, 86:293-300.

10. Kwon YS, Nguyen V-T, Park JG, Gu MB: Detection of Iprobenfos and Edifenphos using a new multi-aptasensor. Anal Chim Acta 2015, 868:60-66. 11. Gu H, Duan N, Wu S, Hao L, Xia Y, Ma X, Wang Z: Graphene oxide-assisted non-immobilized SELEX of okdaic acid aptamer and the analytical application of aptasensor. Sci Rep 2016, 6:21665. 12. Nguyen V-T, Kwon YS, Kim JH, Gu MB: Multiple GO-SELEX for  efficient screening of flexible aptamers. Chem Commun 2014, 50:10513-10516. This paper developed a multi-GO-SELEX method as a high-throughput format of GO-SELEX to screen for flexible aptamers. A group of small molecules was simultaneously selected for generating aptamers that are specific for multiple pesticides with high affinities.

Acknowledgements

13. Chen J, Fang Z, Liu J, Zeng L: A simple and rapid biosensor for ochratoxin A based on a structure-switching signaling aptamer. Food Control 2012, 25:555-560.

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No.

14. Yildirim N, Long F, He M, Shi H-C, Gu AZ: A portable optic fiber aptasensor for sensitive, specific and rapid detection of

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bisphenol-A in water samples. Environ Sci Process Impacts 2014, 16:1379-1386. 15. Zhang C, Wang L, Tu Z, Sun X, He Q, Lei Z, Xu C, Liu Y, Zhang X, Yang J et al.: Organophosphorus pesticides detection using broad-specific single-stranded DNA based fluorescence polarization aptamer assay. Biosens Bioelectron 2014, 55:216-219. 16. Lv Z, Chen A, Liu J, Guan Z, Zhou Y, Xu S, Yang S, Li C: A simple and sensitive approach for ochratoxin a detection using a label-free fluorescent aptasensor. PLoS One 2014, 9:2-6. 17. Zhao Q, Geng X, Wang H: Fluorescent sensing ochratoxin A with single fluorophore-labeled aptamer. Anal Bioanal Chem 2013, 405:6281-6286. 18. Yildirim N, Long F, Gao C, He M, Shi H-CC, Gu AZ: Aptamer-based optical biosensor for rapid and sensitive detection of 17b-estradiol in water samples. Environ Sci Technol 2012, 46:3288-3294. 19. Ragavan KV, Selvakumar LS, Thakur MS: Functionalized aptamers as nano-bioprobes for ultrasensitive detection of bisphenol-A. Chem Commun 2013, 49:5960-5962. 20. Chen X, Huang Y, Duan N, Wu S, Xia Y, Ma X, Zhu C, Jiang Y, Wang Z: Screening and identification of DNA aptamers against T-2 toxin assisted by graphene oxide. J Agric Food Chem 2014, 62:10368-10374. 21. Shim WB, Kim MJ, Mun H, Kim MG: An aptamer-based dipstick assay for the rapid and simple detection of aflatoxin B1.  Biosens Bioelectron 2014, 62:288-294. This paper is one of the first applications for aptamers to be used in a lateral flow assay platform, which is one of the promising formats for realization. However, since this study employed a turn-off signal, which should be avoided if possible, because there are high possibility to be false-positive. 22. Li H, Rothberg L: Colorimetric detection of DNA sequences based on electrostatic interactions with unmodified gold nanoparticles. Proc Natl Acad Sci U S A 2004, 101:14036-14039. 23. Yao D, Liang A, Yin W, Jiang Z: Resonance light scattering determination of trace bisphenol A with signal amplification by aptamer-nanogold catalysis. Luminescence 2014, 29:516-521. 24. Wang C, Dong X, Liu Q, Wang K: Label-free colorimetric aptasensor for sensitive detection of ochratoxin A utilizing hybridization chain reaction. Anal Chim Acta 2015, 860:83-88. 25. Shim WB, Mun H, Joung HA, Ofori JA, Chung DH, Kim MG: Chemiluminescence competitive aptamer assay for the  detection of aflatoxin B1 in corn samples. Food Control 2014, 36:30-35. The paper reported a chemiluminescence aptasensor for detection of aflatoxin B1 using a hemin/G-quadruplex HRP-DNAzyme linked with an aptamer. The aptasensor exhibited a wide dynamic range from 0.1 to 10 ng ml 1 and the recovery from spiked corn samples averaged from 60.4 to 105.5%. 26. Hayat A, Haider W, Rolland M, Marty J-L: Electrochemical grafting of long spacer arms of hexamethyldiamine on a screen printed carbon electrode surface: application in target induced ochratoxin A electrochemical aptasensor. Analyst 2013, 138:2951-2957.

31. Fan L, Zhao G, Shi H, Liu M, Wang Y, Ke H: A femtomolar level  and highly selective 17b-estradiol photoelectrochemical aptasensor applied in environmental water samples analysis. Environ Sci Technol 2014, 48:5754-5761. This is an excellent paper which demonstrated aptasensor performance in real field estradiol detection. A combination aptamer-hybrid to TiO2 nanotubes modified with CdSe nanoparticles was presented, which enabled detection of 17b-estradiol at femtomolar level in water samples. 32. Jiang L, Qian J, Yang X, Yan Y, Liu Q, Wang K, Wang K: Amplified impedimetric aptasensor based on gold nanoparticles  covalently bound graphene sheet for the picomolar detection of ochratoxin A. Anal Chim Acta 2014, 806:128-135. An interesting approach, which combined aptamer-gold nanoparticles bound to reduced graphene oxide as an impedimetric aptasensor. The aptasensor detected ochratoxin in concentrations from 1 pg mL 1 to 50 ng mL 1. 33. Yang X, Qian J, Jiang L, Yan Y, Wang K, Liu Q, Wang K: Ultrasensitive electrochemical aptasensor for ochratoxin A based on two-level cascaded signal amplification strategy. Bioelectrochemistry 2014, 96:7-13. 34. Castillo G, Spinella K, Poturnayova´ A, nejda´rkova´ M, Mosiello L, Hianik T: Detection of aflatoxin B1 by aptamer-based biosensor using PAMAM dendrimers as immobilization platform. Food Control 2015, 52:9-18. 35. Luo P, Liu Y, Xia Y, Xu H, Xie G: Aptamer biosensor for sensitive detection of toxin A of Clostridium difficile using gold nanoparticles synthesized by Bacillus stearothermophilus. Biosens Bioelectron 2014, 54:217-221. 36. Zhan X, Hu G, Wagberg T, Zhan S, Xu H, Zhou P: Electrochemical aptasensor for tetracycline using a screen-printed carbon electrode modified with an alginate film containing reduced graphene oxide and magnetite (Fe3O4) nanoparticles. Microchim Acta 2016, 183:723-729. 37. Cappi G, Spiga FM, Moncada Y, Ferretti A, Beyeler M, Bianchessi M, Decosterd L, Buclin T, Guiducci C: Label-free detection of tobramycin in serum by transmission-localized surface plasmon resonance. Anal Chem 2015, 87:5278-5285. 38. Yao G-H, Liang R-P, Yu X-D, Huang C-F, Zhang L, Qiu J-D:  Target-triggering multiple-cycle amplification strategy for ultrasensitive detection of adenosine based on surface plasma resonance techniques. Anal Chem 2015, 87:929-936. This study describes integrating a target-triggering nicking enzyme signaling amplification and the hybridization chain reaction amplificationbased surface plasmon resonance aptasensor which enabled detection of adenosine down to 4 fM and with high selectivity. 39. Xie S, Chai Y, Yuan Y, Bai L, Yuan R: Development of an electrochemical method for Ochratoxin A detection based on aptamer and loop-mediated isothermal amplification. Biosens Bioelectron 2014, 55:324-329. 40. Yuan Y, Wei S, Liu G, Xie S, Chai Y, Yuan R: Ultrasensitive electrochemiluminescent aptasensor for ochratoxin A detection with the loop-mediated isothermal amplification. Anal Chim Acta 2014, 811:70-75. 41. Park JH, Byun JY, Mun H, Shim WB, Shin YB, Li T, Kim MG: A regeneratable, label-free, localized surface plasmon resonance (LSPR) aptasensor for the detection of ochratoxin A. Biosens Bioelectron 2014, 59:321-327.

27. Hayat A, Andreescu S, Marty J-L: Design of PEG-aptamer two piece macromolecules as convenient and integrated sensing platform: application to the label free detection of small size molecules. Biosens Bioelectron 2013, 45:168-173.

42. Chen X, Huang Y, Ma X, Jia F, Guo X, Wang Z: Impedimetric aptamer-based determination of the mold toxin fumonisin B1. Microchim Acta 2015, 182:1709-1714.

28. Kim YS, Jung HS, Matsuura T, Lee HY, Kawai T, Gu MB: Electrochemical detection of 17beta-estradiol using DNA aptamer immobilized gold electrode chip. Biosens Bioelectron 2007, 22:2525-2531.

43. Chen J, Zhang X, Cai S, Wu D, Chen M, Wang S, Zhang J: A fluorescent aptasensor based on DNA-scaffolded silvernanocluster for ochratoxin A detection. Biosens Bioelectron 2014, 57:226-231.

29. Eissa S, Siaj M, Zourob M: Aptamer-based competitive electrochemical biosensor for brevetoxin-2. Biosens Bioelectron 2015, 69:148-154.

44. Zhu Z, Feng M, Zuo L, Zhu Z, Wang F, Chen L, Li J, Shan G, Luo SZ: An aptamer based surface plasmon resonance biosensor for the detection of ochratoxin A in wine and peanut oil. Biosens Bioelectron 2015, 65:320-326.

30. Elshafey R, Siaj M, Zourob M: DNA aptamers selection and characterization for development of label-free impedimetric aptasensor for neurotoxin anatoxin-a. Biosens Bioelectron 2015, 68:295-302. Current Opinion in Biotechnology 2017, 45:15–23

45. Guo X, Wen F, Zheng N, Luo Q, Wang H, Wang H, Li S, Wang J: Development of an ultrasensitive aptasensor for the detection of aflatoxin B1. Biosens Bioelectron 2014, 56:340-344. www.sciencedirect.com

Aptasensors for small molecule contaminants Nguyen, Kwon and Gu 23

46. Evtugyn G, Porfireva A, Stepanova V, Sitdikov R, Stoikov I, Nikolelis D, Hianik T: Electrochemical aptasensor based on polycarboxylic macrocycle modified with neutral red for aflatoxin B1 detection. Electroanalysis 2014, 26:2100-2109. 47. Istamboulie´ G, Paniel N, Zara L, Granados LR, Barthelmebs L,  Noguer T: Development of an impedimetric aptasensor for the determination of aflatoxin M1 in milk. Talanta 2016, 146:464-469. In this paper, an impedimetric aptasensor was developed for the detection of aflatoxin M1 in milk samples. Both spiked and real milk samples were successfully analyzed with this aptasensor, and AFM1 was detected in a wide range from 20 to 1000 ng/kg.

56. Seo HB, Kwon YS, Lee JE, Cullen D, Noh HM, Gu MB: A novel reflectance-based aptasensor using gold nanoparticles for the detection of oxytetracycline. Analyst 2015, 140:6671-6675. 57. Wang S, Yong W, Liu J, Zhang L, Chen Q, Dong Y: Development of an indirect competitive assay-based aptasensor for highly sensitive detection of tetracycline residue in honey. Biosens Bioelectron 2014, 57:192-198. 58. Xing Y-P, Liu C, Zhou X-H, Shi H-C: Label-free detection of kanamycin based on a G-quadruplex DNA aptamer-based fluorescent intercalator displacement assay. Sci Rep 2015, 5:8125.

48. Soh JH, Lin Y, Rana S, Ying JY, Stevens MM: Colorimetric detection of small molecules in complex matrixes via targetmediated growth of aptamer-functionalized gold nanoparticles. Anal Chem 2015, 87:7644-7652.

59. Emrani AS, Danesh NM, Lavaee P, Ramezani M, Abnous K, Taghdisi SM: Colorimetric and fluorescence quenching aptasensors for detection of streptomycin in blood serum and milk based on double-stranded DNA and gold nanoparticles. Food Chem 2016, 190:115-121.

49. Akki SU, Werth CJ, Silverman SK: Selective aptamers for  detection of estradiol and ethynylestradiol in natural waters. Environ Sci Technol 2015, 49:9905-9913. This paper showed the great selectivity of a new aptamer for capturing 17b-estradiol and 17a-ethynylestradiol. Interestingly, the new aptamers do not lose sensitivity or selectivity in natural waters even in presence of other organic materials.

60. Mohammad Danesh N, Ramezani M, Sarreshtehdar Emrani A, Abnous K, Taghdisi SM: A novel electrochemical aptasensor based on arch-shape structure of aptamer-complimentary strand conjugate and exonuclease I for sensitive detection of streptomycin. Biosens Bioelectron 2016, 75:123-128.

50. Zhou L, Wang J, Li D, Li Y: An electrochemical aptasensor based on gold nanoparticles dotted graphene modified glassy carbon electrode for label-free detection of bisphenol A in milk samples. Food Chem 2014, 162:34-40. 51. Xu S, Yuan H, Chen S, Xu A, Wang J, Wu L: Selection of DNA aptamers against polychlorinated biphenyls as potential biorecognition elements for environmental analysis. Anal Biochem 2012, 423:195-201. 52. Pilehvar S, Mehta J, Dardenne F, Robbens J, Blust R, De Wael K: Aptasensing of chloramphenicol in the presence of its analogues: reaching the maximum residue limit. Anal Chem 2012, 84:6753-6758. 53. Mehta J, Rouah-Martin E, Van Dorst B, Maes B, Herrebout W, Scippo M-L, Dardenne F, Blust R, Robbens J: Selection and characterization of PCB-binding DNA aptamers. Anal Chem 2012, 84:1669-1676. 54. Wu L, Qi P, Fu X, Liu H, Li J, Wang Q, Fan H: A novel electrochemical PCB77-binding DNA aptamer biosensor for selective detection of PCB77. J Electroanal Chem 2016, 771:45-49. 55. Pilehvar S, Ahmad Rather J, Dardenne F, Robbens J, Blust R,  De Wael K: Carbon nanotubes based electrochemical aptasensing platform for the detection of hydroxylated polychlorinated biphenyl in human blood serum. Biosens Bioelectron 2014, 54:78-84. An impedimetric aptasensor for the detection of hydroxylated polychlorinated biphenyls in human blood serum. The aptasensor showed an excellent electrocatalytic activity and was used to detect hydroxylated polychlorinated biphenyl in a human blood serum samples with a limit of detection of 10 nM.

www.sciencedirect.com

61. Shi H, Zhao G, Liu M, Fan L, Cao T: Aptamer-based colorimetric sensing of acetamiprid in soil samples: sensitivity, selectivity and mechanism. J Hazard Mater 2013, 260:754-761. 62. Fei A, Liu Q, Huan J, Qian J, Dong X, Qiu B, Mao H, Wang K: Labelfree impedimetric aptasensor for detection of femtomole level acetamiprid using gold nanoparticles decorated multiwalled carbon nanotube-reduced graphene oxide nanoribbon composites. Biosens Bioelectron 2015, 70:122-129. 63. Li H, Qiao Y, Li J, Fang H, Fan D, Wang W: A sensitive and label free photoelectrochemical aptasensor using Co-doped ZnO diluted magnetic semiconductor nanoparticles. Biosens Bioelectron 2016, 77:378-384. This paper reported a photoelectrochemical aptasensor using Co-doped ZnO diluted magnetic semiconductor nanoparticles. The developed photoelectrochemical aptasensor exhibits high stability, excellent selectivity, and low limit of detection. 64. Guo J, Li Y, Wang L, Xu J, Huang Y, Luo Y, Shen F, Sun C, Meng R: Aptamer-based fluorescent screening assay for acetamiprid via inner filter effect of gold nanoparticles on the fluorescence of CdTe quantum dots. Anal Bioanal Chem 2016, 408:557-566. 65. Zhang Y, Yang L, Lin C, Guo L, Qiu B, Lin Z, Chen G: Fluorescence aptasensor for Ochratoxin A in food samples based on hyperbranched rolling circle amplification. Anal Methods 2015, 7:6109-6113. 66. Pfeiffer F, Mayer G: Selection and biosensor application of aptamers for small molecules. Front Chem 2016, 4:25. 67. Chen A, Yan M, Yang S: Split aptamers and their applications in  sandwich aptasensors. Trends Anal Chem 2016, 80:581-593. This paper is a comprehensive review article on use of split aptamers. It discusses the applications of split aptamers for developing sandwich aptasensors.

Current Opinion in Biotechnology 2017, 45:15–23