biosensors

Review Comparison of Electrochemical Immunosensors and Aptasensors for Detection of Small Organic Molecules in Environment, Food Safety, Clinical and Public Security

Benoit Piro *,†, Shihui Shi †, Steeve Reisberg †, Vincent Noël † and Guillaume Anquetin †

University Paris Diderot, Sorbonne Paris Cité, ITODYS, UMR 7086 CNRS, 15 rue J-A de Baïf, Paris 75205, France; [email protected] (S.S.); [email protected] (S.R.); [email protected] (V.N.); [email protected] (G.A.) * Correspondence: [email protected]; Tel.: +33-1-5727-7224 † These authors contributed equally to this work.

Academic Editors: Nicole Jaffrezic-Renault and Carole Chaix Received: 22 December 2015; Accepted: 19 February 2016; Published: 29 February 2016

Abstract: We review here the most frequently reported targets among the electrochemical immunosensors and aptasensors: antibiotics, bisphenol A, cocaine, ochratoxin A and estradiol. In each case, the immobilization procedures are described as well as the transduction schemes and the limits of detection. It is shown that limits of detections are generally two to three orders of magnitude lower for immunosensors than for aptasensors, due to the highest affinities of antibodies. No significant progresses have been made to improve these affinities, but transduction schemes were improved instead, which lead to a regular improvement of the limit of detections corresponding to ca. five orders of magnitude over these last 10 years. These progresses depend on the target, however.

Keywords: immunosensor; aptasensor; ciprofloxacin; enrofloxacin; ofloxacin; tetracyclines; neomycin; tobramycin; ampicillin; kanamycin; sulfonamides; bisphenol A; cocaine; ochratoxin A; estradiol

1. Introduction There is a need to detect, for environment, security or medical purposes, various small organic molecules including antibiotics, drugs, toxins, pollutants, and so on. Although highly sensitive and selective, conventional chromatographic and spectroscopic analytical methods are time-consuming and laborious. Moreover, these techniques require expensive equipment, trained operators and tedious pretreatments. The need for disposable tools for monitoring these molecules encourages the development of simple, efficient, continuous, reliable, cost-effective and field-portable screening methods for analysis of environmental contaminants. Biosensors, and particularly electrochemical ones, appear to be an optimal technology. Since 1962 and the first enzyme-based glucose sensing device, there has been explosive development in biosensors in terms of scientific publications. The figures of merit for biosensors first depend on the bioprobes which are chosen. Herein we reviewed, among the electrochemical biosensors, the most frequently reported bioprobes (antibodies, aptamers, and peptides, when applicable) for frequently reported targets: antibiotics, bisphenol A, cocaine, ochratoxin A and estradiol. In each case, the immobilization procedure was described, as well as the transduction scheme and the limit of detection. We will compare the results from one probe to another and discuss their efficiency for each target, respectively.

Biosensors 2016, 6, 7; doi:10.3390/bios6010007 www.mdpi.com/journal/biosensors Biosensors 2016, 6, 7 2 of 22 Biosensors 2016, 6, 7 2 of 21

1.1.1.1. Immunosensors Immunosensors ImmunosensorsImmunosensors areare probablyprobably thethe mostmost reportedreported biosensors; they they are are based based on on the the binding binding propertiesproperties of of antibodies antibodies (Ab)(Ab) towa towardrd a a specific specific target, target, called called an anantigen antigen (Ag). (Ag). The Themost mostfrequent frequent Ab Abreported reported in inbiosensors biosensors are are immunoglobulins immunoglobulins G (IgG), G (IgG), with with a typical a typical molecular molecular weight weight of 150 ofkDa 150 and kDa anda mean a mean size size of of14 14 × ˆ9 ×9 4ˆ nm4 nm3. They3. They are are composed composed of oftwo two light light and and two two heavy heavy chains, chains, linked linked by by disulfidedisulfide bonds bonds to to form form a a characteristic characteristic Y-shapeY-shape [[1,2]1,2] (Figure(Figure1 1).).

Figure 1. Immunoglobulin G (IgG) consists of two heavy chains (VH, CH1 and CH2) and two light Figure 1. Immunoglobulin G (IgG) consists of two heavy chains (VH, CH1 and CH2) and two light chains (VL and CL). C are constant regions (which do not bring specificity to a given antigen) whereas chains (VL and CL). C are constant regions (which do not bring specificity to a given antigen) whereas V are variable regions, which bring specificity. As a consequence, antigens bind to the variable regions V are variable regions, which bring specificity. As a consequence, antigens bind to the variable regions VL and VH. In order to decrease the size of IgG, only the Fab (antigen binding fragment, composed VL and VH. In order to decrease the size of IgG, only the Fab (antigen binding fragment, composed of of the VH, VL, CH1 and CL regions) can be employed. Reproduced from [3]. the VH, VL, CH1 and CL regions) can be employed. Reproduced from [3]. 1.2. Aptasensors 1.2. Aptasensors Aptamers are short (15–100 bases) DNA or RNA strands that are able to bind to a specific target molecule.Aptamers Aptamers are short are (15–100usually bases)created DNA by selecting or RNA them strands from that a large are ablerandom to bind sequence to a specific pool target(procedure molecule. called Aptamers SELEX), are but usually naturally created occurring by selecting aptamers them also from exist. a large Aptamers random can sequence be used poolin (proceduresensors but called were SELEX), generally but naturallydeveloped occurring for clinical aptamers purposes, also as exist. drugs, Aptamers for their can interactions be used in sensorswith butexpressed were generally proteins. developed However, forthey clinical can be purposes,selected for as other drugs, targets for theirthan protei interactionsns, such with as natural expressed or proteins.synthetic However, small organic they can molecules. be selected Aptamers for other are targets helpful than in proteins,the detection such asof naturala wide or range synthetic of smallcompounds organic molecules.which are not Aptamers (or too few) are helpful immunogenic in the detection to allow production of a wide range of specific of compounds antibodies, which so arethat not immunosensors (or too few) immunogenic cannot be used to allow[4]. production of specific antibodies, so that immunosensors cannot be used [4]. 1.3. Peptide Sensors 1.3. Peptide Sensors The interest in replacing antibodies with peptides is the same as for aptamers [4]. In recent years, theThe use interestof peptides in replacing as sensing antibodies probes for with constructing peptides iselectrochemical the same as for biosensors aptamers has [4]. received In recent great years, theattention use of peptides because, ascompared sensing to probes antibodies for constructing or aptamers, electrochemical peptides are smaller biosensors and rarely has receiveddenaturized great attentionwhen immobilized because, compared on the sensing to antibodies surface. or However, aptamers, even peptides if various aresmaller peptide and sequences rarely denaturizedexhibiting whenaffinity immobilized to particular on substrates the sensing have surface. been However,found, peptides even ifcapable various of peptide recognizing sequences low molecular exhibiting affinityweight to organic particular compounds substrates with have sufficient been found, affinity peptides have rarely capable been of recognizingreported as of low yet. molecular weight organic compounds with sufficient affinity have rarely been reported as of yet. 2. Substances and Receptors 2. Substances and Receptors The most frequently reported electrochemical biosensing approaches are comprehensively reviewedThe most for each frequently molecule reported of interest electrochemical that we selected. biosensing approaches are comprehensively reviewed for each molecule of interest that we selected. 2.1. Antibiotics 2.1. Antibiotics Immunosensors for drugs detection and quantification have been described for over 30 years. TheImmunosensors most reported drugs for drugs are antibiotics detection and, and quantificationmore particularly, have ciprofloxacin, been described enrofloxacin, for over 30 ofloxacin, years. The mosttetracycline, reported neomycin, drugs are antibioticstobramycin, and, ampicillin, more particularly, kanamycin ciprofloxacin,and sulfonamides enrofloxacin, (Figure 2). ofloxacin, Both tetracycline,antibodies and neomycin, aptamers tobramycin, were used as ampicillin,probes for these kanamycin molecules. and sulfonamides (Figure2). Both antibodies and aptamers were used as probes for these molecules. Biosensors 2016, 6, 7 3 of 21 Biosensors 2016, 6, 7 3 of 22 Biosensors 2016, 6, 7 3 of 21

(a) (b)(c) (a) (b)(c)

(d) (d) ((e)(f) f)

(g) (h)(i) (g) (h)(i) Figure 2. Chemical structures of (a) ciprofloxacin; (b) enrofloxacin; (c) ofloxacin; (d) tetracycline; Figure 2. Chemical structures of (a) ciprofloxacin; (b) enrofloxacin; (c) ofloxacin; (d) tetracycline; Figure(e )2. neomycin; Chemical (f) tobramycin;structures (ofg) ampicillin;(a) ciprofloxacin; (h) kanamycin (b) enrofloxacin;and (i) hydrochlorothiazide (c) ofloxacin; (a sulfonamide). (d) tetracycline; ((e)) neomycin;neomycin; (f) tobramycin; (g) ampicillin; (h) kanamycin and (i) hydrochlorothiazide (a sulfonamide). 2.1.1. Antibodies 2.1.1. Antibodies In 2007, Garifallou et al. [5] described a label-free immunosensor for ciprofloxacin based on Inantibody-modified 2007, Garifallou Garifallou screen-printed etet al. al. [5][5 ]described described carbon aelectr alabel-free label-freeodes (SPEs), immunose immunosensor usingnsor electrochemical for forciprofloxacin ciprofloxacin impedance based based on onantibody-modified antibody-modifiedspectroscopy (EIS) screen-printed screen-printedfor detection. carbon The carbon antibody electr electrodesodes (Ab) (SPEs),was (SPEs), immobilized using using electrochemical through biotin-avidin impedanceimpedance spectroscopyinteractions. (EIS)(EIS) The forfor impedance detection.detection. was The Thereported antibody antibody to increa (Ab) (Ab)se withwas ciprofloxacin immobilized concentration, through biotin-avidinbiotin-avidin with a linearity domain between 1 and 100 ng·mL−1 (3 nM–0.3 nM). The same year, another group proposed interactions. TheThe impedance impedance was was reported reported to increase to increa withse ciprofloxacinwith ciprofloxacin concentration, concentration, with a linearity with a a similar approach, but the Ab was immobilized by peptide coupling, presenting must less steric ´1 −1 domainlinearityhindrance between domain than 1between and the 100biot 1 ngin-avidin and¨ mL 100 coupling, ng·mL(3 nM–0.3 which(3 nM).nM–0.3 allowed The nM). same to Thedecrease year, same another the year, LoD groupanother down proposed to group 10 pg·mL proposed a similar−1 approach,a similar(30 pM) approach, but [6]. the In 2009, Ab but was the the same immobilized Ab group was immobilizedpublished by peptide an innovative by coupling, peptide immunosensor presentingcoupling, presentingwhere must ciprofloxacin less stericmust hindranceless was steric ´ thanhindrance thefirst biotin-avidin grafted than the on thebiot coupling, electrode,in-avidin whichonto coupling, which allowed the which ant toi-ciprofloxacin decrease allowed theto decreaseAb LoD was down complexed the to LoD 10 pg (Figuredown¨ mL to3).1 (30 10In thepg·mL pM) [6−].1 In(30 2009, pM)presence the[6]. sameIn of2009, ciprofloxacin, group the same published groupthe antibody an published innovative was andispla immunosensor innovativeced in solution immunosensor where inducing ciprofloxacin strong where changes ciprofloxacin was first in the grafted was onfirst the graftedimpedance electrode, on theof onto the electrode, electrode; which theonto the anti-ciprofloxacin LoD which was the of caant. 1 i-ciprofloxacinpg·mL Ab− was1 (3 pM) complexed [7]. Ab was (Figurecomplexed3). In (Figure the presence 3). In the of ciprofloxacin,presence of ciprofloxacin, the antibody wasthe antibody displaced was in solution displaced inducing in solution strong inducing changes instrong the impedance changes in of the electrode;impedance the of LoDthe electrode; was of ca. the1 pg LoD¨ mL was´1 (3 of pM) ca. 1 [ pg·mL7]. −1 (3 pM) [7].

Figure 3. Functioning principle of the immunosensor for ciprofloxacin based on anti-ciprofloxacin antibody displacement. Reprinted with permission from [7]. Copyright 2009 American Chemical Society.

Detection of enrofloxacin on SAM-modified electrodes gave similar results. For example, FigureWu et 3.3.al. Functioning, (2009) reported principle EIS ofcharacterization the immunosensor of interactions forfor ciprofloxacinciprofloxacin between based enrofloxacin on anti-ciprofloxacinanti-ciprofloxacin and its Ab antibody displacement. Reprinted Reprinted with with permission permission from from [7]. [7]. Copyright Copyright 2009 2009 American American Chemical Chemical Society. Society.

Detection of enrofloxacinenrofloxacin onon SAM-modifiedSAM-modified electrodeselectrodes gavegave similarsimilar results.results. For example, Wu etet al.al.,, (2009)(2009) reportedreported EISEIS characterizationcharacterization ofof interactionsinteractions betweenbetween enrofloxacinenrofloxacin and itsits AbAb Biosensors 2016, 6, 7 4 of 22 immobilized on a SAM-modified Au electrode; this interaction caused an increase of electron transfer resistance (Rct) with a LoD of 1 ng¨ mL´1 (3 nM). [8] Zhang et al., (2013) described a competitive enzyme-amplified immunosensor (using the horseradish peroxidase enzyme, HRP) based on gold nanoclusters/polypyrrole (PPy)-functionalized electrodes, for sensitive detection of ofloxacin [9], which exhibited a response in the range 0.08–410 ng¨ mL´1, with a LoD of 30 pg¨ mL´1 (0.1 nM). The use of magnetic beads (MBs) can improve sensitivity. For example, Zacco et al. [10] described an electrochemical immunosensor for the detection of sulfonamide antibiotics based on magnetic beads, amplified with the use of HRP as label. The LoD was ca. 1 µg¨ L´1 (6 nM). Centi et al. described a more complicated transduction architecture [11], using protein A immobilized on magnetic microbeads (MBs) to bind the specific Ab. A competition assay between the target analyte and the HRP-labeled sulfonamide target was carried out. The LoD was ca. 1 ng¨ mL´1 (4 nM). Conzuelo et al. (2012) [12] used a selective capture Ab immobilized on the surface of MBs for tetracycline detection. A direct competitive immunoassay using HRP for the enzymatic labeling was performed, with hydroquinone (HQ) as redox mediator. They obtained low LoD (in the ng¨ mL´1 range, i.e., the nM range) for 4 tetracycline antibiotics. The same group described more recently a similar procedure where Abs were immobilized by the diazonium route [13]. Que et al. [14] nanostructured their electrodes for tetracycline detection using platinum NPs deposited on graphene nanosheets (GN). This way, retaining a competitive immunoassay format, they significantly lowered the LoD down to 6 pg¨ mL´1 (13 pM). Even if, as it is generally the case for immunosensors, enzyme-labeling is the dominant strategy for improving sensitivity, it appears that non-enzymatic amplification (through increase of the active surface area or use of catalytic reactions) are often more efficient than classical (HRP) enzymatic amplification.

2.1.2. Aptamers Aptamers have been used for tetracycline detection as well, by Kim et al., in 2009. They used interdigitated gold electrodes modified with a 76-mer thiol-ssDNA aptamer. The dissociation constant (KD) of this aptamer for oxytetracycline was 11 nM. Transduction was explained by the fact that the 3´{4´ redox probe diffusing in solution (Fe(CN)6 ) was influenced by interactions between the aptamers and the target molecules. The dynamic range was 1–100 nM (LoD of 1 nM, i.e., 0.3 ng¨ mL´1)[15]. In 2010, the same group [16] described similar results but using a streptavidin-modified screen-printed gold electrode onto which biotinylated ssDNA aptamers were immobilized. Still using 3´{4´ CV and SWV with Fe(CN)6 as diffusing probe, they found a LoD for tetracycline of 10 nM (i.e., ´1 3´{4´ 3 ng¨ mL ). In another similar example, Zhou et al. [17], still using diffusing Fe(CN)6 as redox probe, described an electrochemical tetracycline aptasensor involving multiwalled carbon nanotubes (MWCNTs), the electroactivity of which being decreased due to the formation of aptamer—tetracycline complexes. The peak current changes obtained by differential pulse voltammetry (DPV) increased linearly with tetracycline concentrations from 5 pM (1.5 ng¨ mL´1) to 50 µM. Chen et al., in 2014 [18], used EIS with a 13 bases-long aptamer; a linear relationship between the log concentration of tetracycline and the charge transfer resistance was found from 5 ng¨ mL´1 to 5 µg¨ mL´1; the LoD ´1 was 1 ng¨ mL (ca. 2 nM). This LoD was significantly lower than the KD determined by the authors using calorimetry (KD of 50 µM). Shen et al. [19] used differential pulse voltammetry (DPV) with Prussian blue immobilized onto AuNPs modified with a 79 bases long aptamer having a KD of 63 nM for tetracycline. The detection range was linear from 10´9 to 10´5 M, with a LoD of 0.3 nM (i.e., 0.1 ng¨ mL´1). The first label-free electrochemical RNA-based aptasensor for detection of an antibiotic was described in 2007 for neomycin (Figure4)[ 20]. The authors described a competitive displacement assay and used EIS. The selectivity of the RNA aptamer allowed determination of neomycin even in biological samples of high protein content. The sensitivity was not excellent, around 1 µM, close to the KD of the aptamers-neomycin complex. Biosensors 2016, 6, 7 5 of 22 Biosensors 2016, 6, 7 5 of 21

FigureFigure 4. Schematic 4. Schematic illustration illustration of of the the modified modified electrode electrodeand and the the competitive competitive assay assay between between complexed complexed neomycinneomycin on the on theelectrode electrode surface surface and and freely freely diffusion diffusion neomycin.neomycin. Reprinted Reprinted with with permission permission from from [20]. [20]. CopyrightCopyright 2007 2007 American American Chemical Chemical Society. Society.

TheThe previous previous examples examples are aremostly mostly based based on onthe the use use of a of redox a redox probe probe diffusing diffusing in solution, in solution, which bringswhich sensitivity brings sensitivity but is obviously but is obviously difficult difficultto integrate to integrate when a when system a system is really is reallyapplied. applied. In 2010, In the group2010, of Plaxco the group showed of Plaxco that showed classical that RNA classical aptamers RNA,aptamers, even if sensitive even if sensitive to nuclease, to nuclease, can be canused in biologicalbe used samples in biological providing samples that providing these samples that these were samples previously were ultra-filtrated. previously ultra-filtrated. They demonstrated They µ µ ´1 the detectiondemonstrated of tobramycin the detection in of the tobramycin range 2–6 in μ theM range(4–10 2–6μg mLM− (4–101) in bloodg¨ mL using) in blooda methylene using a blue methylene blue (MB)-modified RNA aptamer sequence 51-HS-GGG ACU UGG UUU AGG UAA (MB)-modified RNA aptamer sequence 5′-HS-GGG ACU UGG UUU AGG UAA UGA GUC CC-NH- UGA GUC CC-NH-MB-31, having a dissociation constant of 13.2 µM for tobramycin (measured MB-3′, having a dissociation constant of 13.2 μM for tobramycin (measured in homogenous in homogenous conditions) [21]. They have shown that the KD increased at least by one order of conditions)magnitude [21]. in heterogeneousThey have shown conditions. that Athe similar KD increased approach hasat least been reportedby one order in several of magnitude following in heterogeneouspapers, with conditions. variable results. A similar For example, approach Liu haset al. been, in 2014 reported [22], reported in several an electrochemical following papers, DNA with variableaptamer-based results. For tobramycin example, sensorLiu et al. (5,1 -HS-C6-GGGin 2014 [22], reported ACT TGG an TTT electrochemical AGG TAA TGA DNA GTC aptamer-based CC-MB-31 tobramycinsequence, sensor immobilized (5′-HS-C6-GGG on gold microelectrodes). ACT TGG DespiteTTT AGG the electrochemicalTAA TGA GTC deposition CC-MB-3 of dendritic′ sequence, immobilizedgold nanostructures, on gold microelectrodes). they reported a high Despite LoD of ca.the1 mMelectrochemical (KD in the mM deposition range as well). of dendritic gold nanostructures,The reagentless they reported detection a high of small LoD moleculesof ca. 1 mM using (KD aptamers in the mM is notrange straightforward as well). because theThe spatialreagentless reorganization detection triggeredof small molecules by the analyte using aptamers is often small. is not straightforward To address this because issue, the spatialGonzales-Fernandes reorganization ettriggered al. described by athe strategy analyte based is on often competition small. betweenTo addre tobramycinss this issue, immobilized Gonzales- on magnetic microbeads (MBs) and free tobramycin diffusing in solution. Detection was made Fernandes et al. described a strategy based on competition between tobramycin immobilized on by post-labeling aptamers with an enzyme (alkaline phosphatase, AlkP) through the biotin-avidin magnetic microbeads (MBs) and free tobramycin diffusing in solution. Detection was made by post- conjugate. Using a 27-mer anti-tobramycin RNA aptamer (51-GGC ACG AGG UUU AGC UAC ACU labelingCGU aptamers GCC-31), theywith found an enzyme a linear (alkaline response phosphatase, in the range 5–500 AlkP)µM[ through23]. The the same biotin-avidin authors described conjugate. Usinglater a 27-mer a slightly anti-tobramycin modified architecture RNA aptamer for tobramycin (5′-GGC detection ACG AGG using anUUU antibody-antigen AGC UAC ACU association CGU GCC- μ 3′), theyfor introducing found a linear the enzyme response conjugate in the on therange aptamers 5–500 [24 ].M Compared [23]. The with same the authors biotin-avidin described approach, later a slightlythe limitmodified of detection architecture was lowered for tobramycin down to 0.1 µdetectionM. The authors using explained an antibody-antigen this behavior by association the steric for introducinghindrance the of enzyme avidin, which conjugate impeded on detection.the aptamers [24]. Compared with the biotin-avidin approach, the limit Asof detection shown, reported was lowered LoD stay down too highto 0.1 for μM. practical The authors applications explained when this aptamers behavior are by labeled the steric hindrancewith redox of avidin, probes. which Another impeded way detection. to detect a molecule through the use of aptamers is to us themAs shown, as pre-concentrators reported LoD immobilizedstay too high on for the practical electrode applications surface, followed when byaptamers electro-oxidation are labeled or with redoxelectroreduction probes. Another of the way target to detect molecule a molecule directly onthro theugh electrode, the use without of aptamers any other is to intermediate. us them as pre- Zhu et al. [25] proposed such an approach applied to kanamycin and involving AuNPs. The concentrators immobilized on the electrode surface, followed by electro-oxidation or electroreduction of authors used a 22-bases DNA aptamer (51-TGG GGG TTG AGG CTA AGC CGA C-31) showing the target molecule directly on the electrode, without any other intermediate. Zhu et al. [25] proposed a high binding affinity to kanamycin (KD = 79 nM), immobilized onto AuNPs modified with such an approach applied to kanamycin and involving AuNPs. The authors used a 22-bases DNA aptamer (5′-TGG GGG TTG AGG CTA AGC CGA C-3′) showing a high binding affinity to kanamycin (KD = 79 nM), immobilized onto AuNPs modified with poly-[2,5-di-(2-thienyl)-1H-pyrrole-1-(p- benzoic acid)]. Kanamycin detection was achieved by CV and linear sweep voltammetry (LSV). The calibration plot showed a linear range from 0.05 μM to 9.0 μM with a LoD significantly lowered (10 nM, i.e., 5 ng·mL−1) compared to the previously reported architectures. Compared to the other amplification strategies used with aptamers, this approach of preconcentration followed by stripping Biosensors 2016, 6, 7 6 of 22 poly-[2,5-di-(2-thienyl)-1H-pyrrole-1-(p-benzoic acid)]. Kanamycin detection was achieved by CV and linear sweep voltammetry (LSV). The calibration plot showed a linear range from 0.05 µM to 9.0 µM with a LoD significantly lowered (10 nM, i.e., 5 ng¨ mL´1) compared to the previously reported architectures. Compared to the other amplification strategies used with aptamers, this approach of preconcentration followed by stripping (adsorptive stripping voltammetry—ASV) is certainly the one which leads to the highest sensitivities after EIS. Lastly, it should also be stressed that attempts to make all-polymer, all-printed, electrochemical sensors were reported. For example, Dapra et al. [26] presented an all-polymer electrochemical biosensor based on conductive bilayer consisting of tosylate-doped poly(3,4-ethylenedioxythiophene) (PEDOT-TsO) and its hydroxymethyl derivative which was covalently functionalized with two short aptamer probes with affinity to ampicillin or kanamycin A, respectively. The ampicillin DNA aptamer 1 1 sequence was 5 -GCG GGC GGT TGT ATA GCG G-3 (KD = 13 nM) and the kanamycin one was 1 1 5 -TGG GGG TTG AGG CTA AGC CGA-3 (KD = 79 nM). Using EIS, they were able to detect ampicillin in a concentration range from 100 pM to 1 µM and kanamycin A from 10 nM to 1 mM. As discussed by the authors, the low LoD and wide dynamic range of their biosensor could be attributed not only to the high affinity of the DNA probes but also to their device properties. Indeed, for a similar probe applied in different techniques, LoDs can change over three orders of magnitude. All these results are summarized in Table1. As shown, tetracycline is a common target for Abs and aptamers. LoDs in the nM range were obtained for both capture probes, without significant differences. For a same probe, it appears that electrode nanostructuration leads to an improvement of the detection limit of at least one order of magnitude. It is also interesting to note that methods involving enzyme amplifications applied with aptamer probes do not appear to be more sensitive than non-amplified methods.

Table 1. Figures of merit of selected immunosensors and aptasensors for detection of some antibiotics: ciprofloxacin (CF), ofloxacin (OFL), sulfonamides (SA), tetracyclines (TC), neomycin (NEO), tobramycin (TOB), kanamycin (KAN), ampicillin (AMP).

Targets Bioreceptors LoD Transduction & Analytical Methods Ref. CF Ab 3 nM EIS [5] CF Ab 30 pM EIS [6] CF Ab 3 nM EIS [7] CF Ab 3 nM SAM/EIS [8] OFL Ab 0.1 nM HRP/AuNPs/PPy/CV [9] SA Ab 6 nM MBs/HRP/CV [10] TC Ab 4 nM MBs/HRP/CV [11] TC Ab 1 nM HRP/HQ/CV [12] TC Ab 13 pM Platinum-catalyzed HER/CV [14] 3´{4´ TC Aptamer 1 nM Fe(CN)6 /CV [15] 3´{4´ TC Aptamer 10 nM Fe(CN)6 /CV/SWV [16] 3´{4´ TC Aptamer 5 pM CNTs/Fe(CN)6 /DPV [17] TC Aptamer 2 nM EIS [18] TC Aptamer 0.3 nM PB/DPV [19] NEO Aptamer 1 µM EIS [20] TOB Aptamer 2 µM MB label/CV [21] TOB Aptamer 1 mM AuNPs/MB label/CV [22] TOB Aptamer 5 µM MBs/AlkP/CV [23] TOB Aptamer 0.1 µM MBs/AlkP/CV [24] KAN Aptamer 10 nM AuNPs/CV [25] KAN Aptamer 10 nM PEDOT/EIS [26] AMP Aptamer 100 pM PEDOT/EIS [26] Note: LoD (limit of detection); HRP (horseradish peroxidase); PPy (polypyrrole); HER (hydrogen evolution reaction), CV (cyclic voltammetry); SWV (square wave voltammetry); MBs (magnetic beads); CNTs (carbon nanotubes); PB (prussian blue); MB (methylene blue); AuNPs (gold nanoparticles). Biosensors 2016, 6, 7 7 of 22

2.2. BisphenolBiosensors A 2016, 6, 7 7 of 21 Bisphenol-A2.2. Bisphenol (BPA) A (Figure5) is a well-known and much studied contaminant causing a wide range of healthBisphenol-A problems (BPA) to living (Figure beings, 5) is a well-known especially and the much young. studied In 2013,contaminant Ragavan causinget al.a widereviewed the various principles,range of health mechanisms problems to living and performancesbeings, especiallyof the BPA young. biosensors In 2013, Ragavan available et al. reviewed in the literature the [27], various principles, mechanisms and performances of BPA biosensors available in the literature [27], among which are found electrochemical immuno- and apta-sensors. among which are found electrochemical immuno- and apta-sensors.

(a) (b)(c)

Figure 5. Chemical structure of (a) bisphenol A; (b) bisphenol S and (c) bisphenol F. Figure 5. Chemical structure of (a) bisphenol A; (b) bisphenol S and (c) bisphenol F. 2.2.1. Antibodies 2.2.1. AntibodiesThe first electrochemical immunosensor for BPA detection was described very recently, in 2007, by Rahman et al. and Piao et al. (from the same research group) [28,29]. The immunosensor was The firstfabricated electrochemical by covalent binding immunosensor of the Ab onto for BPACOOH-functionalized detection was describedpolythiophene. very It showed recently, in 2007, by Rahmanspecificet al. recognitionand Piao of etBPA al. on(from a linear the dynamic same range research between group) 1 and [10028, 29ng]. mL The−1. The immunosensor LoD was was fabricated0.3 by ng·mL covalent−1 (1.3 binding nM). Wang of the et Abal. ontopublished COOH-functionalized recently a label-free electrochemical polythiophene. competitive It showed specific −1 recognitionimmunosensor of BPA on aable linear to dynamicdetect BPA range directly between with a 1limit and 100of detection ng¨ mL ´of1 .2 The pg·mL LoD was(9 pM). 0.3 ng¨ mL´1 Characterizations made by SWV showed that the polymer film presented a current decrease upon (1.3 nM). Wanganti-BPAet binding al. published and an opposite recently current a label-free increase up electrochemicalon BPA addition in competitive solution [30]. immunosensorThe difference able to detect BPAin directlysensitivity withbetween a limit the results of detection of Rahman of et 2al. pg, Piao¨ mL et ´al.1 and(9 pM). Wang Characterizationset al. could be explained made by by SWV showed thatthe thenature polymer of transduction film presented scheme. aIndeed, current the decrease latter reported upon anti-BPAthe use of binding a competitive and an opposite current increaseimmunodisplacement upon BPA additionapproach wher in solutioneas the two [30 first]. The articles difference reported a in classical sensitivity Ab-Ag betweeninteraction the results where the Ab was covalently immobilized on the electrode. of Rahman et al., Piao et al. and Wang et al. could be explained by the nature of transduction scheme. Indeed, the2.2.2. latter Aptamers reported the use of a competitive immunodisplacement approach whereas the two first articles reportedEven if strategies a classical to develop Ab-Ag antibodies interaction towards wherebisphenol the A, Abincluding was antibodies covalently with immobilized good on the electrode.affinities, have been reported (see above), DNA aptamers specific to BPA were reported to be of higher affinity than antibodies (in the nM range). For this reason, lower LoDs have been reached. 2.2.2. AptamersElectrochemical transduction was investigated by Xue et al. [31]. They used a specific DNA aptamer against BPA (5′-CCG GTG GGT GGT CAG GTG GGA TAG CGT TCC GCG TAT GGC CCA GCG EvenCAT if strategies CAC GGG toTTC develop GCA CCA-3 antibodies′), immobilized towards on the bisphenolsurface of a gold A, includingelectrode via antibodiesself-assembly with good affinities,and have hybridized been reported with its complementary (see above), strand. DNA A aptamers redox intercalator specific was to added. BPA The were detection reported of to be of BPA was based on the competitive recognition of BPA by the immobilized aptamer on the surface of higher affinitythe electrode, than antibodies which dehybridized (in the nMthe range).compleme Forntary this strand reason, and therefore lower LoDs freed havethe redox been reached. Electrochemicalintercalator transduction into solution. wasThe redox investigated signal was byrecorded Xue et by al. CV.[31 The]. LoD They was used 0.3 pg·mL a specific−1 (1 pM). DNA aptamer against BPA (5Graphene-modified1-CCG GTG GGT glassy GGT carbon CAG (GC) GTG electrodes GGATAG were also CGT investigated, TCC GCG which TAT confers GGC higher CCA GCG CAT CAC GGGspecific TTC active GCA areas. CCA-3 Zhou1), et immobilized al. [32] immobilized on the AuNPs surface on GR-modified of a gold electrodeelectrodes and via used self-assembly freely and diffusing Fe(CN)63−/4− as electrochemical probe. The DPV peak current of Fe(CN)63−/4− changed linearly hybridizedwith with the concentration its complementary of BPA in the strand. range from A redox 10 nM intercalatorto 10 μM, with wasa LoD added. of 5 nM (ca. The 1 ng·mL detection−1). of BPA was basedThey on theused competitivethe same aptamer recognition sequence as ofthe BPA previous by thereference. immobilized aptamer on the surface of the electrode, which dehybridized the complementary strand and therefore freed the redox intercalator into solution.2.2.3. ThePeptides redox signal was recorded by CV. The LoD was 0.3 pg¨ mL´1 (1 pM). Graphene-modifiedSpecific peptides glassy were also carbon reported (GC) for electrodesBPA. Yang et wereal. [33] alsodescribed investigated, an electrochemical which sensor confers higher using the peptide sequence Cys-Lys-Ser-Leu-Glu-Asn-Ser-Tyr-Cys (CKSLENSYC) capable of recognizing specific active areas. Zhou et al. [32] immobilized AuNPs on GR-modified electrodes and used freely BPA with high specificity (isolated using a phage display technique). Peptides were immobilized by 3´{4´ 3´{4´ diffusingthiol Fe(CN) adsorption6 onas Au electrochemical electrodes and BPA probe. was sensed The using DPV DPV peak with current Fe(CN)6 of3−/4− Fe(CN)as redox 6probe, changed linearly withwith thea broad concentration detection range of between BPA in1 nM the and range 5 μM from(LoD of 10 1 nM, nM i.e. to, 0.2 10 ng·mLµM,− with1). Kim a et LoD al. also of 5 nM (ca. 1 ng¨ mL´reported1). They very used recently the same the electrochemical aptamer sequence detection as of theBPA previous with a protein reference. immobilized on RGO

2.2.3. Peptides Specific peptides were also reported for BPA. Yang et al. [33] described an electrochemical sensor using the peptide sequence Cys-Lys-Ser-Leu-Glu-Asn-Ser-Tyr-Cys (CKSLENSYC) capable of recognizing BPA with high specificity (isolated using a phage display technique). Peptides were immobilized by thiol adsorption on Au electrodes and BPA was sensed using DPV with Biosensors 2016, 6, 7 8 of 22

3´{4´ Fe(CN)6 as redox probe, with a broad detection range between 1 nM and 5 µM (LoD of 1 nM, i.e., 0.2 ng¨ mL´1). Kim et al. also reported very recently the electrochemical detection of BPA with a Biosensors 2016, 6, 7 8 of 21 protein immobilized on RGO electrodes [34]. The protein was a recombinant one obtained by fusing the disulfide-constrainedelectrodes [34]. The protein high was affinity a recombinant BPA binding one ob peptidetained by (CKSLENSYC, fusing the disulfide-constrained as reported by Yang high et al.) to theaffinity C-terminus BPA binding of the peptide Lac repressor (CKSLENSYC, (Lad), as which reported is a by large Yang protein et al.) to (140 the C-terminus Å ˆ 60 Å ofˆ the45 Å).Lac This modifiedrepressor protein (Lad), was which immobilized is a large protein on RGO (140 by Å × heat-denaturation, 60 Å × 45 Å). This modified so that protein it canadsorb was immobilized by π-stacking interactions.on RGO EISby heat-denaturation, was used (the redox so that reaction it can isadsorb not given by π-stacking in their manuscript)interactions. EIS to probe was used the presence(the of BPAredox down reaction to 100 is fM not (23 given fg¨ mL in ´their1), without manuscript) interference to probe withthe presence bisphenol of SBPA and down bisphenol to 100 F. fM −1 All(23 fg·mL these results), without are interference summarized with in bisphenol Table2. Considering S and bisphenol that F. BPA, Abs, aptamers and peptides All these results are summarized in Table 2. Considering that BPA, Abs, aptamers and peptides have yet been poorly reported in the literature, an unbiased comparison is not possible. have yet been poorly reported in the literature, an unbiased comparison is not possible. Table 2. Figures of merit of selected immunosensors, aptasensors and peptide sensors for detection of Table 2. Figures of merit of selected immunosensors, aptasensors and peptide sensors for detection bisphenol A. of bisphenol A.

BioreceptorsBioreceptors LoD LoD Transduction Transduction and and Analytical Analytical Methods Methods Ref. Ref. Ab 1.3 nM Conducting polymer & EIS [28,29] Ab 1.3 nM Conducting polymer & EIS [28,29] AbAb 9 9 pM pM Conducting Conducting polymer polymer & SWV & SWV [30] [30] Aptamer 1 pM Fe(CN)63−/43/EIS´{4 [31] Aptamer 1 pM Fe(CN)6 /EIS [31] Aptamer 5 nM Graphene/Fe(CN)63−/4/DPV3´{4 [32] Aptamer 5 nM Graphene/Fe(CN)6 /DPV [32] Peptide 1 nM SAM/Fe(CN)63−/4/DPV3´{4 [33] Peptide 1 nM SAM/Fe(CN)6 /DPV [33] PeptidePeptide 100100 fM fM Graphene Graphene oxide/EIS oxide/EIS [34] [34]

2.3. Cocaine2.3. Cocaine Cocaine (Figure 6) is the most frequently used illegal drug after cannabis (ca. 15 million users Cocaine (Figure6) is the most frequently used illegal drug after cannabis ( ca. 15 million users around the world) and directly provokes several thousands of deaths each year. Its detection, e.g., in aroundairports, the world) is routinely and directly made with provokes trained several dogs, thousandsbut more automatic of deaths systems each year. would Its detection,be extremely e.g., in airports,useful. is routinely made with trained dogs, but more automatic systems would be extremely useful.

Figure 6. Chemical structure of cocaine, methyl(1R,2R,3S,5S)-3-(benzoyloxy)-8-methyl-8- Figure 6. Chemical structure of cocaine, methyl(1R,2R,3S,5S)-3-(benzoyloxy)-8-methyl-8-azabicyclo[3.2.1] azabicyclo[3.2.1]octane-2-carboxylate. octane-2-carboxylate. 2.3.1. Antibodies 2.3.1. Antibodies There are very few examples of immunosensors dedicated to narcotics. The two very first articles Therewere published are very fewin 1998 examples and dealt of immunosensors with cocaine detection. dedicated In to1998, narcotics. Bauer et The al. two[35] proposed very first an articles wereautomated published amplified in 1998 flow and immunoassay dealt with cocaine for cocaine detection. using the In alkaline 1998, phosphatase Bauer et al. (AlkP)[35] proposed enzyme an automatedlabel for amplified amplification flow and immunoassay transduction. for The cocaine LoD was using 380 pM. the alkalineThe same phosphatase year, Suleiman (AlkP) et al. [36] enzyme labeldescribed for amplification an amperometric and transduction. immunosensor The for LoD cocaine was using 380 pM.HRP Theand, sameas in the year, case Suleiman of Bauer etet al. al., [36] describedan oxygen an amperometric electrode. Cocaine immunosensor was quantified for in cocainethe range using 10−7–10 HRP−5 M, and, i.e., at as too in high the caseconcentrations of Bauer et al., an oxygenfor being electrode. of practical Cocaine use. was quantified in the range 10´7–10´5 M, i.e., at too high concentrations More than 15 years passed before another example of immunosensor for cocaine or morphine for being of practical use. was published. It is only recently that, considering the good results obtained by aptamer-based Morecocaine than sensors, 15 years researchers passed began before again another to study example cocaine-specific of immunosensor antibodies. for The cocaine literature or morphine remains was published.very limited, It is only however. recently Yang that, et al. considering [37] described the an good immunosensor results obtained based on by a aptamer-based SAM-modified Au cocaine sensors,electrode researchers for detection began of again morphine to study and cocaine-specificmethamphetamine antibodies. (MA), using The EIS, literaturewith a low remains LoD of very limited,10 pg·L however.−1 (33 fM). Yang Foret more al. [37 information,] described a anvery immunosensor recent (2015) book based chapter on a published SAM-modified by Ozkan Au et electrode al., for detectionis available, of dealing morphine with and electrochemical methamphetamine biosensors (MA), for drug using analysis EIS, [38]. with a low LoD of 10 pg¨ L´1 (33 fM). For more information, a very recent (2015) book chapter published by Ozkan et al., is available, dealing with electrochemical biosensors for drug analysis [38]. Biosensors 2016, 6, 7 9 of 22 Biosensors 2016, 6, 7 9 of 21 Biosensors 2016, 6, 7 9 of 21 2.3.2.2.3.2. Aptamers Aptamers 2.3.2. Aptamers ByBy contrast, contrast, aptamer-based aptamer-based sensors sensors for cocainefor cocaine were morewere frequentlymore frequently reported. reported. The first The significant first worksignificant onBy this contrast, work topic on was aptamer-basedthis published topic was inpublished sensors 2006 by for Bakerin 2006cocaineet by al. Baker [were39]. Theyetmore al. [39]. demonstrated frequently They demonstrated reported. a rapid, label-free,Thea rapid, first electrochemicallabel-free,significant electrochemical work method on this for topic method the was detection for published the detection of small in 2006 moleculesof by small Baker moleculesin et general,al. [39]. in They general, based demonstrated on based a target-induced on a atarget- rapid, conformationalinducedlabel-free, conformational electrochemical change, unaffected change, method unaffected by for non-specific the detection by non-specific contaminants, of small moleculescontaminants, and illustrated in general, and illustrated bybased the on detection a by target- the of cocainedetectioninduced in bloodofconformational cocaine serum, in blood saliva, change, serum, and unaffected other saliva, complex and by other non-specific samples. complex Two contaminants, samples. DNA aptamers Two and DNA illustrated were aptamers investigated by were the (GACinvestigateddetection AAG GAAof (GAC cocaine AAT AAG CCTin bloodGAA TCA AATserum, ATG CCT AAG saliva, TCA TGG and ATG GTC other AAG and complex TGG AGA GTC CAA samples. and GGA AGA Two AAA CAA DNA TCC GGA aptamers TTC AAA AAT TCCwere GAA investigated (GAC AAG GAA AAT CCT TCA ATG AAG TGG GTC and AGA CAA GGA AAA TCC GTGTTC GGT AAT CG) GAA with GTG K DGGTof approximately CG) with KD of 90 approximatelyµM each, which 90 allowedμM each, a which LoD below allowed 10 aµ MLoD (Figure below7 ); μ however,10TTC μM AAT(Figure these GAA were7); however,GTG too GGT high these CG) to allow werewith forKtooD practicalof hi ghapproximately to allow applications. for 90prac Mtical each, applications. which allowed a LoD below 10 μM (Figure 7); however, these were too high to allow for practical applications.

(A) (B) (A) (B) Figure 7. (A) Working principle of the aptamer-based cocaine biosensor of Baker et al. MB (methylene FigureFigure 7. (7.A )(A Working) Working principle principle of of the the aptamer-based aptamer-based cocaine cocaine biosensorbiosensor ofof Baker et al. MBMB (methylene (methylene blue) was used as redox label (B) Corresponding calibration curve. Reprinted with permission from blue)blue) was was used used as redoxas redox label label (B) ( CorrespondingB) Corresponding calibration calibration curve. curve. Reprinted Reprinted with with permission permission from from [39 ]. [39]. Copyright 2006 American Chemical Society. Copyright[39]. Copyright 2006 American 2006 American Chemical Chemical Society. Society. The same group reported later [40] a sandwich assay based on single aptamer sequences labeled TheThe same same group group reported reported later later [40 [40]] a a sandwich sandwich assay assay basedbased onon single aptamer sequences sequences labeled labeled with MB and able to detect cocaine. The principle relied on the formation of a complex made of two- withwith MB MB and and able able to to detect detect cocaine.cocaine. The The principle principle relied relied on onthe theformation formation of a ofcomplex a complex made madeof two- of strand aptamers which was stabilized together by the presence of the target (Figure 8). Even though two-strandstrand aptamers aptamers which which was was stabilized stabilized together together by the by presence the presence of the target of the (Figure target (Figure8). Even8 ).though Even the KD was estimated at around 1 mM, the authors reported a significantly lower LoD of ca. 1 μM. the KD was estimated at around 1 mM, the authors reported a significantly lower LoD of ca. 1 μM. thoughThe limit the of K quantificationD was estimated (LoQ), at around however, 1 mM, should the be authors between reported 0.1 and a 1 significantly mM. lower LoD of ca. 1 µTheM. The limit limit of quantification of quantification (LoQ), (LoQ), however, however, should should be between be between 0.1 and 0.1 1 andmM. 1 mM.

(A(A) )

(B(B) ) (C(C))

FigureFigure 8. 8. (A (A) Target) Target binding binding stabilizes stabilizes the the association association of of the the two two aptamer aptamer strands, strands, leading leading to to a a large large Figure 8. (A) Target binding stabilizes the association of the two aptamer strands, leading to a large increaseincrease in in faradaic faradaic current; current; ( B(B) )when when the the target target is is present; present; ( C(C) )Calibration Calibration curve. curve. Reprinted Reprinted withwith increase in faradaic current; (B) when the target is present; (C) Calibration curve. Reprinted with permissionpermission from from [40]. [40]. Copyright Copyright 2009 2009 American American Chemical Chemical Society. Society. permission from [40]. Copyright 2009 American Chemical Society. BiosensorsBiosensors 2016,2016 6, 7, 6, 7 10 of 2210 of 21

On the same principle, Golub et al. [41] described two anti-cocaine aptamer subunits, where one On the same principle, Golub et al. [41] described two anti-cocaine aptamer subunits, where one subunit was assembled on an Au electrode, and the second aptamer subunit was labeled with Pt- subunit was assembled on an Au electrode, and the second aptamer subunit was labeled with Pt-NPs. NPs.The The supramolecular supramolecular Pt-NPs-aptamer Pt-NPs-aptamer subunits-cocaine subunits-cocaine complex complex allowed allowed the detection the detection of cocaine byof cocaine the by the electrocatalyzed reduction of H2O2 on the Pt-NPs. The detection limit was in the´ range6 ´ 5of 10−6– electrocatalyzed reduction of H2O2 on the Pt-NPs. The detection limit was in the range of 10 –10 M 10−5 M(Figure (Figure9). 9).

FigureFigure 9. Supramolecular 9. Supramolecular complex complex of of Pt-NPs-aptamer Pt-NPs-aptamer subunitsubunit(1) (1) and and second second aptamer aptamer subunit subunit immobilizedimmobilized on the on the gold gold surface surface (2). (2). Adapted Adapted with with permissionpermission from from [41 [41].]. Copyright Copyright 2009 2009 American American ChemicalChemical Society. Society.

AgainAgain regarding regarding the the use use of of a atwo-strand two-strand aptamers, DuDuet et al. al.[42 [42]] constructed constructed a label-free a label-free electrochemicalelectrochemical aptasensor aptasensor using using layer-by-layer layer-by-layer se self-assemblylf-assembly of of Fc-modified Fc-modified poly(ethyleneimine), poly(ethyleneimine), withwith a LoD a LoD of 0.1 of 0.1μM.µM. A Asimilar similar approach approach was describeddescribed byby Zhang Zhanget et al. al.[43 [43]] using using a primary a primary immobilizedimmobilized aptamer aptamer and and a secondary a secondary aptamer aptamer labeled labeled with with various various quantum quantum dots dots used used in in the the same way samethan wayin bar-code than in bar-code assays, assays,i.e., differenti.e., different QDs QDs yield yield distinct distinct electrochemical electrochemical signatures. signatures. A A LoD LoD of 50 nMof was 50 nM obtained. was obtained. Another Another approach approach using using multiple multiple stems stems was was described described by byHua Hua et etal. al. [44][44 (with] (with the cocaine DNA aptamer 51-GGG AGA CAA GGA TAA ATC CTT CAA TGA AGT GGG TCT the cocaine DNA aptamer 5′-GGG AGA CAA GGA TAA ATC CTT CAA TGA AGT GGG TCT CCC-3′), CCC-31), where the transduction step was also based on a hairpin structure. The linear range was from where the transduction step was also based on a hairpin structure. The linear range was from 10−9 M 10´9 M to 10´6 M. Again using the multiple stem approach, Zhang et al. [45] investigated the formation −6 to 10of M. the Again corresponding using the supramolecular multiple stem aptamer approach, fragments/cocaine Zhang et al. [45] complex investigated by EIS the in theformation presence of the corresponding3 ´{supramolecular4´ aptamer fragments/cocaine complex by EIS in the presence of of Fe(CN)6 . Cocaine concentrations as low as 100 nM were detected. Very recently, Roushani Fe(CN)and63 Shahdost-Fard−/4−. Cocaine concentrations [46] described a as sensor low basedas 100 on nM the were same principledetected. with Very aptamer-functionalized recently, Roushani and Shahdost-FardAuNPs. The [46] interaction described of cocaine a sensor with thebased aptamer on the caused same the principle AuNPs to with come aptamer-functionalized close to the electrode 3´{4´ AuNPs.surface The and interaction impede diffusion of cocaine of thewith redox the aptamer probe Fe(CN) caused6 the. AuNPs Monitored to come with DPV, close the to detectionthe electrode surfacelimit and was impede 100 pM. diffusion The same of authors the redox described probe a Fe(CN) similar approach63−/4−. Monitored using AgNPs with DPV, [47] and the riboflavin detection as limit was the100 redoxpM. The probe; same the LoDauthors was described equivalent, ai.e. similar, 150 pM. approach using AgNPs [47] and riboflavin as the redox probe;Jiang theet al. LoD[48] was used equivalent, the same supramolecular i.e., 150 pM. aptamer sequences for detection of cocaine using an alkaline phosphatase-modified binding aptamer, leading to a LoD of 1 nM. A similar approach Jiang et al. [48] used the same supramolecular aptamer sequences for detection of cocaine using was used by Zhang et al. [49] but using HRP as the amplifying label. DPV was used to obtain a LoD an alkaline phosphatase-modified binding aptamer, leading to a LoD of 1 nM. A similar approach down to 20 nM. Lastly, Shen et al. [50] added rolling circle amplification (RCA) in addition to alkaline was phosphataseused by Zhang amplification, et al. [49] but and using used DPVHRPfor as detection.the amplifying This dual label. amplification DPV was used strategy to obtain strongly a LoD downdecreased to 20 nM. the Lastly, detection Shen limit et al. down [50] to added 1 nM. rolling circle amplification (RCA) in addition to alkaline phosphataseAll these amplification, results are summarizedand used DPV in Table for3 .detection. Due to very This few examplesdual amplification of Ab sensors strategy for cocaine, strongly decreaseda comparison the detection between limit Abs down and aptamers to 1 nM. cannot be objective. As for other targets, nanostructuration ofAll the these electrodes results led are to summarized a gain in terms in Table of LoD, 3. Due of one to tovery three few orders examples of magnitude. of Ab sensors Again, for as cocaine, for a comparisonantibiotics, between enzymatic Abs amplifications and aptamers applied cannot to be aptamers objective. do notAs bringfor other lower targets, detection nanostructuration limits, as it of thecould electrodes be expected. led to Lastly, a gain even in ifterms it is an of obvious LoD, of observation, one to three these orders results of show magnitude. that the sensitivityAgain, as for antibiotics,largely dependsenzymatic on amplifications the affinity of theapplied aptamer to ap fortamers its target: do thenot lowestbring lower LoD were detection obtained limits, with as it aptamers having low K . could be expected. Lastly,D even if it is an obvious observation, these results show that the sensitivity largely depends on the affinity of the aptamer for its target: the lowest LoD were obtained with aptamers having low KD.

Biosensors 2016, 6, 7 11 of 21

Biosensors 2016, 6, 7 11 of 22 Table 3. Figures of merit of selected immunosensors and aptasensors for detection of cocaine.

Bioreceptors LoD Transduction and Analytical Methods Ref. Table 3. Figures of merit of selected immunosensors and aptasensors for detection of cocaine. Ab 380 pM AlkP-Ab/CV [35] Ab 0.1 μM HRP-Ab/CV [36] Bioreceptors LoD Transduction and Analytical Methods Ref. Ab 33 fM SAM/EIS [37] Ab 380 pM AlkP-Ab/CV [35] Aptamer 10 μM MB-labeled/CV [39] Ab 0.1 µM HRP-Ab/CV [36] AbAptamer 33 fM1 μM Two-strand SAM/EIS aptamer/SWV [40] [37 ] AptamerAptamer 10 µ1M μM PtNPs-aptamer/H MB-labeled/CV2O2/CV [41] [39 ] AptamerAptamer 1 µ0.1M μM Label-free/Two-strand Two-strand aptamer/SWV aptamer/CV [42] [40 ] µ AptamerAptamer 1 50M nM Two-stra PtNPs-aptamer/Hnd aptamer/QDs2O2/CV [43] [41 ] Aptamer 0.1 µM Label-free/Two-strand aptamer/CV [42] AptamerAptamer 50 nM1 nM Two-stra Two-strandnd aptamer/QDs [44] [43 ] AptamerAptamer 1 nM100 nM Two-strand Two-strand aptamer/EIS/[Fe(CN) aptamer/QDs6]3−/4− [45] [44 ] 3´{4´ 3−/4− AptamerAptamer 100 100 nM pM Two-strandTwo-strand aptamer/AuNPs/DPV/[Fe(CN) aptamer/EIS/[Fe(CN)6] 6] [46][45 ] 3´{4´ AptamerAptamer 100 150 pM pM Two-strand Two-strand aptamer/AuNPs/DPV/[Fe(CN) aptamer/AgNPs/CV/riboflavin6] [47][46 ] AptamerAptamer 150 pM1 nM Two-strand Two-strand aptamer/AgNPs/CV/riboflavin aptamer/AlkP/CV [48] [47 ] Aptamer 1 nM Two-strand aptamer/AlkP/CV [48] AptamerAptamer 20 nM20 nM Two-strand Two-strand aptamer/HRP/CVaptamer/HRP/CV [49] [49 ] AptamerAptamer 1 nM1 nM RCA/AlkP/DPV RCA/AlkP/DPV [50] [50 ]

2.4.2.4. OchratoxinOchratoxin AA AA toxintoxin isis aa poisonouspoisonous substancesubstance producedproduced byby livingliving cellscells oror organisms.organisms. ToxinsToxins cancan bebe smallsmall organicorganic molecules,molecules, peptides,peptides, oror proteins.proteins. TheThe mostmost studiedstudied toxintoxin inin electrochemicalelectrochemical biosensorsbiosensors isis ochratoxinochratoxin AA (Figure(Figure 10 10).). ItIt isis oneone ofof thethe mostmost abundantabundant food-contaminatingfood-contaminating mycotoxins. mycotoxins.

FigureFigure 10.10. StructureStructure ofof ochratoxinochratoxin AA (OTA).(OTA).

2.4.1.2.4.1. AntibodiesAntibodies Prieto-SimonPrieto-Simonet et al. al. described,described, inin 20082008 [[51],51], twotwo indirectindirect electrochemicalelectrochemical competitivecompetitive enzyme-linkedenzyme-linked immunosorbentimmunosorbent assaysassays (ELISA)(ELISA) forfor ochratoxinochratoxin AA (OTA).(OTA). PerformancesPerformances ofof polyclonalpolyclonal (pAb)(pAb) andand monoclonalmonoclonal (mAb)(mAb) antibodiesantibodies againstagainst OTAOTA were were compared, compared, showing showing at at leastleast oneone orderorder ofof magnitudemagnitude higherhigher affinityaffinity valuesvalues whenwhen workingworking withwith mAb.mAb. AlkalineAlkaline phosphatasephosphatase (AlkP)(AlkP) andand horseradishhorseradish peroxidaseperoxidase (HRP)-labeled (HRP)-labeled secondary secondary antibodies antibodies were were evaluated. evaluated. Similar Similar limits limits of of detectiondetection of of 0.70.7 andand 0.30.3 ngng·mL¨ mL´−11 (0.8(0.8 nM) nM) were obtainedobtained forfor HRP- HRP- and and AlkP-labeled AlkP-labeled immunosensors, immunosensors, respectively. respectively. Later, Later, in 2009,in 2009, Radi Radiet al. et[52 al.] described[52] described an electrochemical an electrochemical immunosensor immunosensor for detection for detection of OTA of on OTA screen-printed on screen- goldprinted electrodes gold electrodes modified modified by electroreduction by electroreducti ofon 4-nitrophenyl of 4-nitrophenyl diazonium diazonium salts salts and and anti-OTA. anti-OTA. A competitionA competition between between OTA OTA and and HRP-labeled HRP-labeled OTA OTA (OTA-HRP) (OTA-HRP) for for the the immobilized immobilized antibodies antibodies waswas held.held. TheThe activityactivity ofof thethe boundbound OTA-HRPOTA-HRP waswas electrochemicallyelectrochemically measuredmeasured byby chronoamperometrychronoamperometry usingusing 3,3,5,5-tetramethylbenzidine3,3,5,5-tetramethylbenzidine (TMB)(TMB) asas substrate. substrate. A A LoD LoD of of 12 12 ng ng·mL¨ mL´−11 (30 nM) was obtained.obtained. TheThe samesame authorsauthors [ 53[53]] also also investigated investigated a a similar similar architecture architecture using using EIS, EIS, with with a LoDa LoD of of 0.5 0.5 ng ng·mL¨ mL´1−1 (1.3(1.3 nM). nM). ZamfirZamfiret et al. al.described described a label-free a label-free immunosensor immunosensor based onbased magnetic on magnetic nanoparticles nanoparticles for ochratoxin for A[ochratoxin54]. The A OTA [54]. antibodies The OTA wereantibodies attached were to theseattached magnetic to these nanoparticles magnetic nanoparticles (MBs) and afterwards(MBs) and immobilizedafterwards immobilized on a gold electrode on a gold pretreated electrode with pretreated bovine serum with albuminbovine serum (BSA) albumin under a magnetic(BSA) under field. a Themagnetic impedance field. The variations impedance due variations to the specific due to antibody–OTA the specific antibody–OTA interactions inte wereractions correlated were correlated with the OTAwith concentrationthe OTA concentration in the samples. in the sa Themples. increase The increase in electron in electron transfer tr resistanceansfer resistance was proportional was proportional to the concentrationto the concentration of OTA of on OTA a linear on a range linear between range between 0.01 and 0.01 5 ng and¨ mL 5 ´ng1 (25mL pM–13−1 (25 pM–13 nM). nM). Biosensors 2016, 6, 7 12 of 22

An indirect competitive enzyme-linked immunosorbent assay format was also constructed by immobilizing ochratoxin A on gold electrodes [55]. Electrochemical detection was performed by chronoamperometry using TMB and hydrogen peroxide with HRP as label. A LoD of 0.05 µg¨ L´1 (0.12 nM) was achieved. Additionally, boron doped diamond (BDD) was employed [56] for sensitive immunodetection of ochratoxin A using EIS. Abs were grafted through diazonium functionalization. The increase in electron-transfer resistance presented a sigmoidal shape versus log concentration of OTA, with a dynamic range between 7 pg¨ mL´1 and 25 ng¨ mL´1. A LoD of 7 pg¨ mL´1 (17 pM) was obtained. These good results could be attributed to the low background and capacitive currents allowed by BDD compared to other more classical electrode materials. Lastly, a label-free photoelectrochemical sensor was recently reported by Yang et al. [57], by assembly of CdSe nanoparticles on TiO2 electrodes. Ascorbic acid (AA) was used as an electron donor for scavenging photogenerated holes under visible-light irradiation. The photocurrent response of the CdSe NPs-modified electrode was significantly enhanced as a result of the band alignment of CdSe and TiO2 in electrolyte. OTA antibodies were immobilized on CdSe. Even if these values cannot strictly be compared to the previous ones because the transduction mechanism is not of the same nature, one may underline the good sensitivity of this technique, with a LoD of 2 pg¨ mL´1 (5 pM) and a linear dependence with OTA concentration from 10 pg¨ mL´1 to 50 ng¨ mL´1.

2.4.2. Aptamers Aptamers were also frequently reported in the literature, for ochratoxin A detection. A first electrochemical platform was described in 2010 using the DNA sequence 51-GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CGG ACA-31 as aptamer probe [58]. In this article, the aptamer was immobilized on the surface of a glassy carbon electrode treated with PCl5-activated sulfonic acid groups able to bind amino-modified DNA strands. These primary strands were then sandwiched with two other DNA strands carrying methylene blue (MB) as redox label. Binding of the OTA target to the aptamer disassembled the DNA strands and lowered the MB redox current. The low LoD (30 pg¨ mL´1, i.e., 80 pM) could be attributed to the functionalization strategy (direct coupling of the DNA strands on the electrode without any intermediate bulky groups). One year later, a Langmuir–Blodgett (polyaniline (PANI)–stearic acid (SA)) impedimetric OTA aptasensor was developed by Prabhakar et al. [59]. The DNA aptamer was covalently immobilized onto the LB ´7 monolayer deposited onto ITO plates. The dissociation constant (KD) was found to be ca. 0.8 ˆ 10 M and the LoD was 0.1 ng¨ mL´1 (0.25 nM), i.e., higher than with the direct coupling strategy of Kuang et al. Tong et al., in 2011 [60], described an original signal-on aptasensor based on exonuclease-catalyzed target recycling. To construct the aptasensor, a Fc-labeled probe DNA (51-Fc-AAA GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CGG ACA-31-SH), carrying the Fc label at its 51-distal end was immobilized on Au electrode through 31-thiol chemisorption, and hybridized with the complementary ochratoxin A aptamer. In the presence of ochratoxin A, formation of the complex resulted in the transformation of the probe DNA into a hairpin structure where the Fc group comes close to the electrode surface, therefore producing a current increase (signal-on). OTA was then decomplexed by the use of the exonuclease, which digest the aptamer, so that OTA is recycled and can come again to bind to another aptamer. Due to the amplification step, this original method gave a LoD of 1 pg¨ mL´1 (2.5 pM). Zhang et al. [61] described a sensor based on a similar hairpin-shaped aptamer and site-specific DNA cleavage of restriction endonuclease TaqaI, with HRP instead of Fc. In their approach, the endonuclease is able to cut DNA (and release HRP in solution, far from the electrode surface) when OTA is absent; if OTA is present, there is no hairpin structure, and, therefore, the TaqI is inactive and HRP can be used as enzymatic amplification label. Due to this amplification, the reported LoD was 0.4 pg¨ mL´1 (1 pM). Biosensors 2016, 6, 7 13 of 22

Lastly, Hayat et al. reported, in 2013 [62], a strategy for the fabrication of an electrochemical label-free (but needing a redox molecule diffusing in solution) aptasensor where long spacer chains of polyethyleneglycol were used to create diffusion channels for the redox probe, while aptamers acted as gate of the tunnels, depending on their conformation. The LoD was 0.12 ng¨ L´1 (0.3 pM). The aptamer sequence was the same as Tong et al.

2.4.3. Peptides Even though it has not yet been applied to electrochemical sensors, peptides were also described in a few works as a probe for ochratoxin A. Indeed, Bazin et al. [63] reported in 2013 the peptide NF04 (12-mer sequence: N1-Lys-Cys-Cys-Lys-Tyr-Tyr-Lys-Arg-Asn-Met-Tyr-Val-C1) for specific binding to ochratoxin A, which was evaluated using a peptide-based ELISA assay. Heurich et al., described a computational approach to design peptide ligands for Ochratoxin A[64]. Two peptides were identified: N1-Cys-Ser-Ile-Val-Glu-Asp-Gly-Lys-C1 (octapeptide) and N'-Gly-Pro-Ala-Gly-Ile-Asp-Gly-Pro-Ala-Gly-Ile-Arg-Cys-C' (13-mer). SPR analysis confirmed that the peptides bind to ochratoxin A with KD values of 10 to 15 µM. More recently, Solerai et al. [65] reported another peptide conjugated chitosan foam for detection of OTA using enzymatic chemiluminescence. There is therefore no reason for electrochemical OTA sensors based on peptide probes not to appear in the literature in the forthcoming years. All these results are summarized in Table4. It appears that OTA sensors are generally more sensitive than for other targets, in the pM range, for both immunologic and aptamer approaches.

Table 4. Figures of merit of selected immunosensors and aptasensors for ochratoxin A.

Bioreceptors LoD Transduction and Analytical Methods Reference Ab 0.8 nM AlkP- or HRP-Ab/CV [51] Ab 30 nM HRP/TMB/chronoamperometry [52] Ab 1.3 nM HRP/TMB/EIS [53] Ab 25 pM MBs/EIS [54] Ab 0.12 nM HRP/TMB/EIS [55] Ab 17 pM BDD/EIS [56] Ab 5 pM CdSe/TiO2/PEC [57] Aptamer 80 pM Multiple stem/MB-DNA/CV [58] Aptamer 0.25 nM LB/PANI/EIS [59] Aptamer 2.5 pM Fc-labeled aptamer/exonuclease/CV [60] Aptamer 1 pM HRP-labeled aptamer/exonuclease/CV [61] 3´{4´ Aptamer 0.3 pM Fe(CN)6 /EIS [62] Note: TMB (3,31,5,51-tetramethylbenzidine); PEC (photoelectrochemical); BDD (boron doped diamond); MB (methylene blue); LB (Langmuir Blodgett); PANI (polyaniline).

2.5. Estradiol

2.5.1. Antibodies Estradiol (E2, Figure 11) immunosensors were extensively reported in the literature. In 1998, Padeste et al. [66] reported an amperometric immunosensor for estradiol using microperoxidase MP-11 antibody conjugates for amplification. However, in this preliminary work, the authors provided neither LoD nor sensitivity. Five year later, in 2003, Kuramitz et al. [67] described a 17β-estradiol-modified electrode for non-labeled immunoassay. When the antibody was bound to the estradiol self-assembled monolayer on the gold electrode surface, they observed a decrease of the electroactivity of the redox 3´{4´ probes added in solution (BQ or Fe(CN)6 ), attributed to the steric hindrance between the antibody on the electrode surface and the redox marker. The authors detected 17β-estradiol, 17β-estradiol-6-one (1,3,5-estratriene-3,17β-diol-6-one 6-O-carboxymethyloxime) and diethylstilbestrol using competition for the antibody between the analyte in solution and 17β-estradiol immobilized on the electrode Biosensors 2016, 6, 7 14 of 22

surface, and found that the affinities ranked as follow: 17β-estradiol-6-one > 17β-estradiol > DES. The Biosensors LoD2016, for6, 7 17 β-estradiol was ca. 0.1 nM (the KD was estimated around 10´7 M). 14 of 21

FigureFigure 11. 11. StructureStructure ofof estradiol estradiol (E2). (E2).

In 2005,In Pemberton 2005, Pemberton et al.et [68] al. [68 investigated] investigated an electrochemicalelectrochemical immunosensor immunosensor for estradiol for estradiol on on carbon SPEcarbon electrodes. SPE electrodes. A competitive A competitive immunoassa immunoassayy waswas performed performed using using an AlkP-labeled an AlkP-labeled estradiol estradiol conjugate. Electrochemical measurements were performed using differential pulse voltammetry (DPV) conjugate.following Electrochemical the production measurements of 1-naphthol from were 1-naphthyl performed phosphate. using The differential LoD was of pulse 50 pg¨ mLvoltammetry´1 −1 (DPV) following(0.2 nM). In the 2006, production Butler et al. [of69 ]1-naphthol also reported from very similar 1-naphthyl work. Thephosphate. sensor detected The LoD 17-beta-estradiol was of 50 pg·mL (0.2 nM).in In an 2006, environmentally Butler et al. [69] relevant also rangereported with very a LoD similar of 0.25 work. pg¨ mLThe´1 sensor(0.9 pM). detected The same 17-beta-estradiol year, ´ in an environmentallyVolpe et al. [70] reported relevant a similar range work with with a LoD a LoD of of 0.25 40 pg ¨pg·mLmL 1 (0.15−1 (0.9 nM) pM). in bovine The serum.same year, Volpe et al. [70] reportedAs for a similar the other work analytes, with nanostructuration a LoD of 40 pg·mL of the−1 electrodes (0.15 nM) improved in bovine sensitivity serum. and LoD. For example, Liu et al. (2009) [71] reported the use of AuNPs immobilized on gold electrodes in As orderfor the to increaseother analytes, the surface nanost area andructuration therefore to improveof the sensitivity.electrodes For improved amplification, sensitivity they used and a LoD. For example,HRP-labeled Liu et 17al. beta-estradiol(2009) [71] reported conjugate the and use benzoquinone of AuNPs (BQ) immobilized as redox reporter. on gold They electrodes reported a in order to increaseLoD the of 6 pgsurface¨ mL´1 (20area pM). and In 2010,therefore the same to authors improve compared sensitivity. SWV and For EIS onamplification, a SAM-modified they Au used a HRP-labeledelectrode 17 carryingbeta-estradiol AuNPs verticalconjugate bar thiolatedand benzoq proteinuinone G-scaffold (BQ) to as facilitate redox the reporter. immobilization They reported of a an enhanced−1 quantity of an almost uprightly aligned anti-estradiol capture antibody [72]. They used LoD of 6 pg·mL3´{ (204´ pM). In 2010, the same authors compared´1 SWV and EIS on a SAM-modified´1 Au Fe(CN)6 as redox probe. They obtained a LoD of 18 pg¨ mL (65 pM) for SWV, and 26 pg¨ mL electrodefor carrying EIS (95 pM), AuNPs e.g., not vertical better thanbar thiolated for their previous protein work. G-scaffold Again using to facilitate nano/microstructuration, the immobilization of an enhancedMartinez quantityet al. [73 of] developedan almost a competitiveuprightly alig directned immunoassay anti-estradiol using capture magnetic antibody microspheres [72]. and They used Fe(CN)63an−/4− estradiol-HRP as redox probe. conjugate. They Theobtained detection a LoD of estradiol of 18 pg·mL was carried−1 (65 out pM) using for aSWV, competitive and 26 direct pg·mL−1 for EIS (95 immunoassaypM), e.g., not format better using than anti-estradiol for their polyclonal previous Abs work. immobilized Again onusing 3-aminopropyl-modified nano/microstructuration, Martinezmagnetic et al. [73] microspheres. developed Estradiol a competitive present in direct the sample immu competednoassay with using an estradiol-HRP magnetic microspheres conjugate and for the immobilized antibody. The current obtained from the enzymatic reaction was inversely an estradiol-HRP conjugate. The detection of estradiol was carried out using a competitive direct proportional to the amount of estradiol in the sample. The LoD was 0.32 ng L-1 (ca. 1 pM). immunoassayMore format recently, using in2012, anti-estradiol Kim et al. [ 74polycl] describedonal Abs an impedimetric immobilized sensor on 3-aminopropyl-modified by immobilizing a magneticmonolayer microspheres. of estrogen Estradiol receptor-alpha present on in Au the electrodes. sample Thecompeted binding ofwith 17β -estradiolan estradiol-HRP increased the conjugate for the immobilizedelectron-transfer antibody. resistance ofThe the electrodecurrent whichobtain wased directlyfrom the monitored enzymatic by EIS inreaction the presence was ofinversely 3´{4´ proportionalFe(CN) to6 the amount. Despite of the estradiol small size in of the estradiol sample. compared The LoD to thatwas of 0.32 the immobilizedng L-1 (ca. 1 antibody, pM). the authors reported a K of ca. 5 pM and a LoD of ca. 0.1 pM. Liu et al. [75] published an More recently, in 2012, KimD et al. [74] described an impedimetric sensor by immobilizing a original copper monolayer-based sensor to construct a Cu/vertical bar protein G immunosensor β monolayerfor 17of beta-estradiol.estrogen receptor-alpha Copper minimized on Au the non-specificelectrodes. adsorptionThe binding of biological of 17 -estradiol molecules onincreased the the electron-transferimmunosensor resistance surface of and the enhanced electrode the which binding was efficiency directly between monitored immunosensor by EIS surfacein the andpresence of Fe(CN)63protein−/4−. Despite G. SWV the was small employed size to of monitor estradiol the electrochemical compared to reduction that of current the immobilized of ferrocenemethanol; antibody, the SWV current decreased with the increase of estradiol-BSA conjugate concentration. The LoD was authors reported a KD of ca. 5 pM and a LoD of ca. 0.1 pM. Liu et al. [75] published an original copper 12 pg¨ mL´1 (45 pM). monolayer-based sensor to construct a Cu/vertical bar protein G immunosensor for 17 beta-estradiol. In 2012, Ojeda et al., described a more classical approach [76] based on the surface modification of Copper minimizeda carbon SPE withthe non-specific grafted p-aminobenzoic adsorption acid of (PABA), biological followed molecules by covalent on binding the immunosensor of streptavidin surface and enhancedand immobilization the binding of biotinylatedefficiency anti-estradiolbetween immunosensor antibodies labeled surface with HRP. and Hydroquinone protein G. was SWV was employedused to as monitor redox mediator. the electrochemical A LoD of 0.77 pg¨ mL reduct´1 (2.8ion pM) current was achieved. of ferrocenemethanol; Kanso et al. [77] described SWV two current decreased with the increase of estradiol-BSA conjugate concentration. The LoD was 12 pg·mL−1 (45 pM). In 2012, Ojeda et al., described a more classical approach [76] based on the surface modification of a carbon SPE with grafted p-aminobenzoic acid (PABA), followed by covalent binding of streptavidin and immobilization of biotinylated anti-estradiol antibodies labeled with HRP. Hydroquinone was used as redox mediator. A LoD of 0.77 pg·mL−1 (2.8 pM) was achieved. Kanso et al. [77] described two methods using MBs onto which synthetic estrogen derivatives were bound. Using a classical primary antibody and a HRP-labeled secondary antibody, SWV was used for detection of the enzyme product, with LoDs between 1 and 10 ng·L−1 (3.6–36 pM) (Figure 12). Chaisuwan et al. described also an original procedure [78] using estradiol-modified CdSe quantum dots (QDs). A bismuth-coated electrode was used for detecting the cadmium ions (Cd2+) released during the acid dissolution of the QDs, after the recognition step that occurred in solution. The LoD was ca. 52 pg·mL−1 (0.2 nM). Biosensors 2016, 6, 7 15 of 22 methods using MBs onto which synthetic estrogen derivatives were bound. Using a classical primary antibody and a HRP-labeled secondary antibody, SWV was used for detection of the enzyme product, withBiosensors LoDs 2016 between, 6, 7 1 and 10 ng¨ L´1 (3.6–36 pM) (Figure 12). 15 of 21

(A)

(B)

Figure 12. Strategy for (A) estrogen functionalization with magnetic beads; (B) indirect competitive Figure 12. Strategy for (A) estrogen functionalization with magnetic beads; (B) indirect competitive assay for both colorimetric and electrochemical detection based on functionalized MBs. Reproduced from [[77]77] with permission.

Still using nanoparticles, Li et al. [79] described an estradiol immunosensor based on graphene- Chaisuwan et al. described also an original procedure [78] using estradiol-modified CdSe quantum polyaniline (GR-PANI) composites and graphene oxide (GO). HRP-GO-antibody conjugates were dots (QDs). A bismuth-coated electrode was used for detecting the cadmium ions (Cd2+) released used to improve sensitivity, through a competitive immunoassay. The LoD was of 0.02 ng mL−1 (75 during the acid dissolution of the QDs, after the recognition step that occurred in solution. The LoD pM). Very recently (2015), again using nanostructures, Zhang et al. [80] reported a sensor using Cu2S was ca. 52 pg¨ mL´1 (0.2 nM). nanostructures as labels. Cu2S NPs were covalently conjugated to BSA-estradiol. Even without using Still using nanoparticles, Li et al. [79] described an estradiol immunosensor based on enzyme-label and acid dissolution, the Cu2S generated an intense electrochemical SWV signal giving a graphene-polyaniline (GR-PANI) composites and graphene oxide (GO). HRP-GO-antibody conjugates LoD of 7.5 pg·mL−1 (28 pM). The same authors [81] described a graphene-modified electrode modified were used to improve sensitivity, through a competitive immunoassay. The LoD was of 0.02 ng¨ mL´1 with mesoporous Fe3O4 and loaded with Pb2+ or Cd2+ cations and estradiol antibodies. In this case, (75 pM). Very recently (2015), again using nanostructures, Zhang et al. [80] reported a sensor using square wave adsorptive stripping voltammetry (SWASV) was used, in order to detect Pb2+ or Cd2+. Cu S nanostructures as labels. Cu S NPs were covalently conjugated to BSA-estradiol. Even without SWASV2 peak currents were proportional2 to the concentrations of estradiol down to a LoD of 0.015 using enzyme-label and acid dissolution, the Cu S generated an intense electrochemical SWV signal pg·mL−1 (55 fM). Lastly, Jimena et al. [82] developed2 an immunosensor by immobilization of the anti- giving a LoD of 7.5 pg¨ mL´1 (28 pM). The same authors [81] described a graphene-modified electrode 17 beta-estradiol monoclonal antibody on an Au disk modified with AuNPs on a cysteamine SAM. modified with mesoporous Fe O and loaded with Pb2+ or Cd2+ cations and estradiol antibodies. In The limit of detection was 0.843 pg·mL4 −1 (3 pM). To conclude this section and as already remarked in this case, square wave adsorptive stripping voltammetry (SWASV) was used, in order to detect Pb2+ Section 2.1 concerning antibiotics, the most pertinent strategy to reach the lowest limits of detection or Cd2+. SWASV peak currents were proportional to the concentrations of estradiol down to a LoD of is to use adsorptive stripping methods [78,81]. 0.015 pg¨ mL´1 (55 fM). Lastly, Jimena et al. [82] developed an immunosensor by immobilization of the 2.5.2.anti-17 Aptamers beta-estradiol monoclonal antibody on an Au disk modified with AuNPs on a cysteamine SAM. The limit of detection was 0.84 pg¨ mL´1 (3 pM). To conclude this section and as already remarked in SectionComparatively, 2.1 concerning aptamer-based antibiotics, the estradiol most pertinent sensors strategy were less to reach reported the lowest than their limits immunosensor of detection is counterpartsto use adsorptive and strippingtheir use methods appeared [78 ,81more]. recently. Olowu et al. [83] described in 2010 an electrochemical DNA aptasensor for 17-beta-estradiol based on poly(3,4-ethylenedioxythiophene) (PEDOT) doped with AuNPs. Streptavidin was covalently attached to the electrode and the aptamer immobilized via streptavidin-biotin interaction. The electrochemical signal generated from the aptamer-target molecule interaction was monitored by standard CV and SWV using Fe(CN)63−/4− as redox probe. The signal observed showed a current decrease due to steric hindrance from the bound 17 beta-estradiol. The LoD was 0.1 nM. In 2012, Lin et al. [84] reported also a conventional transduction approach based on EIS with Au electrodes modified by a thiolated aptamer. Upon formation of the estradiol/aptamer complex on the electrode surface, the interfacial electron transfer resistance (Ret) increased, with a detection limit of 2 pM. This Ret increased was explained by the fact Biosensors 2016, 6, 7 16 of 22

2.5.2. Aptamers Comparatively, aptamer-based estradiol sensors were less reported than their immunosensor counterparts and their use appeared more recently. Olowu et al. [83] described in 2010 an electrochemical DNA aptasensor for 17-beta-estradiol based on poly(3,4-ethylenedioxythiophene) (PEDOT) doped with AuNPs. Streptavidin was covalently attached to the electrode and the aptamer immobilized via streptavidin-biotin interaction. The electrochemical signal generated from the 3´{4´ aptamer-target molecule interaction was monitored by standard CV and SWV using Fe(CN)6 as redox probe. The signal observed showed a current decrease due to steric hindrance from the bound 17 beta-estradiol. The LoD was 0.1 nM. In 2012, Lin et al. [84] reported also a conventional transduction approach based on EIS with Au electrodes modified by a thiolated aptamer. Upon formation of the estradiol/aptamer complex on the electrode surface, the interfacial electron transfer resistance (Ret) increased, with a detection limit of 2 pM. This Ret increased was explained by the fact that the negatively-charged aptamer probe hindered the electron transfer reaction of the redox probe 3´{4´ Fe(CN)6 on the Au electrode surface. More recently, in 2014, Huang et al. [85] described a more original nanostructured sensor using flower-like vanadium disulfide, showing ordered nanosheets of several nanometers of thickness onto 3´{4´ which the aptamers were immobilized. Very classically, Fe(CN)6 was used as a diffusing redox probe and DPV was used to sense change in apparent diffusion coefficient when 17-beta-estradiol is associated with the grafted aptamers. A detection limit of ca. 1 pM was reported. Again exploiting nanostructuration, Ke et al. [86] reported a dendritic gold microstructure immobilized on boron doped diamond (BDD) electrode. Estradiol aptamers were immobilized on the surface of the dendritic Au/BDD electrode through Au-S interaction. Once estradiol reacts with aptamers, it enlarges the interfacial electron transfer resistance (sensed by EIS, again using the redox probe 3´ 4´ Fe(CN)6 /Fe(CN)6 ) on the electrode surface thus cause the increase of impedance) down to a LoD of 5 fM of estradiol. In 2015, Zhu et al. [87] reported a nanoporous conducting polymer electrode whose surface potential, modified upon interaction between estradiol and its aptamer, was still probed 3´{4´ via EIS using Fe(CN)6 as redox probe. Transduction was explained by the redistribution of negative charges in the electrode double-layer region when the aptamer adopts a folded conformation around the small neutral target molecule. The LoD was in the fM range. Additionally, in 2015, Fan et al. [88] used nickel hexacyanoferrate NPs as signaling probe, immobilized on the electrode. AuNPs were put on the NiFe(CN)6 NPs, and the thiolated aptamer was immobilized. Upon formation of estradiol-aptamer complex, the interfacial electron transfer reaction (still probed via EIS using 3´{4´ Fe(CN)6 ) of the probe was lowered, resulting in the decrease of the electrochemical signal. The LoD was ca. 1 pM. At last, Jimenez et al. [89] described the selection of aptamers having dissociation constants for estradiol in the low nM range (17 nM for the best one). They described an EIS aptasensor based on the conformational change of the aptamer immobilized on a gold electrode by self-assembly. It was shown 3´{4´ that the signal of the Fe(CN)6 redox probe was optimized upon hybridization of the aptamer with a short complementary sequence at some specific sites, which was attributed to the more significant conformational change of the aptamer/DNA duplex than the single-stranded aptamer upon binding with the target. The LoD was 0.90 ng¨ mL´1 (3 nM). All these results are summarized in Table5. LoDs appear to be lower for estradiol than for the other targets dealt with in this review. Conversely to pollutants or other toxic small organic molecules, antibodies are available and lead to low LoDs, even if aptamers allow to obtain the lower ones (for similar transduction methods). If we compared LoD for a same probe, similarly to the others targets discussed above, the enzyme-amplified strategy applied with aptamer probes do not lead to significant improvements; this a obviously not the case for antibodies, for which enzyme-labelling has long proven 3´{4´ its efficiency. Concerning the sensing strategies used with aptamers, EIS coupled to the Fe(CN)6 redox probe is dominant. Efforts could be made to improve this step. Biosensors 2016, 6, 7 17 of 22

Table 5. Figures of merit of selected immunosensors and aptasensors for detection of estradiol.

Bioreceptors LoD Transduction and Analytical Methods Reference Ab 0.1 nM SAM/BQ/CV [67] Ab 0.2 nM AlkP-estradiol/DPV [68] Ab 0.9 pM AlkP-estradiol/amperometry [69] Ab 0.15 nM AlkP-estradiol/amperometry [70] Ab 20 pM AuNPs/HRP/BQ/CV [71] 3´{4´ Ab 65 pM AuNPs/Protein G/[Fe(CN)6] /SWV [72] 3´{4´ Ab 95 pM AuNPs/Protein G/[Fe(CN)6] /EIS [72] Ab 1 pM MBs/HRP-estradiol [73] Ab 0.1 pM HRP-estradiol/Catechol/CV [74] Ab 45 pM Protein G/Fc-MeOH/SWV [75] Ab 2.8 pM PABA/BQ [76] Ab 3.6 pM MBs/AlkP-αIgG/SWV [77] Ab 0.2 nM CdSe QDs/dissolution (SWASV) [78] Ab 75 pM Graphene/HRP-GO [79] Ab 28 pM Cu2S NPs/CV [80] 2+ 2+ Ab 55 fM Pb , Cd /porous Fe3O4/SWASV [81] Ab 3 pM AuNPs [82] 3´{4´ Aptamer 0.1 nM PEDOT/AuNPs/Biot-Avidin/[Fe(CN)6] /CV-SWV [83] 3´{4´ Aptamer 2 pM SAM/Au/[Fe(CN)6] /EIS [84] 3´{4´ Aptamer 1 pM VS2/[Fe(CN)6] /DPV [85] 3´{4´ Aptamer 5 fM Au/BDD/[Fe(CN)6] /EIS [86] 3´{4´ Aptamer 1 fM Nanoporous electrode/[Fe(CN)6] /EIS [87] 3+ 3´ Aptamer 1 pM Ni ,Fe(CN)6] /AuNPs [88] 3´{4´ Aptamer 3 nM SAM/Au/[Fe(CN)6] /EIS [89] Note: BQ (benzoquinone); Fc-MeOH (ferrocenemethanol); PABA (p-aminobenzoic acid); SWASV (square wave adsorptive stripping voltammetry).

3. Conclusions and Perspectives Electrochemical immunosensors and aptasensors for organic molecules have been significantly reported in the literature since the mid-2000s, whereas peptide sensors remain confidential at this time. Limits of detections are generally two to three orders of magnitude lower for immunosensors than for aptasensors due to the highest affinities of antibodies. No significant progresses have been made to improve these affinities, but transduction schemes were improved instead, which led to a regular LoD improvement corresponding to ca. five orders of magnitude over these last 10 years. If we look more closely to each target molecule discussed in this review, one sees that no significant progresses was made for antibiotics, for which immunosensors are still the most efficient compared to aptasensors, with an average LoD of ca. 10´10 M versus 10´8 M, respectively. There are very few immunosensors for cocaine, whereas aptasensors, which appeared in 2006, are frequently reported and show an improvement of their LoD of five order of magnitudes over the last 10 years. Concerning ochratoxin A, there is no difference between immuno- and apta-sensors in terms of limit of detection, which progressed by two to three orders of magnitude over the last 10 years. Lastly, concerning estradiol, there is no significant difference between immuno- and apta-sensors in terms of limit of detection, and very little improvement has been made since the first publications. From the existing literature, we did not identify obvious parameters which could be used to predict affinities of captures probes (antibodies or aptamers) for a given target. As stressed above, generally speaking, antibodies still present higher affinity constants for their targets than aptamers. This not only leads to lower LoD, but also to a better specificity when targets are sensed in complex matrixes. However, aptamers are more pertinent than antibodies when enantioselectivity is necessary, which is particularly pertinent for drug analysis.

Acknowledgments: Shihui Shi thanks China Scholarship Council for a PhD grant. Biosensors 2016, 6, 7 18 of 22

Conflicts of Interest: The authors declare no conflicts of interest.

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