biosensors Article Development of Novel Magneto-Biosensor for Sulfapyridine Detection Talha Jamshaid 1,2,3 , Ernandes Taveira Tenório-Neto 2,4 , Abdoullatif Baraket 1 , Noureddine Lebaz 2 , Abdelhamid Elaissari 2, * , Ana Sanchís 5,6 , J.-Pablo Salvador 5,6 , M.-Pilar Marco 5,6 , Joan Bausells 7 , Abdelhamid Errachid 1, * and Nadia Zine 1 1 Institut des Sciences Analytiques, University Claude Bernard Lyon-1, UMR 5280, 5 rue de la Doua, F-69100 Villeurbanne, France; talhajamshaid007@gmail.com (T.J.); a.baraket@gmail.com (A.B.); Nadia.zine@univ-lyon1.fr (N.Z.) 2 University Claude Bernard Lyon-1, CNRS, LAGEPP UMR-5007, 43 Boulevard du 11 Novembre 1918, F-69100 Villeurbanne, France; tenorioernandes@gmail.com (E.T.T.-N.); noureddine.lebaz@univ-lyon1.fr (N.L.) 3 Faculty of Pharmacy and Alternatives Medicins, The Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan 4 Department of Chemistry, State University of Ponta Grossa, Av. Gen. Carlos Cavalcanti, 4748, CEP 84030-900 Ponta Grossa, Paraná, Brazil 5 Nanobiotechnology for Diagnostics (Nb4D), Department of Chemical and Biomolecular Nanotechnology, Institute for Advanced Chemistry of Catalonia (IQAC) of the Spanish Council for Scientific Research (CSIC), Jordi Girona 18-26, 08034 Barcelona, Spain; asvnqb@iqac.csic.es (A.S.); jpablo.salvador@iqac.csic.es (J.-P.S.); pilar.marco@cid.csic.es (M.-P.M.) 6 CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Jordi Girona 18-26, 08034 Barcelona, Spain 7 Institute of Microelectronics of Barcelona (IMB−CSIC), Universitat Autònoma de Barcelona (UAB), Bellaterra, 08193 Barcelona, Spain; joan.bausells@imb-cnm.csic.es * Correspondence: abdelhamid.elaissari@univ-lyon1.fr (A.E.); abdelhamid.errachid-el-salhi@univ-lyon1.fr (A.E.)  Received: 25 March 2020; Accepted: 17 April 2020; Published: 21 April 2020  Abstract: In this work, we report the development of a highly sensitive biosensor for sulfapyridine detection based on an integrated bio micro-electromechanical system (Bio-MEMS) containing four gold working electrodes (WEs), a platinum counter electrode (CE), and a reference electrode (RE). Firstly, the cleaned WEs were modified with 4-aminophenylacetic acid (CMA). Then, (5-[4-(amino)phenylsulfonamide]-5-oxopentanoic acid (SA2BSA) was immobilized onto the transducers surface by carbodiimide chemistry. The analyte was quantified by competitive detection with SA2BSA immobilized on the WE toward a mixture of Ab155 antibody (with fixed concentration) and sulfapyridine. In order to obtain a highly sensitive biosensor, Ab155 was immobilized onto magnetic latex nanoparticles surface to create a 3D architecture (Ab-MLNp). Using electrochemical impedance spectroscopy (EIS), we investigated the influence of the Ab-MLNp on the sensitivity of our approach. The optimized system was analyzed, as competitive assay, with different concentrations of sulfapyridine (40 µM, 4 µM, and 2 nM) and with phosphate buffer solution. From data fitting calculations and graphs, it was observed that the EIS showed more linearity when Ab-MLNp was used. This result indicates that the magnetic latex nanoparticles increased the sensitivity of the biosensor. Keywords: biosensor; sulfapyridine; SA2BSA; BioMEMS; magnetic nanoparticles; competitive assay Biosensors 2020, 10, 43; doi:10.3390/bios10040043 www.mdpi.com/journal/biosensors Biosensors 2020, 10, 43 2 of 13 1. Introduction Increasing attention has been paid to antibiotics as aquatic micropollutants with their environmental fate and impact to be understood [1]. Sulfonamide antibiotics (SAs), as one of the most important classes of antibiotics, are widely used in aquaculture, livestock husbandry, and human medicine. Recently, SAs were detected ubiquitously in the aquatic environment, which may pose risks toward organisms [2–4]. Among the SAs, sulfapyridine, which is commonly used in aquaculture, was frequently detected in various environmental waters (e.g., wastewater effluents and receiving water bodies as well as fish farms and adjacent water bodies) [5]. For the detection of sulfapyridine, various methods have been used, such as chromatographic methods (likely high-performance liquid chromatography coupled with mass spectrometric detection (HPLC-MS)). Such methods have been applied due to their sensitivity and compound quantification data. Sample preparation is required using commercially available cartridges for solid-phase extraction. Additionally, other techniques have been employed, such as thin layer chromatography, gas chromatography (GC), liquid chromatography (LC) (including their variations coupled with mass spectrometry), and radio-active immune receptor for purpose of foodstuffs [6–8]. However, the above-mentioned approaches are time consuming and require complex sample preparation procedures, expensive laboratory equipment, and skilled professionals to handle these techniques. In this sense, biosensors may offer cost-effective solutions for analyte detection. The biosensor is a compact analytical device or unit incorporating a biological (or biologically) derived sensitive element associated with a physicochemical transducer. They have revolutionized modern analysis due to their technical simplicity, low cost, and the possibility of being employed in field analysis [9,10]. The detection of sulfonamides using biosensors was previously demonstrated in different works [11–13]. However, few examples can be found using impedance spectroscopy for SA detection. To improve the biosensor sensitivity, recently, magnetic nanoparticles (MNP) were produced as labels for biosensing. For the biosensing purpose, different types of biosensors were produced, such as giant-magnetoresistive (GMR) sensors and spin valves (SV) cantilevers [14,15], inductive sensors [16], superconducting quantum interference devices (SQUIDs) [17], anisotropic-magnetoresistive (AMR) rings, and miniature Hall crosses [18]. The detection of biological molecules is usually achieved by using biomolecular recognition between the target molecule and a specific receptor as, for example, an antibody that is tagged with a label. In this context, superparamagnetic iron oxide nanoparticles (SPIONs) have been used as carriers for immobilization of biomolecules, such as peptides, proteins, and antibodies, to enhance the specific capture of the targeted biomolecules [19]. For preparing structured SPIONs with well-defined surface properties, specific functional groups, and better colloidal stability, several approaches have been investigated, including seeded-emulsion polymerization [20,21]. Using such an approach, magnetic latex nanoparticles with high iron oxide content can be obtained. These particles have reactive functional groups to form conjugates with various biomolecules (e.g., proteins, antibodies, DNA, and so forth), making them promising candidates to improve automation. Furthermore, the magnetic latex particles can enhance the diagnosis sensitivity by increasing the concentration of the captured targets [22]. Herein, an alternative procedure for sulfapyridine detection is proposed. Our approach consists of the development of bio-micro-electro-mechanical system (Bio-MEMS) transducers based on four gold micro-working electrodes (µWE) with fully integrated reference (RE) and platinum counter electrodes (CE). The surface of µWE was modified with coating antigen (5-[4-(amino)phenylsulfonamide]-5-oxopentanoic acid (SA2BSA), and the quantification of sulfapyridine was achieved through competitive assay toward antibodies Ab-155 deposited onto the magnetic latex nanoparticles surface. In addition, to evidence our proof of concept, we also compared the results obtained from our approach with those obtained from the competitive detection without magnetic latex particles. Biosensors 2020, 10, 43 3 of 13 2. Materials and Methods 2.1. Reagents and Apparatus The 4-aminophenylacetic acid 98%, the N-Hydroxysuccinimide (NHS), and the 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) were purchased from Acros Organics. Sodium nitrite was acquired from Fisher Scientific. Triton X-405 and phosphate buffer saline tablet were acquired from Sigma, ethanolamine from Fluka analytical, and hydrochloric acid 37% (HCl) from VWR, France. Iron (II) chloride tetrahydrate (FeCl2 ·4H2 O), iron (III) chloride hexahydrate (FeCl3 ·6H2 O), and oleic acid were purchased from Merck. Octane, ammonium hydroxide, and chlorhydric acid were from Prolabo. The preparation of the described bioconjugates and antibodies (5-[4-(amino)phenylsulfonamide]-5-oxopentanoic acid (SA2BSA) and Ab155) was performed with the support of the ICTS (Infraestructuras Científico-Tecnológicas Singulares “NANBIOSIS” Spain), more specifically by the Custom Antibody Service (CAbS, CIBER-BBN, IQAC-CSIC). The immunoreagents for sulfapyridine (SPy) detection used for the development of the biosensor were described before [23]. All other chemicals were purchased from Sigma-Aldrich and were used as received. All electrochemical measurements were performed on a VMP3 electrochemical workstation. 2.2. Preparation of Oil-in-Water Magnetic Emulsion The oil-in-water magnetic emulsion (ME) consisted of magnetite nanoparticles stabilized with oleic acid, octane, and dodecyl sodium sulfate and was prepared according to our previous work [20,24,25]. Before obtaining the ME, the superparamagnetic iron oxide nanoparticles (SPIONs) were synthetized by coprecipitation method. Firstly, a solution containing iron chloride salts (ratio [Fe3+ ]/[Fe2+ ] = 2) was prepared. Then, concentrated ammonia was carefully added to this solution to produce magnetic nanoparticles. When the solution pH was around 9, oleic acid (stabilizing agent) was added to the dispersion under vigorous stirring. The SPIONs were extracted by introducing octane (organic phase) in the medium. Finally, the organic ferrofluid was collected by magnetic separation and filtered on a 1 mm porous nylon filter in order to eliminate aggregates, and it was concentrated by octane evaporation under vacuum until it achieved a solid content of about 70% wt. Afterward, the organic ferrofluid was emulsified in an aqueous solution of a nonionic surfactant (Triton X-405, at least 25 wt% in water) and sheared in a couette emulsifier in order to target submicron ferrofluid droplets. The sheared emulsion was recovered and then diluted in a water solution containing sodium dodecyl sulphate (SDS) (2 g L−1 ). Then, the oil-in-water magnetic emulsion was sorted under magnetic field in order to collect narrowly sized distributed droplets. Lastly, the magnetic emulsion was diluted until it achieved a total solid content of about 4% wt. 2.3. Electrochemical Measurements The electrochemical measurements were made by using a multi-channel potentiostat (VMP3 Biologic Science Instrumentation, France). Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were applied to characterize the surface of WEs before and after bio-functionalization using K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ] (5 mM) as the electrolyte in phosphate buffered saline (PBS, pH 7.4). CV measurements were made at potentials of 0.5 V to −0.3 V, scan rate of 80 mV s−1 , and 3 cycles. EIS measurements were made at a potential of 0.228 V, frequency of 200 kHz to 200 mHz, and sinus amplitude of 25 mV. 2.4. Fabrication of the Biosensor The Bio-MEMS, based on gold microelectrodes, was fabricated in collaboration with the National Centre of Microelectronic (CNM) in Spain. The detailed process of fabrication and the characterization of the microlectrode were previously published in our group [26]. Briefly, a silicon wafer of 100 mm diameter was used as a substrate. Silicon dioxide (SiO2 ) was then grown up to 800 nm by wet thermal oxidation. Then, gold working microelectrodes were defined by deposition and patterning of a metal Biosensors 2020, 10, 43 4 of 13 tri-layer of Ti (50 nm), Ni (50 nm), and Au (200 nm). For the counter microelectrode, a bilayer of Ti (15 nm) plus Pt (150 nm) was deposited. Finally, for the reference integrated microelectrode, a bilayer of Ti (15 nm) plus Ag (150 nm) was deposited and patterned by lift-off. All microelectrodes dimensions and Biosensors 2020, 10, x FOR PEER REVIEW 4 of 13 the process of fabrication were detailed in our previous work [26]. After the fabrication process, the wafer was diced to individual devices 4 × 7 mm and was then microelectrode, a bilayer of Ti (15 nm) plus Pt (150 nm) was deposited. Finally, for the reference glued to the PCB board (Figure 1b). Micro pads of all microelectrodes were then electrically connected integrated microelectrode, a bilayer of Ti (15 nm) plus Ag (150 nm) was deposited and patterned by to the PCB board (Figure 1c) and then were passivated with epoxy resin (Ep-Tek H70E-2LC) to protect lift-off. All microelectrodes dimensions and the process of fabrication were detailed in our previous all electrical connections (Figure 1c) [26]. work [26]. Figure Figure 1. Optical 1. Optical microscope microscope images images of (a) of (a) printed printed circuit circuit board board (PCB) (PCB) dimensions, dimensions, (b) (b) transducers transducers glued glued ontoonto thethe PCBPCB board, board, (c) (c) electrical electrical connections, connections, andand (d)(d) passivation passivation with with epoxy epoxy resin resin Working After electrodesprocess, the fabrication (WEs) were cleaned the wafer was bydiced rinsing to them with ethanol individual devicesfor 4 ×5 7minutes, mm andand wasthen they then were glued toproperly rinsed (Figure the PCB board with deionized 1b). Microwater. padsThey of allwere then dried under microelectrodes weresteam of nitrogen.connected then electrically The surface WEs were then cleaned from organic contaminants by using to the PCB board (Figure 1c) and then were passivated with epoxy resin (Ep-Tek UV/O 3 (UV/Ozone ProCleanerTM, H70E-2LC) to protect all BioForce electricalNanosciences) for 30 min. connections (Figure The device was connected to the potentiostat VMP3. Analysis was 1c) [26]. monitored Working and modeled electrodes using (WEs) the EC-Lab were cleaned software. by rinsing them with ethanol for 5 minutes, and then Electrochemical deposition of 4-aminophenylacetic they were properly rinsed with deionized water. They were acid then98% (CMA) dried underwas madeofby steam using 5 The nitrogen. mM of CMA WEs surface in water withthen were sodium nitritefrom cleaned 15 mM and HCl organic 15 mM. Both by contaminants sodium usingnitrite UV/Oand HCl solutions 3 (UV/Ozone were added to the solution of CMA and kept in an ice bath for 15 min. ProCleanerTM, BioForce Nanosciences) for 30 min. The device was connected to the potentiostat This electrode preparation approach VMP3. Analysiswaswasadapted from that monitored andreported modeledby Allongue using et al. (1997) the EC-Lab [27]. The final solution was kept software. 0 ◦ C and immediately atElectrochemical used.ofThe deposition reductive absorption 4-aminophenylacetic acidof98% the (CMA) salt onto wasthemade electrode was achieved by using 5 mM by four repetitive scans of CV between 0.3 and −1.0 V at 200 mV s −1 . Figure 2 shows a schematic of CMA in water with sodium nitrite 15 mM and HCl 15 mM. Both sodium nitrite and HCl solutions wereillustration added to of theCMA deposition solution of CMAonto andgold keptWEs. in an ice bath for 15 minutes. This electrode preparation approach was adapted from that reported by Allongue et al. (1997) [27]. The final solution was kept at 0 °C and immediately used. The reductive absorption of the salt onto the electrode was achieved by four repetitive scans of CV between 0.3 and −1.0 V at 200 mV s−1. Figure 2 shows a schematic illustration of CMA deposition onto gold WEs. of CMA in water with sodium nitrite 15 mM and HCl 15 mM. Both sodium nitrite and HCl solutions were added to the solution of CMA and kept in an ice bath for 15 minutes. This electrode preparation approach was adapted from that reported by Allongue et al. (1997) [27]. The final solution was kept at 0 °C and immediately used. The reductive absorption of the salt onto the electrode was achieved byBiosensors four repetitive scans of CV between 0.3 and −1.0 V at 200 mV s−1. Figure 2 shows a schematic 2020, 10, 43 5 of 13 illustration of CMA deposition onto gold WEs. Figure 2. Schematic representation of surface gold working electrode modification with 4-aminophenylacetic acid (CMA). WEs, CE, and RE are working electrode, counter electrode, and reference electrode, respectively. 2.5. Process for Biosensor Bio-Functionalization A solution of EDC 0.4 M and NHS 0.1 M was made up in 1 mL of anhydrous ethanol, and the device was incubated in a 2 mL Eppendorf for 1 h at room temperature. After this time, the device was rinsed with HCl 0.1 M and dried with nitrogen stream carefully. Immediately, the CMA modified WEs were incubated in 60 µL of SA2BSA antigens at 100 µg mL−1 for 1 h at room temperature. Then, the microelectrodes were rinsed with PBS and incubated in 0.1% solution of ethanolamine (ETA) in PBS for 20 min at room temperature. This deactivated all remaining carboxylic activated sites. This step was necessary in order to prevent non-specific binding. At this stage, the biosensors were ready for antibodies detection. 2.6. Preparation of Magnetic Nanoparticles The magnetic nanoparticles (MNPs) were prepared using the strategy that consisted of an oil-in-water magnetic emulsion as seed in the emulsion polymerization of styrene (St) and divinylbenzene (DVB). The polymerization was carried out in a 60 mL three-necked double wall glass reactor made up of glass anchor type stirrer, a reflux condenser, and a nitrogen inlet. The temperature was controlled at 70 ◦ C by using a thermal bath. Firstly, the supernatant of the magnetic emulsion (50 mL) was removed after 5 min of magnetic separation. After that, the remaining magnetic particles were stabilized with the same volume of 1 g L−1 aqueous solution of dodecyl sodium sulfate (SDS) and introduced into the reactor. Then, the ME was purged with nitrogen for 1 h under stirring of 300 rpm to remove the residual oxygen. Afterward, a total of 1.2 mL of monomers (with ratio of 20 wt.% St and 80 wt.% DVB) was charged at once into the reactor containing the ME being kept under continuous stirring during 60 min. The radical polymerization started after adding 2 wt.% (with respect to the total weight of monomers) of 4,40 -azobis(4-cyanopentanoic acid) (ACPA) solubilized in a solution of 0.1 mol L−1 sodium hydroxide with SDS. The radical polymerization was conducted at constant mechanical stirring (300 rpm) and was left overnight for 24 h. A schematic representation of magnetic nanoparticles preparation is shown in Figure 3. the reactor containing the ME being kept under continuous stirring during 60 min. The radical polymerization started after adding 2 wt.% (with respect to the total weight of monomers) of 4,4′- azobis(4-cyanopentanoic acid) (ACPA) solubilized in a solution of 0.1 mol L−1 sodium hydroxide with SDS. The radical polymerization was conducted at constant mechanical stirring (300 rpm) and was left overnight Biosensors for4324 h. A schematic representation of magnetic nanoparticles preparation is shown 2020, 10, 6 ofin 13 Figure 3. COOH COOH St / DVB / ACPA COOH 70 °C polymerization COOH COOH Oil in water magnetic emulsion Magnetic latex particle containing carboxylic groups on the surface Figure Figure3.3.Schematic Schematicrepresentation representationof ofmagnetic magneticnanoparticles nanoparticlespreparation. preparation. 2.7.Preparation 2.7. Preparationofofthe theMNP-Ab MNP-AbLabels Labels Inorder In orderto toincrease increasethe thesensitivity sensitivityofofthe thebiosensor, biosensor,the theantibodies antibodiesAb155 Ab155were wereimmobilized immobilizedonto onto MNP by the carbodiimide chemistry (Figure 4). The general procedure MNP by the carbodiimide chemistry (Figure 4). The general procedure was based on our previous was based on our previous work[23]. work [23].Briefly, Briefly,aavial vialofof 11 mL mL of of MNP MNP waswas placed placedin in aa magnetic magneticrack rackfor for55 min mintotoimmobilize immobilizethe the MNP and to remove the support solution. Then, 1 mL of PBS was added MNP and to remove the support solution. Then, 1 mL of PBS was added in order to wash the MNP. in order to wash the MNP. Thisoperation This operationwas wasrepeated repeated three three times times to to ensure ensure that that all all surfactants surfactants were were removed. removed. The carboxylic groups from the MNP were activated The carboxylic groups from the MNP were activated after treatment with after treatment with a mixture a mixture of EDC of EDC 0.1 0.1 M and M and2020, Biosensors NHS NHS 10,0.1 0.1 M PEER x FOR M and and prepared prepared in anhydrous ethanol for 90 min REVIEW in anhydrous ethanol for 90 min with continuous stirring atwith continuous stirring13 at room 6 of room temperature. temperature. After thisAfter time,thisthetime, MNPsthe MNPs were rinsedwere by rinsed using aby using arack magnetic magnetic rack with three times threeHCl times 1 with HCl 1 mM in order to keep the activity of carboxylic acid. Then, 500 mM in order to keep the activity of carboxylic acid. Then, 500 µL of a purified antibody Ab155 (100 µL of a purified antibody −1 in PBS 10 mM) was added to the activated MNP with stirring (500 rpm at room µgAb155 mL−1 in(100 PBSµg 10mL mM) was added to the activated MNP with stirring (500 rpm at room temperature) fortemperature) 2–3 hours. The for 2–3 hours. Ab155 wasThe Ab155 was previously previously purified frompurified from the the antisera antisera As155 As155 by ammonium by ammonium sulfate precipitation at 40% of total saturation. Finally, the vial of Ab155-modified MNP was washed washed sulfate precipitation at 40% of total saturation. Finally, the vial of Ab155-modified MNP was with PBS with PBSusing 3 times 3 timestheusing the magnetic magnetic rack and rack and stored stored in PBS at 4in °C.PBS 4 ◦ C. The Theatantibody antibody reacted withreacted with the the activated COOHactivated by theCOOH by the free free accessible accessible lysines of thelysines of the antibody, asantibody, shown inas shown Figure 4. in Figure 4. Figure Figure 4. Chemical 4. Chemical strategy strategy for the for the biofunctionalization biofunctionalization of Ab155 of Ab155 using using carbodiimide carbodiimide chemistry chemistry to the to the magnetic magnetic nanoparticles nanoparticles (MNPs). (MNPs). 2.8. Magneto-ELISA for MNP-Ab155/SA7-HRP 2.8. Magneto-ELISA for MNP-Ab155/SA7-HRP Horseradish peroxidase (HRP) labeled with a specific enzymatic tracer SA7-HRP for Ab155 Horseradish peroxidase (HRP) labeled with a specific enzymatic tracer SA7-HRP for Ab155 (2 (2 µg−1 mL−1 in PBS-tween) was mixed with a suspension of different concentration of the biomodified µg mL in PBS-tween) was mixed with a suspension of different concentration of the biomodified −1 of Ab155-MNP). The immunological reaction was magnetic beads (100 µL, from 100 to 0 µg mL magnetic beads (100 µL, from 100 to 0 µg mL of Ab155-MNP). The immunological reaction was −1 allowed to proceed for 30 min at RT with vigorous shaking. The magnetic beads were then washed allowed to proceed for 30 min at RT with vigorous shaking. The magnetic beads were then washed with PBST (150 µL, three times), and the substrate solution (100 µL, 0.01% tétraméthylbenzidine (TMB) with PBST (150 µL, three times), and the substrate solution (100 µL, 0.01% tétraméthylbenzidine and 0.004% H2 O2 in citrate buffer 0.05M pH 5.5) was added and incubated again for 30 min at RT. (TMB) and 0.004% H2O2 in citrate buffer 0.05M pH 5.5) was added and incubated again for 30 min at The enzymatic reaction was stopped by adding 2 M H2 SO4 (50 µL). Finally, the supernatants were RT. The enzymatic reaction was stopped by adding 2 M H2SO4 (50 µL). Finally, the supernatants were removed from the magnetic beads and then added to a different plate for measuring the absorbance at removed from the magnetic beads and then added to a different plate for measuring the absorbance 450 nm. at 450 nm. 3. Results 3.1. Characterization of the Magnetic Nanoparticles The characterization of the produced nanoparticles was performed by transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential measurement, and thermal gravimetric analysis (TGA). Before polymerization, the average particle size of ME was approximately 237 nm [20]. As we expected, after polymerization, the average particle size increased Biosensors 2020, 10, 43 7 of 13 3. Results 3.1. Characterization of the Magnetic Nanoparticles The characterization of the produced nanoparticles was performed by transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential measurement, and thermal gravimetric analysis (TGA). Before polymerization, the average particle size of ME was approximately 237 nm [20]. As we expected, after polymerization, the average particle size increased up to 296 nm due to addition of polymer shell (See Figure 5). However, due to the presence of such a polymer shell, the values of zeta potential decreased. In a pH ranging from four to nine, the particles showed zeta potential values of −40 to −50 mV and −28 to −35 mV for magnetic emulsion and core-shell particles, respectively. The highest zeta potential values for ME was associated with the presence of the strong acid sulfate groups (−SO4 − ) emanating from SDS (stabilizing agent) of the magnetic emulsion. However, after polymerization, the ME surface was covered by non-charged molecules, and only ACPA could contribute to the surface charge due 2020, Biosensors to the 10,presence ofREVIEW x FOR PEER COOH moieties. 7 of 13 (A) (B) Figure 5. TEM images of: (A) magnetic emulsion, (B) samples (with core-shell like morphology Figure 5. TEM images of: (A) magnetic emulsion, (B) samples (with core-shell like morphology containing COOH moieties from 4,4′-azobis(4-cyanopentanoic acid) (ACPA) obtained after seed- containing COOH moieties from 4,40 -azobis(4-cyanopentanoic acid) (ACPA) obtained after polymerization using 20 wt.% St and 80 wt.% divinylbenzene (DVB). seed-polymerization using 20 wt.% St and 80 wt.% divinylbenzene (DVB). Once the final bioconjugates were purified, a characterization of the biofunctionality was Once theThe required. finalbest bioconjugates way to know were thepurified, a characterization functionality of the conjugate of the Ab-MNPs biofunctionality was bywas required. magneto- TheELISA best way (Figure 6). This method consisted of testing the Ab-MNPs conjugate with an enzymatic6). to know the functionality of the Ab-MNPs conjugate was by magneto-ELISA (Figure Thistracer method thatconsisted had an of testingfor affinity thethe Ab-MNPs conjugate immobilized with anAfter antibody. enzymatic tracerofthat a period had an affinity incubation and for washing, the immobilized antibody. After a period of incubation and washing, a substrate a substrate was added in order to obtain the colorimetric signal from the enzyme, was added in order to obtain the colorimetric signal from the enzyme, which was bound to the which was bound to the antibody. The antibody amount immobilized onto the surface was antibody. The antibody amount immobilized onto the −1 for batch preparation. quantified and found to surface be 100 was µg quantified and found mL−1 for batch to be 100 The preparation. µg mL magneto-ELISA let us Theconclude magneto-ELISA let us conclude that not only was the immobilization was that not only was the immobilization was successful but also the antibody was successful but also the antibody was functional and the obtained signal functional and the obtained signal was specific. was specific. tracer that had an affinity for the immobilized antibody. After a period of incubation and washing, a substrate was added in order to obtain the colorimetric signal from the enzyme, which was bound to the antibody. The antibody amount immobilized onto the surface was quantified and found to be 100 µg mL−1 for batch preparation. The magneto-ELISA let us conclude that not only was the immobilization was successful but also the antibody was Biosensors 2020, 10, 43 8 of 13 functional and the obtained signal was specific. Figure 6. Representation of a magneto-ELISA and the results obtained for MNPs coupled with Ab155 Figure 6. Representation of a magneto-ELISA and the results obtained for MNPs coupled with Ab155 (specific for SA7-HRP and Sulfonamides). (specific for SA7-HRP and Sulfonamides). 3.2. Characterization of CMA-Modified Electrode. 3.2. Characterization of CMA-modified Electrode. The reductive adsorption of the salt onto the electrode was achieved by four repetitive scans of CV The between Biosensors 2020, 10,reductive 0.3 x FOR adsorption −1.0 andPEER V at 200 mV REVIEW s−1 salt of the . Theonto thecycle initial electrode was7A in Figure achieved shows aby four and broad repetitive 8 ofscans irreversible 13 of CV between cathodic wave with 0.3 and −1.0 V peak a potential at 200atmV −0.9s V,. The −1 whichinitial cycle diazotized indicates in Figure 7A CMAshows a broad onto attachment and irreversible the gold cathodic successive surface wave byscans. diazonium withsalt These a potential were peak attributed reduction. at Theto −0.9 large the cathodic V, current which indicates passivation diazotized area was remarkably of the CMA attachment microelectrode. weakened onto the This in the successive goldThese statement scans. surface was werebyattributed diazonium confirmed by CV to salt the reduction. before largeand Thegold after passivation cathodic WEs area current was remarkably modification of the (Figure microelectrode. 7B), This weakened where an statement in the was important confirmed decrease in peak-to-peak by CV before and after goldof the WEsCV cycle was (Figure modification seen when compared 7B), where to the bare an important gold in decrease microelectrodes. peak-to-peak of the CV cycle was seen when compared to the bare gold microelectrodes. Figure 7. (A) Figure Cyclic 7. (A) voltammetry Cyclic voltammetry (CV) forfor (CV) electrodeposition electrodeposition of CMA. Four of CMA. repetitive Four potential repetitive cycles potential cycles from 0.30.3 from VV to to −1.0 V with −1.0 V witha scan rate a scan rate 0.20.2V Vs−1s.−1(B) ofof Cyclic . (B) voltammetry Cyclic scan, voltammetry (C)(C) scan, electrochemical electrochemical impedance impedance spectroscopy spectroscopyforfor bare gold bare WE gold WE (black (blackline), and line), CMA and CMA immobilized onto immobilized thethe onto WEWE (red line). (red line). These results were confirmed by EIS analysis before and after CMA deposition (Figure 7C). Therefore, the first semi-circle of Nyquist plot diagram corresponded to impedance of gold WEs, which was too weak when compared to CMA modified WEs. This was related to the passivation of WEs with the CMA layer, which increased the transfer charge resistance (Rtc) between the Biosensors 2020, 10, 43 9 of 13 These results were confirmed by EIS analysis before and after CMA deposition (Figure 7C). Therefore, the first semi-circle of Nyquist plot diagram corresponded to impedance of gold WEs, which was too weak when compared to CMA modified WEs. This was related to the passivation of WEs with the CMA layer, which increased the transfer charge resistance (Rtc ) between the modified WEs and the electrolyte solution. 3.3. Titration Assay for Ab155 to SA2BSA Coated Wes by EIS Electrochemical impedance spectroscopy (EIS) was made for the coated-antigens SA2BSA gold WEs (Figure 8). Here, the first semi-circle of Nyquist plot (black line) corresponded to the coating antigens without any detection. In order to detect Ab155 antibodies, the biosensors were rinsed with PBS and incubated in 60 µL of antibodies aqueous solution of Ab155 at different concentrations ranging from 1 µg mL−1 to 50 µg mL−1 for 30 min, then rinsed with PBS and analyzed with EIS. The subsequent Nyquist plot semi-circles were shifted from the first, indicating an increase of the transfer charge resistance (Rtc ). As the Rtc became higher, the greater the concentration of antibodies Ab1552020, Biosensors was.10, x FOR PEER REVIEW 9 of 13 Figure 8. Electrochemical Figure impedance 8. Electrochemical impedancespectroscopy (EIS) spectroscopy measurements (EIS) of different measurements concentrations of different of of concentrations Ab155 with fixed concentration of SA2BSA. Ab155 with fixed concentration of SA2BSA. 3.4. 3.4. Competitive Competitive Assay Assay forfor thethe Detection Detection of of Sulfapyridine Sulfapyridine (SPY) (SPY) Usin Usin SA2-BSA SA2-BSA CoatedElectrode Coated Electrodewith with Ab155 Ab155 After showing the interactivity of the coating antigens with different concentrations of Ab155 antibodies, the same After showing theprinciple of EIS interactivity of detection the coating was appliedwith antigens in this section concentrations different by using Ab155ofantibodies Ab155 with another antibodies, antigen the same sulfapyridine principle (SPY). The of EIS detection was SPY is also applied specific in this sectiontobyAb155 usingantibodies. Here, a Ab155 antibodies mixture of 20 µL of fixed concentration of Ab155 antibodies (50 µg mL −1 ) was added to 20 µL of with another antigen sulfapyridine (SPY). The SPY is also specific to Ab155 antibodies. Here, a different mixture of concentrations 20 µL of fixed of SPY 40 µM, 4ofµM, concentration 2 nM, Ab155 and finally antibodies (500 M µg(only mL−1)PBS), was respectively. added to 20 For µL each of concentration, different the twoofsolutions concentrations SPY 40 µM,(20 4µLµM,of Ab 2 nM,155and andfinally 20 µL0of MSPY) (onlywere PBS),then allowed to respectively. Forincubate each the SA2BSA the concentration, modified biosensor two solutions (20for µL30 of min Ab 155andandkept20atµL4 ◦ofC.SPY) Afterwere eachthenincubation, allowed the surface of to incubate microelectrodes was rinsed with PBS and analyzed by EIS measurements. Here, the SA2BSA modified biosensor for 30 min and kept at 4 °C. After each incubation, the surface of there was a competition between SPY and microelectrodes wasSA2BSA rinsed to detect with PBSAb155and antibodies. analyzed by EIS measurements. Here, there was a Figure 9 shows the Nyquist plot semi-circles competition between SPY and SA2BSA to detect Ab155 obtained for 50 µg mL−1 of Ab155 in the presence of antibodies. different Figure concentrations of SPY.plot 9 shows the Nyquist Thesemi-circles first and theobtained second semi-circles for 50 µg mL corresponded −1 of Ab155 into thepresence the bare gold of different concentrations of SPY. The first and the second semi-circles corresponded to the bare gold of (black line) and the coating antigens SA2BSA (blue line), respectively. After the first incubation the biosensor (black within line) and the Ab155 coating and 40 antigens µM of (blue SA2BSA SPY (the line),third Nyquist After respectively. plot semi-circle), we observed the first incubation of the a shift showing biosensor withinaAb155 detection andof40Ab155 µM ofantibodies by SA2BSA. SPY (the third Nyquist Inplot this semi-circle), case, the concentration we observed of SPY was a shift showing a detection of Ab155 antibodies by SA2BSA. In this case, the concentration of SPY was toothe too high, which meant a large amount of Ab155 reacted with SPY, while the rest was attached to coating high, which antigens meantSA2BSA. By decreasing a large amount of Ab155 the reacted concentration with SPY,of SPY, there while thewere restmore antibodies was attached toAb155 the coating antigens SA2BSA. By decreasing the concentration of SPY, there were more antibodies Ab155 free in the solution, which have may reacted with SA2BSA. This was confirmed by the increase of Nyquist plot semi-circles. Finally, when the biosensor was incubated only in Ab155 antibodies with PBS, the totality of the antibodies reacted with the coating antigen SA2BSA, thus giving a big shift between Nyquist plot semi-circles. Biosensors 2020, 10, 43 10 of 13 free in the solution, which have may reacted with SA2BSA. This was confirmed by the increase of Nyquist plot semi-circles. Finally, when the biosensor was incubated only in Ab155 antibodies with PBS, the totality of the antibodies reacted with the coating antigen SA2BSA, thus giving a big shift between Biosensors Biosensors Biosensors Biosensors Nyquist 2020, 2020, 2020, 2020, 10, 10,xxx 10, 10, plot FOR xFOR FOR FOR semi-circles. PEER PEER PEER PEER REVIEW REVIEW REVIEW REVIEW 10of 10 10 of 13 of13 13 13 Figure Figure9.9. Figure Figure 9. EIS 9.EIS EIS measurements EISmeasurements measurementsof measurements measurements of of of Ab155 ofAb155 Ab155 Ab155 with Ab155with with with different withdifferent different different concentrations differentconcentrations concentrations concentrations of concentrationsof of of SPY ofSPY SPY SPY (40 SPY(40 (40 (40 µM, µM,4444µM, (40µM, µM, µM, µM,2222nM) µM, nM) nM) and nM)and and and phosphate-buffered phosphate-buffered phosphate-buffered saline saline saline phosphate-buffered saline phosphate-buffered (PBS) (PBS) (PBS) saline (PBS) only. only. only. (PBS) only. only. 3.5. 3.5. 3.5. CompetitiveAssay Competitive 3.5.Competitive Competitive Competitive Assay Assay for Assayfor for the forthe the Detection Detectionofof theDetection Detection of Sulfapyridine ofSulfapyridine Sulfapyridine (SPY) Sulfapyridine(SPY) (SPY) Usin (SPY)Usin UsinSA2-BSA Usin SA2-BSA SA2-BSA Coated SA2-BSACoated Coated Electrode Electrode CoatedElectrode with Electrodewith with with MNP-Ab155 MNP-Ab155 MNP-Ab155 MNP-Ab155 MNP-Ab155 Firstly, the the immobilization of the coating antigen with 100 µg mL −1 solution of SA2BSA was Firstly, Firstly, Firstly,the Firstly, the immobilization theimmobilization immobilizationof immobilization of of the ofthethe coating thecoating coating antigen coatingantigen antigenwith antigen with with100 with 100 100µg 100 µg µgmL µg mL mL −1 solution mL−1solution −1 −1 solutionof solution of of SA2BSA ofSA2BSA SA2BSAwas SA2BSA was was was made made made made onto onto onto onto WEs WEs WEs WEs surface, surface, surface, surface, as as as as explained explained explained explained in in in in Section Section Section Section 2.4. 2.4. 2.4. 2.4. Afterward, Afterward, Afterward, Afterward, the the the the Ab155-modified Ab155-modified Ab155-modified Ab155-modified MNPwith MNP MNP MNP withfixed with with fixed fixed fixed made onto WEs surface, as explained in Section 2.4. Afterward, the Ab155-modified MNP with fixed quantity quantity quantity quantity of 20 µL was mixed µL was mixed withwith different different concentrations concentrations of SPY (40 µM, 4 µM, and 2 nM) µM, and nM)and andfinally finally quantityof of of 20 20 of20 µL 20µL was µLwas mixed wasmixed mixedwith with different withdifferent concentrations differentconcentrations concentrationsof of of SPY SPY ofSPY (40 (40 SPY(40 µM, µM,4444µM, µM, (40µM, µM, and and2222nM) µM,and nM) and nM)and finally andfinally finally only with only only with PBS. Here, the same principle of competitive detection between SPY and SA2BSA was onlywith only withPBS. with PBS. PBS. Here, PBS.Here, Here, the Here,thethe same thesame same principle sameprinciple principleof principle of of competitive ofcompetitive competitive detection competitivedetection detection between detectionbetween betweenSPY between SPY SPY and SPYand and SA2BSA andSA2BSA SA2BSAwas SA2BSA was was was applied applied applied applied (Figure (Figure (Figure (Figure 10). 10). 10). 10). By By By By decreasing decreasing decreasing decreasing thethe the concentration the concentration concentration concentration of SPY, of of of the SPY, SPY, SPY, shift the the the between shift shift shift between betweenNyquist between plot Nyquist Nyquist Nyquist semi-circles plot plot plot semi- semi- semi- applied (Figure 10). By decreasing the concentration of SPY, the shift between Nyquist plot semi- increased. circles circles However, when whencompared to the to competitive detection without MNP,MNP, this shift was more circles increased. circlesincreased. increased. However, increased.However, However,when However, when compared whencompared comparedto compared to the tothe the competitive thecompetitive competitive detection competitivedetection detection without detectionwithout withoutMNP, without MNP,this MNP, this this shift thisshift shift was shiftwaswas was significant, more more especially for the concentrations 40 µM, 2 µM, and 2 nM. This result was associated with more significant, moresignificant, significant, especially significant,especially especiallyfor especially for for the forthe the concentrations theconcentrations concentrations40 concentrations 40 40 µM, 40µM,µM,2222µM, µM, µM, µM,and µM, and and2222nM. and nM. nM. This nM.This This result Thisresult result was resultwaswas associated wasassociated associated associated the high with with number of antibodies present on the MNP surface. with the withthethe high thehigh high number highnumber numberof number of of antibodies ofantibodies antibodies present antibodiespresent presenton present on onthe on the the MNP theMNP MNP surface. MNPsurface. surface. surface. Figure Figure Figure 10. Figure10. 10. EIS 10.EIS EIS EIS measurements EISmeasurements measurementsof measurements measurements of of of fixed offixed fixed fixed quantity fixedquantity quantity quantity of quantityof of of Ab ofAb Ab Ab 155 Ab155 155 155 modified 155modified modified modified magnetic modifiedmagnetic magneticlatex magnetic magnetic latex latex latex particles latexparticles particles particles (20 particles(20 (20µL) (20 (20 µL) µL) µL) µL) against against against different againstdifferent different concentrations differentconcentrations concentrations of concentrationsofof sulfapyridine ofsulfapyridine sulfapyridine (SPY) sulfapyridine(SPY) (SPY) (40 (SPY)(40 (40 µM, µM,4444µM, (40µM, µM, µM, µM,2222nM) µM, nM) nM)and nM) and andPBS and PBS PBS only. PBSonly. only. Bare only.Bare Bare gold Baregold gold gold (), (), (), (), SA2BSA(), SA2BSA SA2BSA (),SA2BSA SA2BSA (N),MNP-Ab (), (), (), MNP-Ab MNP-Ab 155+40 155+40 MNP-Ab155+40 155+40µM µM µM µMSPYSPY SPY (H),MNP-Ab155+ (), (), SPY(), (), MNP-Ab155+ MNP-Ab155+ MNP-Ab155+4444µM MNP-Ab155+ µM µM SPY SPY µMSPY SPY(♦); ();MNP-Ab (♦); (♦); (♦); MNP-Ab MNP-Ab MNP-Ab155+2155+2 155+2 155+2 155+2nM nM nM nMSPYSPY SPY SPY ((), ), (), (), and and and MNP-Ab MNP-Ab MNP-Ab 155+Only 155+Only 155+Only 155+Only PBS PBS PBS (), and MNP-Ab 155+Only PBS (). ( ). (). (). (). EIS EIS EISmeasurements EIS measurements were measurements were repeated were were repeatedat repeated atatleast at leastfour least least four four four times, times, times, times, as as asas wewe we we had had had hadfour fourfour four Wes, Wes,Wes, Wes, and and andand Nyquist Nyquist Nyquist Nyquist plots plots plotsplots were were were fitted for both Figures 9 and were fitted for both Figures 9 and 10 concerning the detection of Ab155 with and without MNP by fitted for both Figures 99 and and 10 10 concerning concerning the the detection detection of of Ab155 Ab155 Ab155 with with with and and and without without without MNPMNP MNP by using using by the using equivalent the equivalent (Figure circuit 11A by using the equivalent circuit (Figure 11A inset). Here, Rs indicated electrolyte solution Q the equivalent circuit circuit (Figure 11A (Figure inset). inset). 11A Here, Here, inset). Rs Rs indicated indicated Here, Rs electrolyte electrolyte indicated solution solution electrolyte resistance, resistance, solution Q was was the constant the resistance, resistance, constant Q Q was phase phase was the the constant constantelement elementphase phase that that corresponded corresponded element element that to the to that corresponded correspondedthe charge charge to to the distribution the distribution charge charge at the at distribution distributiontheat interface atinterface the the electrode/electrolyte, electrode/electrolyte, interface RR (which electrode/electrolyte, ct ct R ct (which parallel interface electrode/electrolyte, Rct (which was in parallel with Q) was the resistance of charge and (which was was in in parallel was inwith with Q) Q) parallel was was with the theQ) resistance resistance was the of of charge charge resistance oftransfer, transfer, charge and Biosensors 2020, 10, 43 11 of 13 EIS measurements were repeated at least four times, as we had four Wes, and Nyquist plots were fitted for both Figures 9 and 10 concerning the detection of Ab155 with and without MNP by using the equivalent circuit (Figure 11A inset). Here, Rs indicated electrolyte solution resistance, Q Biosensors was the2020,constant 10, x FORphase PEER element REVIEW that corresponded to the charge distribution at the interface 11 of 13 electrode/electrolyte, Rct (which was in parallel with Q) was the resistance of charge transfer, and finally, W corresponded transfer, and finally, W to corresponded Warburg impedance, which to Warburg is related to impedance, the diffusion which is relatedphenomenon to the diffusionat the electrode–electrolyte interface [10,28]. phenomenon at the electrode–electrolyte interface [10,28]. After Aftercalculating calculating fitting parameters fitting parametersfromfrom the applied equivalent the applied circuit,circuit, equivalent it was clearly it wasevidenced clearly that there wasthat evidenced morethere linearity was for morethelinearity biosensorfor with theAb155 modified biosensor withMNPAb155 (Figure 11B) when modified MNP compared (Figure to 11B) competitive detection without when compared magnetic to competitive nanoparticles detection without (Figure 11A).nanoparticles magnetic This result indicated (Figure that 11A).the sensitivity This resultof the biosensor indicated increased that and provides the sensitivity of thea better understanding biosensor increased ofand the provides Ab155 interaction a better in theunderstanding presence of SA2BSA and SPY at the same time. of the Ab155 interaction in the presence of SA2BSA and SPY at the same time. In In this this study, study, wewe hadhad thethe reverse reverse system system of of normal normal detection detection of of biosensors. biosensors. Here, Here, as as thethe response response of of thethebiosensors biosensors was washigher, higher,the theless lesscontamination contamination therethere was, andand vice viceversa. versa.Indeed, Indeed, when when thethe response response of the biosensor was too weak, this meant that there was an important concentration of of the biosensor was too weak, this meant that there was an important concentration SPYof that SPYsaturated all theall that saturated antibodies Ab155,Ab155, the antibodies such assuch in theas case ofcase in the the sample contaminated of the sample with SPY. contaminated Onwith the other SPY. hand, On theif other the response hand, ifofthe theresponse biosensorofwasthetoo high, this biosensor wasmeant that allthis too high, themeant antibodies were that all collected with the coating antigens SA2BSA, such as in the presence of a the antibodies were collected with the coating antigens SA2BSA, such as in the presence of asample without contamination or sample one thatwithout contained a weak amount contamination or oneof SPY. that contained a weak amount of SPY. Figure Graphical 11. 11. Figure representation Graphical of fixed representation concentration of fixed of SA2BSA concentration andand of SA2BSA Ab155 without Ab155 magnetic without magnetic nanoparticles (A) and with magnetic nanoparticles (B). nanoparticles (A) and with magnetic nanoparticles (B). 4. Conclusions 5. Conclusions In this article, we reported the sensitivity of the synthetized biosensor using the application of In this article, we reported the sensitivity of the synthetized biosensor using the application of competitive assay by immobilized fixed concentration of SA2BSA (100 µg mL−1 ) and fixed concentration competitive assay by immobilized fixed concentration of SA2BSA (100 µg mL−1) and fixed of Ab155 (50 µg mL−1 ) against different concentrations of SPY, such as 40 µM, 4 µM, 2 nM, and finally concentration of Ab155 (50 µg mL−1) against different concentrations of SPY, such as 40 µM, 4 µM, 2 only with PBS, with and without use of magnetic nanoparticles. Using CV and EIS, the microelectrodes nM, and finally only with PBS, with and without use of magnetic nanoparticles. Using CV and EIS, were characterized for the bare gold, surface-functionalized CMA, immobilized SA2BSA, and fixed the microelectrodes were characterized for the bare gold, surface-functionalized CMA, immobilized concentration of MNP containing immobilized Ab155 with different concentrations of SPY. SA2BSA, and fixed concentration of MNP containing immobilized Ab155 with different The Nyquist plots showed a stepwise increase of Rct in both cases (Ab155 with and without concentrations of SPY. magnetic nanoparticles that contain immobilized Ab155 with different concentrations of SPY). However, The Nyquist plots showed a stepwise increase of Rct in both cases (Ab155 with and without after calculations from fitting parameters and drawing graphs, it could be clearly observed that there magnetic nanoparticles that contain immobilized Ab155 with different concentrations of SPY). was more linearity while using magnetic nanoparticles compared to that without their use. This However, after calculations from fitting parameters and drawing graphs, it could be clearly observed indicated the increase in sensitivity of the biosensor as compared to competitive assay without that there was more linearity while using magnetic nanoparticles compared to that without their use. magnetic nanoparticles. This indicated the increase in sensitivity of the biosensor as compared to competitive assay without magnetic Author nanoparticles. Contributions: T.J. designed, planned, performed and analyzed the major experiments. E.T.T.-N. and A.B., A.S., J.-P.S., J.B. and M.-P.M. Marco were in charge of characterization and provision of the immunoreagents Author Contributions: T. Jamshaid designed, planned, performed and analyzed the major experiments. E. T. Tenório-Neto and A. Baraket, A. Sanchís, J. P. Salvador, J. Bausells, and M. P. Marco were in charge of characterization and provision of the immunoreagents for SA detection. N. Lebaz. and A. Elaissari revised and assisted in editing the manuscript. A. Errachid and N. Zine are project supervisors and overview the research progress. All authors have read and agreed to the published version of the manuscript. Funding: The authors acknowledge the financial support from the projects European Communities Seventh Biosensors 2020, 10, 43 12 of 13 for SA detection. N.L. and A.E. (Abdelhamid Elaissari) revised and assisted in editing the manuscript. A.E. (Abdelhamid Errachid) and N.Z. are project supervisors and overview the research progress. All authors have read and agreed to the published version of the manuscript. Funding: The authors acknowledge the financial support from the projects European Communities Seventh Framework Program: SEA-on-a-CHIP (FP7-OCEAN-2013) under the grant agreement N◦ 614168, and funding from the European Union’s Horizon 2020 research and innovation program entitled KardiaTool under grant agreement N◦ 643694, and the fellowship from Science Without Borders program—Conselho Nacional de Desenvolvimento Ciêntífico Tenológico (CNPq)–Brazil grant N◦ 249056/2013-5. Conflicts of Interest: The authors declare no conflict of interest. References 1. Rosi-Marshall, E.J.; Kelly, J.J. Antibiotic Stewardship Should Consider Environmental Fate of Antibiotics. Environ. Sci. Technol. 2015, 49, 5257–5258. [CrossRef] [PubMed] 2. Ge, L.; Zhang, P.; Halsall, C.; Li, Y.; Chen, C.-E.; Li, J.; Sun, H.; Yao, Z. 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