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A “shoot and sense” Janus micromotors-based strategy for the simultaneous degradation and detection of persistent organic pollutants in food and biological samples Daniel Rojas, Beatriz Jurado-Sanchez, and Alberto Escarpa Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00574 • Publication Date (Web): 03 Mar 2016 Downloaded from http://pubs.acs.org on March 8, 2016

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Analytical Chemistry

A “shoot and sense” Janus micromotors-based strategy for the simultaneous degradation and detection of persistent organic pollutants in food and biological samples D. Rojas, B. Jurado-Sánchez* and A. Escarpa* Department of Analytical Chemistry, Physical Chemistry and Chemical Engineering, University of Alcala, Madrid, Spain ABSTRACT: A novel Janus micromotor-based strategy for the direct determination of diphenyl phthalate (DPP) in food and biological samples is presented. Mg/Au Janus micromotors are employed as novel analytical platforms for the degradation of the non-electroactive DPP into phenol, which is directly measured by difference pulse voltammetry on disposable screen-printed electrodes. The self-movement of the micromotors along the samples result in the generation of hydrogen microbubbles and hydroxyl ions for DPP degradation. The increased fluid transport improves dramatically the analytical signal, increasing the sensitivity while lowering the detection potential. The method has been successfully applied to the direct analysis of DPP in selected food and biological samples, without any sample treatment and avoiding any potential contamination from laboratory equipment. The developed approach is fast (~5 min) and accurate with recoveries of ~100%. In addition, efficient propulsion of multiple Mg/Au micromotors in complex samples has also been demonstrated. The advantages of the micromotors assisted technology i.e., disposability, portability and the possibility to carry out multiple analysis simultaneously hold considerable promise for its application in food and biological control in analytical applications with high significance.

Persistent organic pollutants are toxic chemicals that can accumulate to hazards levels in living organism and in the food chain.1 Phthalate esters (PAEs), used as plasticizers in many food packaging materials, are considered as a new class of “indirect food” additives.2-5 The carcinogenic potential of PAEs is a controversial issue, however, it has been clearly demonstrated that they are potent endocrine disruptors. As such, PAEs act as natural hormones mimics, interfering thus with hormone receptors and inducing inappropriate biological responses. In particular, di-n-octyl-ortho phthalate and diphenyl phthalate have been shown to exhibit strong inhibition towards drug-metabolizing enzymes (glucuronosyltransferases).6,7 The US Environmental Protection Agency have included eight phthalates in the “Toxic substances control act” and the FDA have limited its use as plasticizers in some pharmaceuticals products.8,9 The European Food Safety Authority have established tolerable daily intakes of some PAEs in foods and the European Union set specific migration levels for several PAEs in food contact materials of up to 60 mg/kg.10,11 China Ministry of Health limited the use of 17 PAEs as food additives and as food contacts materials.12 Current progress in the field of artificial micromotors13-19 hold considerable promise for novel “real-time” analytical measurements of phthalates in different samples, avoiding existing drawbacks of current methodologies. The selfpropulsion capability of bubble-propelled micromotors induces efficient fluid transport-mixing, enhancing the yield of classical chemical processes while obviating the need for tedious sample preparation procedures.20 For example, the movement of receptor-functionalized micromotors around complex samples improve analyte-receptor interactions for the

direct isolation of biological targets (nucleic acids, circulating tumor cell, bacteria) without preparatory or washing steps.21 Micromotors also show considerable potential in environmental remediation, in connection with pollutant degradation, owing to motion-induced mixing. In this context, chemical environment exerts strong influence on the micromotors motion, allowing for their use as sensors and actuators to chemical leakage.22,23 As such, new motion-based sensing approaches, based on “analyte-induced” changes in the motor movement, have been developed for the determination of heavy metals24 and DNA.25 Microengines incorporating fluorescent quantum dots in its surface26 or Janus micromotors loaded with fluorescent reagents27 have been used as novel mobile sensors for the selective determination of mercury and nerve agents, respectively. Motion accelerated binding of trace target analytes quenches the fluorescent emission of the modified micromotors, offering thus real-time optical visualization of analyte-recognition events. Similarly, Marangoni-driven motors offer electrochemical/optical detection of hydrogen peroxide in raw clinical samples.28 Polyaniline/Pt micromotors can assist the transport of target molecules (protein biomarkers) within the sample solution toward an immobilized receptor (antibodies microarrays) for enhanced surface biosensing.29 Magnetic Janus-Mg microengines has been confined onto the surface of sensor strips to serve as ‘artificial’ enzymes toward the alkaline hydrolysis of paraoxon into readily detectable pnitrophenol.30 Food intake is the common exposure pathway for PAEs in humans.5 It is thus highly desirable the development of fast and novel methodologies for routine analysis of such compounds in foodstuffs and biological samples. Most

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methods reported in the literature, however, are mainly concerned with migration studies of PAEs in simulants rather than in food analysis. The complexity of food samples, as compared with simulants, demands for time-consuming sample extraction procedures, mainly solvent extraction,31,32 solid-phase extraction and solid-phase microextraction.33 Next, complex instrumental techniques such as gas or liquid chromatography with mass spectrometric detection are used for the determination of PAEs in the resulting extracts.33 Due to the ubiquitous presence of PAEs in laboratory equipment and glassware, the above mentioned methods are prone to sample contamination, leading to “false-positive” results. In summary, existing limitations in current methodologies for PAEs analysis, i.e., requirement for expensive equipment, trained personnel and high background contamination contact prevent its application for routine analysis. Analytical microfluidics, nano science and nano technologies are of paramount significance in food analysis.34 Indeed, the possibility to carry out laboratory operations on a small scale using miniaturized devices in very appealing since the amount of reagents, samples and chemical waste can be dramatically reduced. More important is that at micro-and nanoscale levels, greater control of molecular interactions is achieved. These valuable features constitute a great opportunity to revolutionize “in-situ” food analysis.34 Some progress have been made towards the challenge of developing “in-situ” analytical techniques for PAEs analysis. Various sensors based on molecularly-imprinted polymers (MIPs) have been applied for the direct analysis of such compounds in food samples. For example, MIP/nano-Fe3O4/silica nanoparticles has been used in combination with a carbon electrode for the determination of dibutyl phthalate in milk samples.35 Similarly, graphene oxide@gold-molecular imprinted polymer particles have been explored as electrochemical sensor material for dibutyl phthalate in wines.36 Herein we describe a new micromotor-based strategy for the direct determination of diphenyl phthalate (DPP) in a wide variety of food and biological samples, without any sample treatment. Mg/Au Janus micromotors are employed as novel disposable analytical platforms for the degradation of the nonelectroactive DPP into phenol, which can be directly measured by differential pulse voltammetry (DPV). Mg microspheres, coated with a thin gold layer, efficiently propel within the samples using NaCl (which also act as supporting electrolyte) as fuel. Upon contact with the chloride-enriched samples, Mg surface is readily oxidized (by combination of galvanic and pitting corrosion processes), resulting in the generation of hydrogen microbubbles and hydroxyl ions for DPP degradation into phenol. We will demonstrate in the following section the critical role of the bubbles generated and micromotors’s movement upon the improvement of the analytical signal, increasing the sensitivity while lowering the detection potential. Such increased fluid transport imparted by the micromotors allow for the fast, direct determination of DPP in viscous samples. Samples are directly dropped into the navigating Janus Mg/Au electrode solution, avoiding thus any potential contamination from laboratory equipment. We will demonstrate also the applicability of the developed platform in the determination of DPP in water, milk, whiskey and raw human serum samples with fast analysis time, good reproducibility and excellent recoveries.

MATERIALS AND METHODS

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Chemicals. Phenol (cat. P1037-256), diphenyl phthalate (cat. 105880) and sodium chloride (cat. 793566) were purchased from Sigma-Aldrich. DPP stock solution (10 mM) was prepared daily in methanol (Sigma-Aldrich, cat. 34860). Working solutions were made by simply diluting the stock solution at the desired concentration with the sample. The micromotors were fabricated using magnesium microparticles (catalog #FMW20, TangShan WeiHao Magnesium Powder Co.; 20 ± 5 µm) as the base particles. Magnesium particles were cleaned in absolute ethanol (Sigma-Aldrich, cat. 459844) and subsequently filtered through a 10 µm filter membrane. The particles were then spread onto glass slides and coated with a 20 nm Au layer using a desktop sputter coater system (Denton Desk V). The deposition was performed at room temperature with a DC power of 25 W for 120 s. In order to get a uniform gold coating over the surface of the magnesium, rotation speed was turned off. The microengines were removed from the glass slide and dispersed in ethanol, at a concentration of 200 mg motors/mL. To further characterize the motor, scanning electron microscopy and energydispersive X-ray mapping analysis images were taken using a JEOL JSM 6335F instrument. Screen-printed carbon electrodes (DS-110) were purchased from DropSens (Spain). The strips compromise an alumina substrate using heat curing carbon composite inks, a carbon working electrode and a silver pseudo-reference electrode. An insulating layer serves to delimit the working area and electric contacts. Samples. Human plasma was kindly donated by Dr. Goya Clinical Laboratory (Alcalá de Henares). Milk and whiskey samples were purchased at local supermarkets. Tap water samples were obtained from the laboratory at the University of Alcalá. The autonomous motion of the micromotors in the different media was observed and recorded using an inverted optical microscope (Nikon Eclipse Instrument Inc. Ti-S/L100), coupled 20X objective and Zyla sCMOS camera controlled by NIS Elements AR 3.2 software. The speed of the micromotors was tracked using a NIS Elements tracking module. In all experiments, 0.1% Triton X-100 (Fisher Scientific, Fair Lawn, NJ) was used as surfactant. Commercial polystyrene particles of 3 µm at 5.68 X 109 particles/mL (ca. 19814, Polysciences, USA) were used as tracers. A fresh stock solution was prepared daily by dissolving 1 µL of commercial solution in 99 µL of D.I water. Electrochemical detection of diphenyl phthalate using Mg Janus micromotors. 4 µL of the Mg Janus microengines dispersion were added onto the working area of the electrode. Then, a 50 µL drop of the different samples (milk, water, whiskey or human plasma) containing 0.1 M NaCl and 0.1% Triton X-100 were added. Calibration curves were obtained using fortified samples analysed under identical conditions. DPV measurements were performed after 5 min reaction, allowing the degradation of diphenyl phthalate by the moving microengines. DPV was performed using an Autolab PGSTAT 12 (Eco Chemie, The Netherlands). The potential window scanned was 0 to 1,2 V vs Ag/AgCl at 5 mV/s. Unless otherwise stated, the measurement parameters were as follows: step potential 10 mV, modulation amplitude 50 mV, pulse width (modulation time) 5 ms, pulse period (interval) 100 ms. The peak height was measured after introduction of a baseline to take account of the shift in background current. Mg micromotor-enhanced transport of passive microparticles. The enhanced fluid motion in the presence of Janus micromotors and its role in the improvement of the


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analytical performance is characterized by analysis of the mean squared displacement of passive microsphere tracers. For experiments with moving micromotors, 1 µL of each particles solutions, 0.1 % Triton X-100, 0.01 M NaCl (as fuel) prepared either in water or whiskey and Mg/Au Janus micromotors (initial concentration, 50 mg motors/mL) were placed onto a glass slide. To investigate particle motion, we captured movie clips of the particles (7 s, 16 frames per sec). Following particle tracking and analysis of the video clips with NIS element software, the mean square displacement () for each particle was calculated as a function of time.

RESULTS AND DISCUSSION Figure 1A shows a schematic representation of the micromotor-assisted detection platform, where Mg/Au selfpropelled Janus micromotors are used in combination with screen-printed electrodes for the simultaneous degradation/detection of phtalates in food and biological samples. The concept was proved here using diphenyl phthalate (DPP) as model compound. At basic pH (~11) the non-electroactive DPP readily degrade into phenol which can be detected by differential pulse voltammetry (DPV). This principle has from the basis for the detection mechanism reported here. Thus, under the presence of NaCl (supporting electrolyte) Mg microengines generate hydrogen bubbles and OH‾ ions, increasing both the pH for DPP degradation and inducing mixing to assist mass transport and improve the sensing performance of the detection system (see Figure 1B). No electrochemical signal/response is obtained in similar control experiments performed on bare electrodes, which reveal the crucial role of the micromotors for the detection of DPP. Micromotors were first prepared by coating Mg microparticles (average diameter 20 µm) with an Au layer by sputter deposition. Such gold coating is essential for the efficient hydrogen production since the surface of the Mg microparticles is readily passivated by the formation of a Mg(OH)2 layer.

Figure 1. (A) Schematic of the Mg/Au Janus micromotorsbased strategy for the simultaneous degradation/detection of DPP. The generation of hydroxyl ions during the Janus micromotors movement increased the pH (~11) for the degradation of the non-electroactive DPP into electroactive phenol, which can be detected by differential pulse voltammetry. (B) Reactions involved in the degradation of DPP by Mg/Au Janus micromotors.

Figure 2. (A) Scanning-electron microscopy and energy dispersive X-ray analysis images showing the Janus structure and the distribution of Mg (white) and Au (red) on the Janus micromotors. (B) SEM images of several Mg-Au Janus micromotors on a screen-printed electrode. The Mg(OH)2 passivation layer can be easily dissolved by solutions containing chloride ions, which under the presence of the gold layer allow the reaction to proceed by a combination of galvanic and pitting corrosion effects.35 Subsequently, the spontaneous redox oxidation between Mg and water is dramatically promoted to generate directional hydrogen bubble propulsion (for enhanced fluid mixing) increasing at the same time the pH for DPP degradation into phenol. The scanning electron microscopy (SEM) and energydispersive X-ray spectroscopy (EDX) images of Figure 2A show the morphology of the resulting micromotors, with an Au layer covering most part, with a 5 µm of the Mg core exposed to facilitate directional H2 bubble thrust. To achieve such motor design, sample slides were placed directly in front of the target, which lead to a uniform Au coating while still leaving an opening at the contact point of the sphere to the glass slide. As will be described later, such configuration avoid the vigorous production of H2 bubbles allowing thus for the use of the microengines in the working electrode area without suppressing the electrochemical signal of interest. The presence of the Au coating is also confirmed by the corresponding EDX image mapping of Au and Mg displayed in the right part of the figure. Figure 2B also shows SEM images of several Mg-Au Janus micromotors over the working area of the electrode. The localized OH‾ ions generation and fluid mixing related to the movement of the Mg/Au autonomous micromotors enhance dramatically the analytical performance (towards enhancing the sensitivity while lowering the oxidation/detection potential) for DPP detection. The effect of such dual action of the micromotor is summarized in Figure 3. First, the pH induced increase not only promoted the degradation of DPP into readily detectable phenol, but also induce a shift in the oxidation/detection peak to lower potentials for higher selectivity. Thus, Figure 3A shows the DPV voltammograms for the detection of 0.1 mM of phenol under the presence (a) and absence (c) of Mg/Au microengines.



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Figure 3. Micromotor enhanced-mixing effect on the improvement of the analytical signal. (A) DPV detection of 0.1 mM of phenol with (black line, a) or without (red line, c) Mg/Au Janus micromotors in 0.1 M NaCl. For comparison, blue line (b) shows the analytical signal of phenol alone (without micromotors) at pH 12. (B) DPV scans obtained for 1 mM of DPP (blue line, a) after 5 min treatment with Mg/Au Janus micromotors or after 30 min treatment with 0.01 M NaOH under static conditions (black line, b). The potential window scanned was 0 to +1.2 V vs Ag/AgCl at 5 mV/s. (C) Mean-squared displacement obtained by averaging over 10 passive tracers (3 µm), whose trajectories are subject to the effect of active Mg micromotors swimming for 7s in water (a) and milk (b) samples, with respect to that governed only by brownian motion (c) upon time. Inset show time-lapse images (taken from video S1) illustrating typical trajectories of particle tracers undergoing Brownian motion (left) and a mixture of Brownian motion and convection (right) using Mg micromotors in the absence and presence of NaCl, respectively. Scale bars, 3 µm.

As can be seen, a shift in the detection potential from +0.70 to +0.40 V is produced. Such pH-dependent electrochemical behavior of phenol can be attributed to its particular oxidation mechanism at carbon electrodes. Thus, under the conditions used in this work, phenol is oxidized to phenoxy radical via the release of one proton and one electron. Two phenol oxidation intermediate products (o-quinone and o-phenol) are generated, due to the delocalization of the electron in the aromatic ring. As can be seen in scheme 1, the reaction is pH dependent: an increase in the pH facilitate the abstraction of H+ from phenol, which bring down thus the overpotential for the oxidation reaction.38 Scheme 1. Electrochemical oxidation of phenol


concentrations up to 0.01 M NaCl, with a subsequent sharp decrease due to the interference of hydrogen bubbles. For further experiments, 4 µL of Mg/Au micromotors (0.8 mg) and 0.01 M NaCl as fuel were chosen as optimal.

20

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To corroborate the above mentioned hypothesis, Figure 3A (blue line, b) shows the DPV voltammogram obtained for 0.1 mM of phenol on bare carbon SPE at pH 12 (similar to that produced by the Mg/Au micromotors). The detection potential is similar to that obtained with the micromotors. Although a similar peak potential is observed, however, a great decrease in the intensity can be noted, which testifies the role of the mixing effect of the Mg/Au micromotors in the improvement of the analytical signal. Figure 3B also display the critical role of such mixing for DPP degradation. A well-defined, intense peak is obtained after 5 min treatment of 1 mM DPP solution with Mg/Au micromotors (a), whereas negligible peak is observed after 30 min treatment of 1 mM of DPP with 0.01 M NaOH (pH=12) under static conditions (b). In order to check the nature of the micromotors’s mixing effect, the displacement of passive microparticles tracers in NaCl-rich solutions were studied in the presence and absence of Mg/Au Janus micromotors, mimicking the experimental conditions held on the electrodes. The enhanced fluid motion in the presence of Mg/Au Janus micromotors is characterized by the analysis of the mean squared displacement (MSD) of the “passive microtracers”. Experiments were performed by placing equal volumes of solutions containing passive microparticles, surfactant, Mg/Au micromotors and 0.01 M NaCl on a glass slide. As depicted in Figure 3C, both the movement of the micromotors and the hydrogen bubbles generated flow field that caused the displacement of the passive tracers. The microscopy images of the inset in Figure 3C and the corresponding SI Video 1 demonstrate the significant movement of two microparticles due to the bubbles generated by Mg/Au Janus micromotors; as compared with the negligible displacement of particle undergoing Brownian motion (in the absence of micromotors). The transport is quantified in terms of the MSD after a fixed time interval ∆t, as previously described in the literature.20 The plot of versus time obtained for bead microtracers (in water) in the presence (a) and absence (c) of Mg/Au micromotors of Figure 3C indicate a dramatically larger MSD of the tracers as compared with pure Brownian motion (c). In order to check the effect of the viscosity of the medium on the mixing event, similar experiments were performed in milk (b). MSD values were similar to that obtained in water, testifying thus the applicability of the method for the direct analysis of complex samples. On overall, this data demonstrate the critical role of Mg/Au micromotors to induce greatly enhanced mass transport for improving the degradation yield of DPP into readily detectable phenol. We optimize the effect of the amount of micromotors and NaCl concentration upon the signal-to-noise-ratio (S/N). As displayed in the plot of Figure 4A, the amount of micromotors exert a strong influence on the pH of the solution, which is directly related to the degradation of DPP to phenol. Thus, solution pH increase from 9 to 12 as the volume (amount) of Mg/Au micromotors increase from 1 to 4 µL, respectively. Such pH increase accelerate the rate of DPP degradation, with the corresponding increase in the S/N ratio. At higher micromotors volumes, although optimal pH for DPP degradation is also achieved, the increased amount of micromotors results in the vigorous production of hydrogen bubbles which in turn suppress the electrochemical signal of phenol, with poor S/N ratios. A similar effect is observed when using different concentrations of NaCl as fuel (Figure 4B), with a dramatic improvement in the S/N ratio at

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[NaCl] (M) Figure 4. (A) Influence of the amount of micromotors on the pH (a) and on the signal-to-noise ratio (b, S/N) (B) Influence of NaCl concentration on the S/N.

Figure 5 A, B and C illustrate the well-defined DPV responses for different DPP concentrations in the different media studied. Calibration plots were constructed by plotting the peak current against DPP concentration (see the bottom part of Figure 5). In all cases, linearity over the range 0.12‒1 mM (water), 0.12‒1 mM (milk) and 0.50‒2 mM (whiskey) was observed, with correlation coefficients higher than 0.990. The limits of detection and quantification, calculated as S/N=3 and S/N=10 criteria, respectively were 0.039 and 0.13 mM (water); 0.040 and 0.13 mM (milk) or 0.15 and 0.50 mM (whiskey). The precision (as relative standard deviation, RSD) was evaluated by analyzing 12 individual water, milk or whiskey samples fortified with 0.5 mM of DPP and was found to be satisfactory with RSD of 6 (water), 8 (milk) and 9% (whiskey). Also, good recoveries of 99 ± 7, 97 ± 8 and 105 ± 12 %, (n=3, 0.50 mM DPP) for water, milk and samples were obtained; revealing the excellent accuracy of the method for the determination of DPP in the different samples assayed.


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S/N

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Figure 5. Analytical performance of DPP determination in real samples using Mg/Au Janus micromotors. DPVs corresponding to different concentrations of DPP in (A) water: 0.25 (black, a), 0.5 (red, b) and 1 mM (blue, c), (B) Milk: 0.125 (black, a), 0.25 (red, b) and 1 mM (blue, c) and (C) whiskey: 1 (blue, a) and 2 mM (red, b). Voltammograms were recorded after 5 min addition of the sample into the electrode. Bottom part shows the corresponding calibration plots obtained for DPP under the same conditions in each sample. Other conditions, see Figure 2. Measurements were performed in triplicate at each concentration.

Figure 6. Real sample analysis. DPV voltammograms obtained for milk (A) and whiskey (B) samples fortified with 1mM or 2.5 mM of DPP, respectively and (C) Human plasma fortified with 0.25 or 0.50 mM of DPP. Voltammograms were recorded after 5 min addition of the sample into the electrode. Green lines shows the responses obtained for each sample prior fortification. Other conditions, see Figure 2. Measurements were performed in triplicate at each concentration. (D) Time lapse images (taken from videos S2 and S3) showing the efficient motor movement in milk (a), water (b) whiskey (c) and (d) human serum. Top part shows tracking line images illustrating the motor’s propulsion over 1 s period in the different samples. Scale bars, 20 µm.


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In order to evaluate the suitability of the Janus micromotorbased strategy in the analysis of real samples, selected food samples with different viscosity (water, whisky and milk) were assayed. In order to expand the applicability of our method to biological samples, human serum samples were also analyzed as representative matrix. Indeed, humans are exposed to phthalates by multiple routes. In general population, exposure can be oral through contaminated food. Main exposure of workers, however, is inhalation and dermal. As such, phthalates and its metabolites have been measured in many body fluids including urine and serum.39 Figure 6 shows the voltammograms (selected) obtained for milk (A) and whiskey (B) samples before and after fortification with 1 mM or 2.5 mM of DPP, respectively or human plasma (C) fortified with 0.25 and 0.50 mM of DPP. Well-defined responses were observed in all cases. Interestingly as demonstrated in the time-lapse microscopy images of Figure 6D and corresponding SI Videos 2 and 3, Mg/Au micromotors can efficiently operate in food and biological samples. A long tail of bubbles and a directional, prolonged movement is observed, with average speed of 108 ± 18, 296 ± 40, 223 ± 38 and 40 ± 8 µm/sec in milk, water, whiskey and serum samples, respectively. Considering the viscosity of the different media evaluated (µ) and based on the drag force equation F=6πµrv (were r is the radius of the micromotor and v the speed), similar driving forces of over 150-180 pN are observed in all samples. In addition, the corresponding tracking line images at the top part of Figure 6D shows the efficient motor’s propulsion over 1 s period in the different samples. Directional, straight trajectories over the different samples are observed. This fact further support the practical operation of the motor in real media for highly efficient determination of DPP.

CONCLUSIONS In conclusion, we have developed a new micromotor-based platform for the direct determination of persistent organic pollutants in raw food and biological samples without any sample treatment. We have demonstrated how the mixing induced by the motion of Mg/Au Janus micromotors along with the hydroxyl ions generated makes possible the rapid conversion of non-electroactive DPP into electroactive phenol. Thus, both micromotor movement and hydrogen bubble generation induce enhanced mass transport (for improved sensitivity) and increase the pH of the solution lowering the detection potential (for improved selectivity). The critical role of the motor movement on the improvement of the analytical signal (towards lowering the oxidation potential) and enhancement of sensitivity has been characterized and modelled. The method has been successfully applied to the analysis of direct, viscous food and biological samples, under 5 min analysis time, avoiding any sample treatment and the existing drawbacks associated with labware contamination of common methods for phthalates determination. In addition, efficient propulsion of multiple Mg/Au micromotors in the complex samples (as exemplified in SI Video 4) lead to an increase in the sensitivity of DPP detection, up to ~20 fold, as compared to experiments conducted in static conditions. Thus, without any sample treatment, an excellent precision (RSDs< 8 %) and quantitative recoveries (~100%) were obtained. To the best of our knowledge, this is the first time that micromotors are used for food-control related applications. The advantages of the micromotors assisted technology such

as disposability, portability and the possibility to carry out multiple analysis simultaneously make it particularly promising for its application in food and biological control of a plethora of non-electroactive analytes. The only limitation of the methods lies in the relative short lifetime of the motor, which dissolve after 5 min movement in the sample. This fact limits thus the possibility to increase further the sensitivity of DPP detection through the generation of more electroactive phenol (i.e. higher degradation yield). In addition, the concept can be extended for the detection of trace environmental pollutants such as pesticides or chemical warfare agents. For example, carbofuran, a nearly no electroactive compound, can be degraded at alkaline pH (following a similar principle to that used in our Mg micromotor Janus strategy) to electroactive carbofuran phenol, increasing thus the sensitivity in the detection for environmental monitoring applications. Similarly, m-parathion, a deadly toxic pesticide, can be degraded into readily detectable p-nitrophenol. Finally, it can be also used in alkaline-assisted electrocatalytic oxidation reactions for the enhanced detection or carbohydrates and dopamine, in combination with different electrodes. Such combination hold considerable promise in medical diagnosis, food industries, industrial wastewater treatment, etc. No question about the potency of micromotor-based strategies in contemporary analytical scene applications.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. SI Video 1 (wmv) SI Video 2 (wmv) SI Video 3 (wmv) SI Video 4 (wmv)

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]; [email protected]

Notes The authors declare no competing financial interest. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT B. J-S acknowledges support from the People Programme (Marie Curie Actions) of the EU 7th Framework Programme (FP7 20072013) under REA Grant PIOF-GA-2012-326476. AE acknowledges financial support from the Spanish Ministry of Economy and Competitiveness (CTQ2014-58643-R) and the NANOAVANSENS program (S2013/MIT-3029) from the Community of Madrid.

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