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Highly monodisperse Fe3O4@Au superparamagnetic nanoparticles as reproducible platform for genosensing genetically modified organisms Maria Cristina Castro Freitas, Maria Manuela Sá Couto, Maria Fátima Barroso, Clara Pereira, Noemí delos-Santos-Álvarez, Arturo J. Miranda-Ordieres, María Jesús Lobo-Castañón, and Cristina Delerue-Matos ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.6b00182 • Publication Date (Web): 26 Jul 2016 Downloaded from http://pubs.acs.org on July 27, 2016

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Highly monodisperse Fe3O4@Au superparamagnetic nanoparticles as reproducible platform for genosensing genetically modified organisms

Maria Freitas1,2, Maria Sá Couto3, Maria Fátima Barroso1,*, Clara Pereira3, Noemí de-los-Santos-Álvarez2, Arturo J. Miranda-Ordieres2, María Jesús Lobo-Castañón2, Cristina Delerue-Matos1 1

REQUIMTE/LAQV, Instituto Superior de Engenharia do Instituto Politécnico do Porto, Rua Dr. António

Bernardino de Almeida, 4200-072 Porto, Portugal 2

Departamento de Química Física y Analítica, Universidad de Oviedo, Av. Julián Clavería 8, 33006 Oviedo,

Spain 3

REQUIMTE/LAQV, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto,

4169-007 Porto, Portugal

Maria Freitas: [email protected] Maria Sá Couto: [email protected] Maria Fátima Barroso*: [email protected] (*corresponding autor; Phone: 00 351 228 340 537) Clara Pereira: [email protected] Noemí-de-los-Santos-Álvarez: [email protected] Arturo J. Miranda-Ordieres: [email protected] Maria Jesús Lobo-Castañón: [email protected] Cristina Delerue-Matos: [email protected]

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ABSTRACT Several routes have been developed to prepare magnetic core-shell Fe3O4@Au nanoparticles (MNPs). However, only highly monodisperse MNPs are suitable for analytical applications. Herein, we describe the detection of GMO through the use of gold-coated MNPs with fine-tuned properties as platforms. The MNPs were prepared through a procedure that involves the preparation of Fe3O4 cores by thermal decomposition and their coating through reduction of a gold precursor. Different Fe3O4:Au precursor molar ratios (1:1; 1:4; 1:7) were tested on the Fe3O4 encapsulation. Monodisperse quasi-spherical core-shell Fe3O4@Au were obtained for the 1:4 and 1:7 ratios, in contrast, the 1:1 ratio did not lead to complete encapsulation of Fe3O4 cores. Therefore, the Fe3O4@Au obtained from higher Fe3O4:HAuCl4 ratios were tested as platforms for an electrochemical genoassay to detect MON810. The best performance was achieved with the Fe3O4@Au prepared from 1:4 ratio (10.0±1.7 nm). A DNA probe covalently linked to a carboxylated self-assembled monolayer and a fluorescein isothiocyanate (FITC) signaling probe were used in a sandwich assay format. Labeling with anti-FITC-peroxidase Fab fragment conjugate allowed chronoamperometric measurements of the enzyme activity captured on Fe3O4@Au placed on screen-printed electrodes upon hybridization event. The genoassay provided a linear range from 0.25-2.5 nM, LOD of 0.15 nM, with a reproducibility < 4%. Certified samples containing the transgenic event were measured without further purification after PCR amplification. The results highlight the efficiency of the genoassay for the MON810 detection, opening new horizons to achieve a low-cost, out of large laboratory facilities analysis to verify the compliance of GMO regulations. Key words: Core-shell Fe3O4@Au magnetic nanoparticles, Genoassay, MON810 maize, Genetically modified organisms, Chronoamperometry, Screen-printed electrodes


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Technological advancements in the area of materials science and nanotechnology have made possible the synthesis of nanoparticles with desirable properties not exhibited by the bulk counterparts.1 In all perspectives, nanoparticles with magnetic properties are very attractive due to their easy manipulation with an external magnetic field and large surface area to immobilize as many biomolecules as possible. Superparamagnetic nanoparticles, typically with diameter lower than 20 nm, are the ideal magnetic materials. They rapidly respond to an external magnetic field and can be easily redispersed upon magnet removal due to the absence of remanent magnetization and coercivity. These features make them less prone to aggregation than their ferromagnetic counterparts, so they have been extensively used as contrast agents in Magnetic Resonance Imaging (MRI) and, more recently, for hyperthermia treatment of cancer.2 However, uncoated nanoparticles are inherently unstable over prolonged periods of time due to their large surface area to volume ratio and low surface charge at physiological pH, so they tend to aggregate.3 Iron oxide nanoparticles, the most widely used superparamagnetic nanoparticles, namely magnetite (Fe3O4), are also easily oxidized in air or water and/or can suffer degradation phenomena when exposed to harsh environments. To overcome these drawbacks, iron oxide nanoparticles are often coated with protective organic or inorganic layers such as polymers and surfactant stabilizers, precious metals or silica, among others.4 This coating step is unavoidable for most applications and frequently advantageous because it provides the mean to functionalize the nanoparticles, paving the way to innovative designs for a myriad of applications such as magnetically-guided drug delivery, magnetically-modulated controlled drug release through heating, catalysis and biotechnology.2,4,5 Core-shell iron oxide magnetic nanoparticles can thus show enhanced properties and functionalities that promote biocompatibility. In complex matrices such as those often faced in bioanalytical applications, a separation of certain biological components from their native environment is advantageous or even essential, and magnetic separation is one of the easiest ways of performing that step in order to concentrate the component of interest or avoid fouling of the transducer surface. Commercial availability of magnetic microparticles (beads) with different functionalities has fueled the development of a myriad of bioassays.6 Comparatively, the use of nanometer size core-shell magnetic nanoparticles is more recent and much less explored.7,8 This nanomaterial can be integrated in electrochemical devices to benefit from the synergistic effect of building a biocompatible high-binding capacity nanostructure electrode surface.9 3 ACS Paragon Plus Environment

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Core-shell gold-coated iron oxide magnetic nanoparticles (FexOy@Au MNPs) are emerging as suitable platforms for such devices due to the easy functionalization of the gold shell with thiolated molecules, which facilitates bioconjugation reactions through well-established chemistry, as well as to their long-term stability, biocompatibility and optical properties.10 However, the deposition of Au on the surface of iron oxide nanoparticles is not straightforward due to the dissimilarity of both surfaces.5,11 Nevertheless, in the last decades significant progress has been achieved in the synthesis of core-shell FexOy@Au MNPs namely by reverse microemulsion method,12,13 iterative hydroxylamine seeding11 and wet chemical reduction.14,15 In particular, the coating of Fe3O4 MNPs prepared by thermal decomposition through the reduction of a Au(III) salt on their surface under mild conditions has been reported as a promising strategy to produce monodisperse core-shell Fe3O4@Au MNPs with tunable shell thickness and plasmonic properties.14 The latest advances in synthetic routes have encouraged the recent use of Fe3O4@Au or γ-Fe2O3@Au MNPs for direct electrochemistry,16 immuno-17 and aptamer-based assays18,19 and genoassays.20,21 On the other hand, Au-Fe3O4 nanocomposites are a distinct class of materials that have also been used in bioassays.22-24 The high surface area of these magnetic nanoparticles along with the gold coating allow the easy immobilization of most biological molecules through several binding strategies, enabling to achieve the optimum loading to maximize the sensitivity of the bioassay.15,16 In this work, an electrochemical genoassay using core-shell MNPs as platform is presented. The Fe3O4@Au MNPs were synthesized by a sequential strategy involving the formation of iron oxide cores and subsequent coating with a gold shell through reduction of HAuCl4 on the surface of the MNPs in the presence of an organic capping agent. Three different Fe3O4:HAuCl4 molar ratios were tested in the coating procedure, 1:1, 1:4 and 1:7. Several techniques were used to characterize the uncoated and gold-coated MNPs, namely transmission electron microscopy (TEM), UV-VIS spectroscopy, X-ray diffraction (XRD) and SQUID magnetometry. By ligand-exchange, the Fe3O4@Au nanoparticles surface was covered with a mixture of thiolated compounds containing carboxyl groups for further bioconjugation of a DNA probe. A DNA sandwich assay was performed on this solid support to recognize a specific fragment of the transgenic construct from MON810 maize, an EU-authorized event that is frequently found in food and feed. The electrochemical detection was carried out after enzymatic labeling of the duplex formed on the surface and deposition of the modified MNPs on a screen4 ACS Paragon Plus Environment

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printed carbon electrode (SPCE). We show that this synthetic route leads to highly monodisperse and reproducible core-shell MNPs easily converted into a water-soluble platform. These high-quality features are a key element to obtain reproducible analytical results as it is shown for the first time for the sensitive electrochemical detection of specific sequences of DNA. The genoassay was successfully applied to the detection of real samples after PCR amplification.

EXPERIMENTAL SECTION Reagents. Iron(III) acetylacetonate ([Fe(acac)3], 97%), hydrogen tetrachloroaurate(III) trihydrate (HAuCl4·3H2O, ≥99.9%, Au 48.5–50.25%), oleylamine (70%), 1-hexadecanol (95%), oleic acid (90%), 1-methyl-2-pyrrolidinone (NMP, ≥99.0%), 6-mercapto-1-hexanol (MCH), thioctic acid (TOA), anhydrous toluene (99.8%), Nhydroxysuccinimide (NHS, >97%), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), albumin from bovine serum (BSA, >99%), enzyme substrate 3,3´,5,5’-tetramethylbenzidine (TMB, Neogen K-blue enhanced activity substrate containing H2O2), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES, >99.5%), 20× saline sodium phosphate-EDTA (20× SSPE) pH 7.4 and Tween 20 were purchased from Sigma-Aldrich. Absolute ethanol (analytical grade) and casein 1% (w/v) in 1× PBS buffer were obtained from Thermo Scientific. Anti-fluorescein-peroxidase Fab fragments (antiFITC-POD) were obtained from Roche Diagnostics GmbH (Mannheim, Germany). Ultra-pure water was used throughout this work, purified with a Milli-Q system. Different washing buffers were used: (i) SSPE-T (2× SSPE, 0.01% of Tween 20); (ii) HEPES-T (0.1 M HEPES, 0.01% Tween 20); (iii) PBS-C (1× PBS solution containing 1% casein), and (iv) HEPES (0.1 M, pH 7.4). For the homogeneous hybridization reaction step a SSPE-BSA (2× SSPE, 2.5 % of BSA) was used. All buffers were used in the aforementioned concentrations and pH, except when stated otherwise. The oligonucleotide sequences, shown in Table 1, were obtained as lyophilized desalted salts from Sigma-Aldrich. Stock solutions were prepared in Milli-Q water and stored at –20 ºC until use. Certified reference materials (CRM) of genetically modified maize event MON810 2 and 10 % (w/w) were obtained from the Institute of Reference Materials and Measurements through Sigma-Aldrich. Instrumentation and Measurements. TEM images were obtained with a MET-JEOL-2000-ExII transmission electron microscope. The samples were prepared by dilution of the Fe3O4 and Fe3O4@Au ferrofluids with 5 ACS Paragon Plus Environment

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toluene, followed by the immersion of a carbon-coated 400 mesh copper grid in the diluted dispersions and subsequent air-drying. The average particle sizes and size distributions were calculated from the diameters of at least 200 particles randomly selected from the TEM micrographs. UV-Vis spectra were acquired on a Thermo Scientific Evolution 3000 UV-Vis spectrophotometer using a glass cell with an optical path length of 1 cm. XRD measurements were performed at room temperature over the 2θ range of 7−80°, at the Departamento de Química and CQ-VR, Universidade de Trás-os-Montes e Alto Douro (UTAD), Portugal, with a PW 3040/60 X’Pert Pro Röntgen diffractometer using Cu Kα radiation (λ = 1.5406 Å) and the Bragg−Brentano θ/2θ configuration. The system includes the ultrafast PW3015/20 X’Celerator detector and a secondary monochromator. The magnetic properties of the dried MNPs were studied at IFIMUP-IN, Physics and Astronomy Department, Faculty of Sciences of Porto University (FCUP), Portugal, using a commercial SQUID magnetometer. The magnetization as a function of applied magnetic field (M(H)) was performed at 300 and 5 K for a maximum applied magnetic field of 30 kOe. Temperature-dependent zero-field-cooled (ZFC) and field-cooled (FC) measurements were performed over the range of 5−370 K with an applied magnetic field of 100 Oe. Electrochemical measurements were carried out with SPCE (DropSens-110, Spain), by using a μ-Autolab type II potentiostat with GPES 4.9 software (EcoChemie, The Netherlands). The SPCEs were cleaned before the measurements with ethanol and deionized water and dried with nitrogen flow. No electrochemical pre-treatment was required. A 12-tube mixing wheel (Dynal MX1) for solution mixing in eppendorf tubes and the magnet (DynaMag-2) for magnetic separation were purchased from Thermo Fischer Scientific (Spain). PCR amplifications were run in a thermal cycler (GeneAmp® PCR System2700 thermocycler (Applied Biosystems, Spain).

Synthesis of Fe3O4 and Fe3O4@Au MNPs. Monodisperse core-shell Fe3O4@Au MNPs were synthesized by a two-step procedure consisting on: (i) the synthesis of Fe3O4 MNPs by thermal decomposition method and (ii) subsequent coating with a gold shell, by chemical reduction of Au(III) on the surface of the Fe3O4 cores, induced by oleylamine, which acts both as reducing and capping agent. Three Fe3O4:HAuCl4 molar ratios were tested to prepare the core-shell MNPs nanoparticles (1:1; 1:4; 1:7). An illustration of the preparation of the core-shell 6 ACS Paragon Plus Environment

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Fe3O4@Au MNPs is shown in Scheme 1A. The Fe3O4 magnetic cores were synthesized by thermal decomposition,25 following a procedure adapted from reference.26 Firstly, 35 mL of NMP solution containing 0.3 M of 1-hexadecanol, 0.3 M of oleic acid and 0.3 M of oleylamine were heated to 200 ºC, under vigorous stirring and inert atmosphere. When that temperature was reached, 10 mL of a 0.15 M solution of [Fe(acac)3] in NMP was quickly added, and stirring was continued at that temperature for 1 h under an inert atmosphere. After that, the reaction mixture was cooled to room temperature and left under stirring for ~18 h. Afterwards the resulting black nanomaterial was precipitated by adding 50 mL of ethanol to the reaction mixture and then magnetically separated. Finally, the precipitate was washed with ethanol several times and redispersed in 5 mL of anhydrous toluene. The core-shell Fe3O4@Au MNPs were prepared using three different Fe3O4:HAuCl4 molar ratios – 1:1, 1:4 and 1:7 – by a procedure adapted from reference.17 Briefly, 1.25 mL of the colloidal dispersion of Fe3O4 MNPs in anhydrous toluene were diluted with 20 mL of anhydrous toluene and heated to 100 ºC under an inert atmosphere. Afterwards, a solution of HAuCl4·3H2O (0.0489, 0.1956 or 0.3423 g for 1:1, 1:4 and 1:7 Fe3O4:HAuCl4·3H2O molar ratios, respectively) and oleylamine (1.22, 4.89 or 8.56 mL) in anhydrous toluene (5, 20 or 35 mL) was slowly added, dropwise, under vigorous stirring. The reaction mixture was stirred at 100 ºC for 1 h, developing a dark purple color. After that, the system was cooled to room temperature and 50 mL of ethanol were added in order to precipitate the resulting nanomaterial. Finally, the gold-coated Fe3O4 MNPs were magnetically separated, washed several times with ethanol, and redispersed in 10 mL of anhydrous toluene, giving rise to dark purple dispersions. The prepared samples will be denoted as Fe3O4@Au 1:1, Fe3O4@Au 1:4 and Fe3O4@Au 1:7, where 1:1, 1:4 and 1:7 are the molar ratios of Fe3O4:Au. Genoassay protocol. All the genoassay steps were performed at room temperature and protected from light under delicate stirring in the mixing wheel to allow a proper contact between the solution and the MNPs. After each step, the supernatant was easily removed by trapping the core-shell MNPs with a magnet. Functionalization of the Fe3O4@Au MNPs surface. Before the functionalization of the Fe3O4@Au surface with thiol groups, a solvent exchange step was carried out from toluene to ethanol. This step should be carried out very carefully to avoid the aggregation of the core-shell MNPs. To this aim, 3 mL of Fe3O4@Au MNPs in toluene were slowly added, dropwise, to 20 mL of absolute ethanol to allow the dispersion of the MNPs in the ethanolic medium. Afterwards, the MNPs were magnetically separated, subsequently washed with 10 mL of ethanol at least 7 ACS Paragon Plus Environment

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2 times and finally redispersed in 10 mL of ethanol. The final Fe3O4@Au MNPs concentration in ethanol was calculated as follows. The MNPs colloidal dispersion was stirred for at least 1 min, and 1 mL of the MNPs dispersion were put in eppendorf tubes and the organic solvent evaporated to dryness at room temperature by a nitrogen stream. The difference between the vials weight (with and without containing the dried MNPs) was used to determine the Fe3O4@Au MNPS concentration in mg of F3O4@Au MNPs per mL. The functionalization of the core-shell MNPs (4 mg) surface was carried out by using a mixture of thiols containing 0.75 mM 6-mercapto-1hexanol (MCH) and 0.25 mM thioctic acid (TOA) in ethanol (Scheme 1B). After removing the supernatant ethanol solvent from the core-shell MNPs by magnetic separation, 4 mL of the ethanolic thiols mixture were slowly added. This mixture remained under stirring in the mixing wheel for 24 h and subsequently, the functionalized MNPs were washed with ethanol and dispersed in 0.1 M HEPES buffer, pH 7.4 (the addition was also carried out very slowly) to obtain a final Fe3O4@Au MNPs concentration of 1 mg mL-1. Immobilization of DNA capture probe on Fe3O4@Au MNPs. 0.0625 mg of Fe3O4@Au MNPs functionalized with MCH-TOA in HEPES buffer were subsequently bioconjugated with DNA by removing the supernatant and then adding a solution of 200 mM EDC and 50 mM NHS prepared in 500 μL of HEPES buffer. After mixing in a mixing wheel for 15 min to activate the surface carboxylic groups, the modified Fe3O4@Au MNPs were washed twice with 500 μL of HEPES-T, followed by incubation with 1 μM aminated capture probe (CP) in 500 μL HEPES buffer during 1 h. The CP-modified MNPs (CP-MNPs) were again magnetically separated and washed twice with HEPES-T. Finally, 1 M ethanolamine was put into contact with the CP-MNPs for 10 min under stirring to block the unreacted carboxylic groups, followed by washing with HEPES-T and SSPE-T. The CPmodified Fe3O4@Au MNPs were finally dispersed in a certain volume of 2× SSPE buffer (50 µl for each assay) and immediately used to carry out the sandwich assay (Scheme 1C). Sandwich assay and electrochemical detection. The bioconjugated Fe3O4@Au MNPs were used in a sandwichtype assay with two consecutive hybridization steps. A detailed scheme of the sandwich assay is provided in Scheme 1C. Firstly, for homogeneous hybridization, 0.5 µM of signaling probe labeled with FITC (FITC-SP) and the desired DNA target concentrations were incubated for 30 min (final volume of 500 µL SSPE-BSA). Subsequently, 50 µL of the CP-modified Fe3O4@Au MNPs were added to each eppendorf tube to allow the heterogeneous hybridization reaction for 1 h, under stirring. The DNA-conjugated MNPs were then washed with 8 ACS Paragon Plus Environment

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SSPE-T and PBS-C buffers. The core-shell nanoparticles functionalized with the DNA duplex were resuspended in a solution of anti-FITC-POD conjugate in PBS-C buffer (0.25 U mL-1) and, after 30 min, washed twice with SSPE-T and resuspended in 50 µL SSPE 2×. The electrochemical measurements were performed in disposable SPCE. A magnet was placed under the working electrode and the modified MNPs were deposited and magnetically attracted for 2 min. The liquid in excess was carefully removed and 40 µL of TMB-H2O2 K-Blue reagent solution was added to cover the electrodes for 1 min. The detection of the enzymatically oxidized product was performed by chronoamperometry at 0 V, for 60 s. Three replicates were carried out for all measurements. Real sample preparation. Certified materials were extracted and purified using a Nucleospin® food kit (Macherey-Nagel, Düren, Germany) as previously described.25 Extracted DNA quantity and purity were determined spectrophotometrically. Conventional PCR was carried out for DNA quality control using a vegetaluniversal primer pair designed on chloroplast rbcL gene.26 Specific PCR amplifications of transgenic event were carried out as previously reported.27 Amplification of the endogenous gene for negative control was carried out in 25 µL of total volume containing 5 µL of DNA extract, 1.25 U of ImmolaseTM DNA polymerase (Ecogen, Spain)., 1 × buffer, 200 µM of each dNTP, 6.5 mM of MgCl2, 0.3 µM of each primer (forward: 5’-TTG GAC TAG AAA TCT CGT GCT GA-3’ and reverse: (5’-GCT ACA TAG GGA GCC TTG TCC T-3’). The amplification protocol was as follows: initial denaturation at 95 ºC for 10 min, 45 cycles at 95 ºC for 15 s, 60 ºC for 60 s and 72 ºC for 30 s; with a final extension at 72 ºC for 5 min. In both types of amplifications, the success of the amplification reaction was verified by electrophoresis (1× TBE running buffer) on 2 % (w/v) agarose gel and ethidium bromide staining (0.5 µg mL-1) at 80 V. RESULTS AND DISCUSSION Nanoparticles synthesis and characterization. High-quality Fe3O4 MNPs with uniform particle size and narrow particle size distribution can be synthesized by thermal decomposition of metal complexes, such as [Fe(acac)3], in high-boiling organic solvents containing surfactants such as oleic acid and oleylamine.28,29 In order to prevent the oxidation of the Fe3O4 MNPs and allow their use as platforms for the development of genoassays, they were coated with a gold shell. The coating with gold was performed by slow deposition of a gold(III) salt on the surface of the Fe3O4 cores with subsequent reduction of Au(III) to Au(0) induced by oleylamine, under controlled temperature.14,15 Oleylamine has a dual function in this process, as reducing agent 9 ACS Paragon Plus Environment

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and stabilizer of the resulting core-shell MNPs. The influence of the amount of gold precursor added to the reaction mixture on the complete encapsulation of Fe3O4 cores and thickness of the resulting gold shell was evaluated using three Fe3O4:HAuCl3 molar ratios (1:1; 1:4 and 1:7) in the coating procedure. In order to confirm the encapsulation of the Fe3O4 MNPs with gold, both the parent (Fe3O4 MNPs) and core-shell MNPs were characterized by TEM and UV-Vis spectroscopy. In Figure 1 are presented the TEM images of the parent and gold-coated Fe3O4 MNPs prepared with different Fe3O4:HAuCl3 molar ratios as well as the particle size distribution histograms. As can be observed from Figure 1A, the parent Fe3O4 sample is composed of nanometer-sized particles with an average particle size of 4.8±0.5 nm and a Gaussian-type size distribution. Upon the coating of Fe3O4 with gold, in the case of the Fe3O4@Au 1:1 sample, the TEM images (Figure 1B) mostly show the presence of uncoated Fe3O4 cores, indicating their incomplete coating due to an insufficient amount of gold when using an equimolar ratio of Fe3O4:HAuCl3 (1:1). As the Fe3O4:HAuCl3 ratio used in the coating procedure increases to 1:4 and 1:7, it can be observed the presence of quasi-spherical nanoparticles with a darker contrast as well as an increase of the particle size to 10.0±1.7 and 10.4±1.7 nm for Fe3O4@Au 1:4 and 1:7, respectively, which confirms the successful formation of core-shell nanoparticles; furthermore, the nanoparticles are well-dispersed with no signs of aggregation and no free iron oxide cores are detected, indicating the complete encapsulation of the Fe3O4 cores. The UV-Vis spectra of the core-shell MNPs dispersed in toluene exhibit the expected surface plasmon resonance band (SPR) centered between λ = 520 nm and λ = 540 nm (Figure 2A). In contrast, the spectrum of the supernatant solution resulting from the last washing step of the core-shell MNPs (Fe3O4@Au 1:4) before redispersion in toluene showed the absence of any absorption band, which rules out the presence of free Au(0) nanoparticles and strongly indicates the formation of a Au shell on the surface of the Fe3O4 MNPs. As expected, the spectrum of the Fe3O4 dispersion also did not show any SPR band. The larger the amount of gold precursor added in the coating procedure, the higher the wavelength of the SPR band, which is centered at λ = 523, 529 and 532 nm for the Fe3O4@Au 1:1, Fe3O4@Au 1:4 and Fe3O4@Au 1:7 samples, respectively. This red shift of the SPR absorption band ongoing from Fe3O4@Au 1:1 to Fe3O4@Au 1:7 is due to the progressive increase of the thickness of the gold shell, being in accordance with the TEM results and with the literature.14


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The TEM and UV-Vis spectroscopy characterization of the nanomaterials thus suggest that the core-shell samples prepared with 1:4 and 1:7 Fe3O4:HAuCl3 molar ratios are suitable for further assays. The ratio 1:1 is not sufficient to successfully coat all the core nanoparticles, and an inefficient coating will not allow an effective binding between the capture probe and the nanoparticles. Furthermore, since the Fe3O4@Au 1:4 sample led to the best genoassay performance (results presented below), an additional characterization was performed by X-ray diffraction and SQUID magnetometry in order to obtain complementary information about structure, composition, particle size and magnetic properties before and after coating with gold. In Figure 2B the diffractograms of the parent Fe3O4 and core-shell Fe3O4@Au 1:4 MNPs. The diffractogram of the parent Fe3O4 MNPs presents the characteristic Bragg reflections of ferrites, (111), (220), (311), (400), (422), (511), (440) and (533), revealing the crystalline nature of the nanomaterial and the expected cubic spinel structure (Fd3m space group).30,31 The peak broadening is due to the small size of the crystalline domains. In order to identify the nature of the iron oxide, the lattice parameter a of the cubic unit cell was determined. The obtained a value was 8.407 Å, which is close to the value of bulk Fe3O4 (8.396 Å, JCPDS card No. 19-0629), confirming the type of iron oxide phase as being magnetite and the lack of oxidation. In the diffractogram of the core-shell Fe3O4@Au 1:4 sample are detected additional diffraction peaks that can be indexed to the (111), (200), (220) and (311) planes of gold with a cubic structure (Fm3m space group),30 indicating its presence in the sample, in accordance with the results from UV-Vis spectroscopy. Furthermore, a decrease of the intensity of the Bragg reflections associated with magnetite is observed, which confirms the successful coating of the Fe3O4 cores with gold. The average particle size of the uncoated Fe3O4 MNPs, dXRD, estimated from the full width at half-maximum (FWHM) of the (311) reflection using the Debye–Scherrer equation,32 was 3.3 nm. In the case of the Fe3O4@Au 1:4 sample it was not possible to estimate the diameter of the magnetic cores after the gold coating since the Bragg reflections characteristic of gold are detected at 2θ values very close to those associated with magnetite, leading to the partial peak overlapping. The magnetization (M) of the nanoparticles as a function of temperature (T) and applied magnetic field (H) was performed by SQUID magnetometry. In Figure 3A are presented the M(T) curves of Fe3O4 and Fe3O4@Au 1:4, which provide information about the magnetic state of the samples at room temperature. 11 ACS Paragon Plus Environment

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For both nanomaterials, there is a complete overlap of the ZFC and FC curves at 300 K, which indicates that the nanomaterials are superparamagnetic at room temperature. The blocking temperature (TB) values, which correspond to the temperature that separates the unblocked from the blocked state, were determined through the temperature derivative of the difference between the FC and ZFC magnetization curves and are presented in Table 2. The obtained TB values were 14.0 and 12.6 K for Fe3O4 and Fe3O4@Au 1:4, respectively. The slight decrease of the TB value upon the coating of the Fe3O4 MNPs with gold indicates that the gold coating leads to a reduction of the dipolar interactions between the magnetic cores.11 The average diameter of the Fe3O4 cores before and after the coating with gold, dSQUID, can also be estimated from the ZFC-FC studies, using the Stoner-Wohlfarth relation.33 య

଻ହ௞ ்ా

݀ୗ୕୙୍ୈ = ට ସ஠௄ా

౛౜౜

(1.1)

where Keff is the effective anisotropy constant (Keff = 5.00 × 104 J m-3 for magnetite prepared by a synthesis process similar to the one used in this work29) and kB is the Boltzmann constant. The dSQUID values only take into account the magnetic component of the samples and are therefore only related with the Fe3O4 cores, not taking into account the gold shell thickness. The obtained dSQUID values were 5.7 nm for Fe3O4 and 5.5 nm for Fe3O4@Au 1:4, which can be considered similar (within the measurement error), confirming that the gold coating does not induce changes on the diameter of the magnetic cores, i.e., the magnetic cores size is preserved upon their coating with gold. The M(H) curves of the samples at 300 and 5 K, presented in Figures 3B and 3C, respectively, provide additional information about the magnetic properties of the samples. For both Fe3O4 and Fe3O4@Au 1:4 samples, the M(H) curves at 300 K exhibit negligible coercive field values (Figure S-1 in the Supporting Information and Table 2), confirming their superparamagnetic state at room temperature, in accordance with the results obtained from ZFC-FC studies. In contrast, at 5 K the M(H) curves of both materials present a hysteretic behavior (Figure 3C), with coercive values (HC) of 207 and 103 Oe for Fe3O4 and Fe3O4@Au 1:4, respectively (Table 2), which indicates that the MNPs are blocked at 5 K. The saturation magnetization (MS) of the Fe3O4 sample at 300 K is 42.6 emu g-1, slightly decreasing to 41.0 emu g-1 upon the coating with gold (Table 2). On the other hand, at 5 K, the MS values are 56.1 and 51.1 emu g-1 for Fe3O4 and Fe3O4@Au 1:4, respectively (Table 2). For both temperatures, the MS value of the uncoated 12 ACS Paragon Plus Environment

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Fe3O4 MNPs is higher than that of the core-shell Fe3O4@Au 1:4 sample, which confirms the successful encapsulation of the Fe3O4 cores with gold, corroborating the results obtained by TEM, UV-Vis spectroscopy and XRD. Genoassay performance. The successful conversion of the oleylamine-capped Fe3O4@Au MNPs into waterdispersible MNPs and the feasibility of using them as immobilization support for DNA hybridization assays were evaluated for both 1:4 and 1:7 ratios. 0.250 mg of each type of core-shell MNPs were allowed to react with 1 µM CP solution as described above and the entire assay was carried out in the absence of target and with a concentration of 50 nM of target MON810 DNA. The largest currents were obtained with the 1:4 Fe3O4@Au MNPs (Figure 4A), so they were selected for further assays. We have previously reported that the amount of magnetic microbeads plays a crucial role in the performance of the electrochemical measurement,34,35 so the effect of this parameter when using nanometer-sized particles was also studied. When the amount of Fe3O4@Au MNPs subjected to the entire assay was decreased from 0.250 mg to 0.0625 mg, the blank current in the absence of target clearly decreased (Figure 4B). Simultaneously, the current for a 5 nM target concentration also decreases but in a milder fashion down to 0.125 mg and then it remained constant within the experimental error. Consequently, the signal to blank ratio (S/B) monotonically increased when decreasing the amount of nanoparticles on the electrode, so the lowest amount of nanoparticles was used in further experiments. In genosensor or genoassay protocols, the addition of a blocking agent such as bovine serum albumin (BSA) in the hybridization step is usually advantageous to improve the S/B ratio. In our case, the presence of BSA pushed down the blank signal, enhancing the S/B ratio from 5 to 18.5 at the lowest amount of MNPs, so it was included in further experiments. Interestingly, the reproducibility of the measurements is remarkable especially when BSA is added. Under the optimized conditions, the response to increasing concentrations of MON810 target DNA was evaluated by measuring the chronoamperometric currents from 0.25 to 25 nM. As it can be seen in Figure 5, a linear correlation between the blank-subtracted current intensity Inet and the target concentration was found in the interval from 0.25 to 2.5 nM. The regression equation is the following: Inet (nA) = 935(±9) [MON810 ssDNA] (nM) + 37(±12)

r = 0.9998 13

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The limit of detection (LOD) and the limit of quantification (LOQ) calculated as three times and ten times the standard deviation of the blank experiment divided by the slope of the calibration plot were 0.15 nM and 0.50 nM, respectively. The precision of this genoassay was evaluated by using 1 nM of MON810 target DNA. Thus, the repeatability was determined by inter-electrode measurements and the reproducibility was evaluated by performing four inter-day measurements. The repeatability and reproducibility expressed as relative standard deviation were 1.2% and 3.6%, respectively. We have previously reported on the analytical performance of two genosensor platforms; the classical screen-printed Au electrode and a novel 3D-nanostructure.27 The superior sensitivity of the latter was partially obscured by the somehow limited reproducibility mainly associated to the home-made manufacture of the sensor surface. By using our synthetic Fe3O4@Au NPs an outstanding reproducibility was found maintaining detectability and dynamic range. The reproducibility of the MNPs is also much higher than that found for similar approaches with microparticles.34 This might be attributed to the better stability and homogeneous distribution of the colloidal dispersion.36 They are though less sensitive probably due to the low background currents achieved with microsized particles. Different routes of synthesis have been proposed to prepare gold coated MNPs for bioassays. Direct chemisorption of thiolated DNA was proposed but this procedure requires 2 days only for NPs aging with some of them taking several days. A sandwich assay with a signaling DNA probe labeled with AuNPs caused the aggregation of the nanoparticles, achieving a sensitive detection by UV-Vis absorption spectroscopy.37 Simpler and shorter synthesis procedures have been proposed in the two electrochemical approaches reported so far to the best of our knowledge. A direct hybridization assay that requires the labeling of the target DNA fragment was proposed with detectability in the pM range.20 However, the target labeling step makes the measurement of extracted DNA cumbersome if realistic. The reproducibility of our assay is better probably due to the high quality of our MNPs that are well dispersed in the solvent. As partially oxidized Fe3O4 MNPs are less prone to be covered by gold, γ-Fe2O3 NPs were also tested for gold deposition and used for genosensing. The hybridization step was carried out at high temperature to achieve a low detectability and good reproducibility.21 The method herein shown does not require labeling nor temperature so it is the easiest and amenable to on-site analysis.


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To demonstrate the applicability of the method described, CRMs of maize MON810 were extracted, amplified by PCR and tested with the genoassay. Several volumes of the amplicon were added to the solution containing the signaling probe. As shown in Figure 6, even using as low as 1 µL of the amplicon the signal measured was distinguishable from the blank experiment. Likewise a minimal dilution (1:10) also allows the direct detection of the transgenic event. These results indicates that the genoassay responds to different concentrations of transgenic DNA from real samples without interferences from reagents used in the amplification step, that is, purification is not needed even using high volumes of PCR mixture. No false positive results were obtained when transgenic maize DNA was tested after amplification for an endogenous gene (Supplementary Information Figure S-3). Current intensities were identical to the blank sample (no DNA) within the experimental error for each dilution assayed. This indicates that there is no cross-reactivity with other short ssDNA present in the sample.

Conclusions A disposable magnetogenoassay for the MON810 maize detection was developed by using core-shell Fe3O4@Au MNPs as effective platform easily deposited on SPCE for electrochemical detection. Superparamagnetic Fe3O4@Au MNPs were successfully synthetized. Full Au coating was achieved when the Fe3O4:HAuCl4 ratio was equal or higher than 1:4. A larger ratio such as 1:7 did not significantly increase the thickness of the Au shell, but the electrochemical behavior of the resulting core-shell nanomaterial in the genoassay was worse than that obtained with 1:4 the core-shell MNPs prepared with a 1:4 ratio. The Fe3O4@Au 1:4 particles, with an average particle size of 10.0 nm, presented a high separation efficiency, which associated with their biocompatibility allowed a well oriented DNA immobilization and contributed to the sensitivity and reproducibility of the developed genoassay. The electrochemical genoassay showed good analytical performance, with remarkable high repeatability and reproducibility, which is significantly better than that of genosensors performed on screen-printed Au electrodes or 3D-nanostructure electrodes or genoassays on magnetic microbeads. The ease of handling makes it a promising low-cost tool for the monitoring of transgenic maize in food and feed. In fact, detection of DNA


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extracted from real samples was readily achieved at several levels of dilutions without any purification step minimizing loses and time. Supporting

Information

Available:

The

following

files

are

available

free

of

charge.

Supp Info. Figure S-1 showing the M(H) curves at 300 K near the coercive field, Figure S-2 showing the chronamperograms for the calibration linear range and Figure S-3 showing the assay selectivity. Acknowledgements: The work was funded by Fundação para a Ciência e a Tecnologia (FCT)/MEC and FEDER under Program PT2020 (project UID/QUI/50006/2013 - POCI/01/0145/FEDER/007265), and by Marie Curie Actions FP7PEOPLE-2013-IRSES through the project no. 612545 entitled “GMOsensor–Monitoring Genetically Modified Organisms in Food and Feed by Innovative Biosensor Approaches”. M. F. Barroso and Maria Freitas are grateful to FCT for the grants (SFRH/BPD/78845/2011 and SFRH/BD/111942/2015, respectively) financed by POPHQREN (subsidized by FSE and MCTES). The authors thank Dr. R. Miranda-Castro for preparation of real samples, Prof. P. Tavares and MSc. L. Fernandes from UTAD (Vila Real, Portugal) for the XRD measurements and Prof. J. P. Araújo from IFIMUP-IN, FCUP (Porto, Portugal) for access to the SQUID magnetometer.


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(14) Xu, Z. C.; Hou, Y. L.; Sun, S. H. Magnetic core/shell Fe3O4/Au and Fe3O4/Au/Ag nanoparticles with tunable plasmonic properties. J. Am. Chem. Soc. 2007, 129, 8698-8699. (15) Wang, L.; Park, H.-Y.; Lim, S. I. I.; Schadt, M. J.; Mott, D.; Luo, J.; Wang, X.; Zhong, C.-J. Core@shell nanomaterials: gold-coated magnetic oxide nanoparticles. J. Mater. Chem. 2008, 18, 2629-2635. (16) Liu, Y.; Han, T.; Chen, C.; Bao, N.; Yu, C. M.; Gu, H. Y. A novel platform of hemoglobin on core-shell structurally Fe3O4@Au nanoparticles and its direct electrochemistry. Electrochim. Acta 2001, 56, 3238-3247. (17) Freitas, M.; Viswanathan, S.; Nouws, H. P. A.; Oliveira, M. B. P. P.; Delerue-Matos, C. Iron oxide/gold core/shell nanomagnetic probes and CdS biolabels for amplified electrochemical immunosensing of Salmonella typhimurium. Biosens. Bioelectron. 2014, 51, 195-200. (18) Zhao, J.; Zhang, Y. Y.; Li, H. T.; Wen, Y. Q.; Fan, X. Y.; Lin, F. B.; Tan, L. A.; Yao, S. Z. Ultrasensitive electrochemical aptasensor for thrombin based on the amplification of aptamer-AuNPs-HRP conjugates. Biosens. Bioelectron. 2011, 26, 2297-2303. (19) Jie, G. F.; Zhang, J.; Jie, G. X.; Wang, L. A novel quantum dot nanocluster as versatile probe for electrochemiluminescence and electrochemical assays of DNA and cancer cells. Biosens. Bioelectron. 2014, 52, 69-75. (20) Loaiza, O. A.; Jubete, E.; Ochoteco, E.; Cabanero, G.; Grande, H., Rodriguez, J. Gold coated ferric oxide nanoparticles based disposable magnetic genosensors for the detection of DNA hybridization processes. Biosens. Bioelectron. 2001, 26, 2194-2200. (21) Li, K.; Lai, Y. J.; Zhang, W.; Jin, L. T. Fe2O3@Au core/shell nanoparticle-based electrochemical DNA biosensor for Escherichia coli detection. Talanta 2011, 84, 607-613. (22) Jing, P.; Xu, W. J.; Yi, H. Y.; Wu, Y. M.; Bai, L .J.; Yuan, R. An amplified electrochemical aptasensor for thrombin detection based on pseudobienzymic Fe3O4-Au nanocomposites and electroactive hemin/G-quadruplex as signal enhancers. Analyst 2014, 139, 1756-1761. (23) Bai, Y. H.; Li, J. Y.; Xu, J. J.; Chen, H. Y. Ultrasensitive electrochemical detection of DNA hybridization using Au/Fe3O4 magnetic composites combined with silver enhancement. Analyst 2010, 135, 1672-1679.


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(24) Yang, X.; Wu, F.; Chen, D. Z.; Lin, H. W. An electrochemical immunosensor for rapid determination of clenbuterol by using magnetic nanocomposites to modify screen printed carbon electrode based on competitive immunoassay mode. Sens. Actuat. B-Chem. 2014, 192, 529-535. (25) Moura-Melo, S.; Miranda-Castro, R.; de-los-Santos-Álvarez, N.; Miranda-Ordieres, A. J.; Ribeiro Dos Santos Junior, J.; da Silva Fonseca, R. A.; Lobo-Castañón, M. J. Targeting helicase-dependent amplification products with an electrochemical genosensor for reliable and sensitive screening of genetically modified organisms. Anal. Chem. 2015, 87, 8547−8554. (26) Han, J.; Wu, Y.; Huang, W.; Wang, B.; Sun, C.; Ge, Y.; Chen, Y. PCR and DHPLC methods used to detect juice ingredient from 7 fruitsFood Control 2012, 25, 696-703. (27) Barroso, M. F.; Freitas, M.; Oliveira, M. B. P. P.; de-los-Santos-Álvarez, N.; Lobo-Castañón, M. J.; Delerue-Matos, C. 3D-nanostructured Au electrodes for the event-specific detection of MON810 transgenic maize. Talanta 2015, 134, 158-164. (28) Sun, S.; Zeng, H.; Robinson, D. B.; Raoux, S.; Rice, P. M.; Wang, S. X.; Li, G. Monodisperse MFe2O4 (M = Fe, Co, Mn) Nanoparticles. J. Am. Chem. Soc. 2004, 126, 273-279. (29) Verma, S.; Pravarthana, D. One-Pot Synthesis of Highly Monodispersed Ferrite Nanocrystals: Surface Characterization and Magnetic Properties. Langmuir 2011, 27, 13189-13197. (30) Robinson, I.; Tung, L. D.; Maenosono, S.; Walti, C.; Thanh, N. T. K. Synthesis of core-shell gold coated magnetic nanoparticles and their interaction with thiolated DNA. Nanoscale 2010, 2, 2624-2630. (31) Pereira, C.; Pereira, A. M.;

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superparamagnetic Fe3O4 nanoparticles for application as MRI contrast agents: mastering size and magnetism for enhanced relaxivity. J. Mater. Chem. B 2015, 3, 6261-6273. (32) Dinnebier, R. E.; Billinge S. J. L. Powder Diffraction: Theory and Practice, RSC Publishing, Cambridge, UK, 2008. (33) Coey, J. M. D. Magnetism and magnetic materials, Cambridge University Press, Cambridge, UK, 2009. (34) Manzanares-Palenzuela, C. L.; de-los-Santos-Álvarez, N.; Lobo-Castañón, M. J.; López-Ruiz, B. Multiplex electrochemical DNA platform for femtomolar-level quantification of genetically modified soybean. Biosens. Bioelectron. 2015, 68, 259-265. 19 ACS Paragon Plus Environment

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(35) González-Álvarez, M. J.; Pérez-Ruiz, E.; Miranda-Castro, R.; de-los-Santos-Álvarez, N.; MirandaOrdieres, A. J.; Lobo-Castañón, M. J. Effect of tags and labels on the performance of enzyme-emplified electrochemical genomagnetic assays. Electroanalysis 2013, 25, 147-153. (36) Vergauwe, N.; Witters, D.; Ceyssens, F.; Vermeir, S.; Verbruggen, B.; Puers, R.; Lammertyn, J. A versatile electrowetting-based digital microfluidic platform for quantitative homogeneous and heterogeneous bio-assays. J. Micromech. Microeng. 2011, 21, 054026. (37) Zhou, H.; Lee, J.; Park, T. J.; Lee, S. J.; Park, J. Y.; Lee, J. Ultrasensitive DNA monitoring by Au-Fe3O4 nanocomplex. Sens. Actuat. B-Chem. 2012, 163, 224-232.


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CAPTIONS FOR TABLES AND FIGURES

Table 1. Oligonucleotide sequences

Table 2. Magnetic properties of Fe3O4 and Fe3O4@Au 1:4 samples

Scheme 1. General scheme of the genoassay development, divided into three principal steps: (A) Core-shell Fe3O4@Au MNPs synthesis; (B) Fe3O4@Au MNPs surface functionalization with MCH and TOA; and (C) genoassay procedure that generically consisted on: i) aminated DNA capture immobilization onto Fe3O4@Au MNPs via EDC/NHS reaction; ii) homogenous hybridization reaction; iii) heterogeneous hybridization reactions and iv) enzymatic labeling and electrochemical detection by using chronoamperometry. Figure 1. TEM images of the synthesized MNPs: (A) parent Fe3O4 MNPs; (B), (C) and (D) core-shell Fe3O4@Au MNPs prepared from Fe3O4@HAuCl4 molar ratios of 1:1, 1:4 and 1:7, respectively. Insets: Particle size distribution histograms (the solid line represents the Gaussian distribution fit). Figure 2. (A) UV-Vis spectra of bare Fe3O4 MNPs and Fe3O4@Au MNPs synthesized from different Fe3O4:HAuCl4 ratios; the spectrum of the supernatant solution from the last washing step of the core-shell MNPs before redispersion in toluene is also included for comparison. (B) X-ray diffractograms of Fe3O4 and Fe3O4@Au 1:4 samples. Figure 3. Upper panel: (A) Temperature dependence of the magnetization (ZFC and FC) over the temperature range 5−370 K with H = 100 Oe for Fe3O4 and Fe3O4@Au 1:4. Bottom panel: M(H) curves between –30 kOe and 30 kOe for for Fe3O4 and Fe3O4@Au 1:4 MNPs at (B) 300 K and (C) 5 K. Figure 4. (A) Current intensities obtained with 0.250 mg of Fe3O4@Au 1:4 and Fe3O4@Au 1:7 MNPs in the absence (blank) and in the presence of 50 nM target DNA (gray). (B) Effect of the amount of Fe3O4@Au 1:4 MNPs used on the current intensity with or without BSA in the absence (blank) and in the presence of 5 nM target DNA (gray). Secondary axis: Signal to blank ratio for each condition.


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Figure 5. (A) Dependence of the blank subtracted current and the target concentration for the quantification of MON810 specific fragment with Fe3O4@Au 1:4 MNPs. (B) Plot of the linear range The corresponding chronoamperograms are shown as additional Figure S-2 in Supporting Information.

Figure 6. Dependence of the blank subtracted current and dilution of amplified DNA from 2% maize certified material with specific primers for the transgenic event.


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TABLES

Table 1. Oligonucleotide sequences DNA strand name

Length

Capture probe (CP)

21

Sequence (5´- 3´) TTA GAG TCC TTC GTC CTT CGA- NH2 FITC-TCT TCA CAA TAA AGT GAC AGA TAG CTG GGC AAT GGC

Signaling probe 51 (FITC-SP)

AAA GGA TGT TAA ACG TCG AAG GAC GAA GGA CTC TAA CGT TTA ACA TCC TTT GCC ATT

Target (T)

72 GCC CAG CTA TCT GTC ACT TTA TTG TGA AGA

Table 2. Magnetic properties of Fe3O4 and Fe3O4@Au 1:4 samples Magnetic properties TB

MS300 K

MS5 K

HC300 K

HC5 K

(K)

(emu g-1)

(emu g-1)

(Oe)

(Oe)

Fe3O4

14.0

42.6

56.1

5.6

207

Fe3O4@Au 1:4

12.6

41.0

51.1

10.7

103

Sample


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Scheme 1. General scheme of the genoassay development, divided into three principal steps: (A) Core-shell Fe3O4@Au MNPs synthesis; (B) Fe3O4@Au MNPs surface functionalization with MCH and TOA; and (C) genoassay procedure that generically consisted on: i) aminated DNA capture immobilization onto Fe3O4@Au MNPs via EDC/NHS reaction; ii) homogenous hybridization reaction; iii) heterogeneous hybridization reactions and iv) enzymatic labeling and electrochemical detection by using chronoamperometry.


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FIGURE 1 (A)

(B)

(C)

(D)

Figure 1. TEM images of the synthesized MNPs: (A) parent Fe3O4 MNPs; (B), (C) and (D) core-shell Fe3O4@Au MNPs prepared from Fe3O4@HAuCl4 molar ratios of 1:1, 1:4 and 1:7, respectively. Insets: Particle size distribution histograms (the solid line represents the Gaussian distribution fit).


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FIGURE 2

Figure 2. (A) UV-Vis spectra of bare Fe3O4 MNPs and Fe3O4@Au MNPs synthesized from different Fe3O4:HAuCl4 ratios; the spectrum of the supernatant solution from the last washing step of the core-shell MNPs before redispersion in toluene is also included for comparison. (B) X-ray diffractograms of Fe3O4 and Fe3O4@Au 1:4 samples.


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FIGURE 3

(A)

6

Fe 3O 4 Fe 3O 4@Au 1:4

-1

M (emu g )

5 4 3 2 1 0 0

50

100

150

200

250

300

350

400

T (K) 60

(B)

Fe3O4

40

60

Fe3O4@Au 1:4

(C)

Fe3O4 Fe3O4@Au 1:4

40 20

20

M (emu g )

T = 300 K

T=5K

-1

-1

M (emu g )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sensors

0 -20

0 -20 -40

-40 -60 -60 -30000

-20000

-10000

0

10000

20000

30000

-30000

-20000

-10000

0

10000

20000

30000

H (Oe)

H (Oe)

Figure 3. Upper panel: (A) Temperature dependence of the magnetization (ZFC and FC) over the temperature range 5−370 K with H = 100 Oe for Fe3O4 and Fe3O4@Au 1:4. Bottom panel: M(H) curves between –30 kOe and 30 kOe for for Fe3O4 and Fe3O4@Au 1:4 MNPs at (B) 300 K and (C) 5 K.


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FIGURE 4

Figure 4. (A) Current intensities obtained with 0.250 mg of Fe3O4@Au 1:4 and Fe3O4@Au 1:7 MNPs in the absence (blank) and in the presence of 50 nM target DNA (gray). (B) Effect of the amount of Fe3O4@Au 1:4 MNPs used on the current intensity with or without BSA in the absence (blank) and in the presence of 5 nM target DNA (gray). Secondary axis: Signal to blank ratio for each condition. 28 ACS Paragon Plus Environment

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Figure 5. (A) Dependence of the blank subtracted current and the target concentration for the quantification of MON810 specific fragment with Fe3O4@Au 1:4 MNPs. (B) Plot of the linear range. The corresponding chronoamperograms are shown as additional Figure S-2 in Supporting Information. 29 ACS Paragon Plus Environment

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FIGURE 6

Figure 6. Dependence of the blank subtracted current and dilution of amplified DNA from 2% maize certified material with specific primers for the transgenic event.


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TOC


Highly Monodisperse Fe3O4@Au ... - ACS Publications

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