Development and applications of a DNA labeling method with ...

Environ Sci Pollut Res (2015) 22:20322–20327 DOI 10.1007/s11356-015-5614-0

RESEARCH AND EDUCATION HIGHLIGHTS

Development and applications of a DNA labeling method with magnetic nanoparticles to study the role of horizontal gene transfer events between bacteria in soil pollutant bioremediation processes J. Pivetal 1 & M. Frénéa-Robin 1 & N. Haddour 1 & C. Vézy 1 & L. F. Zanini 2,3,4 & G. Ciuta 2,3 & N. M. Dempsey 2,3 & F. Dumas-Bouchiat 2,3,6 & G. Reyne 4 & S. Bégin-Colin 5 & D. Felder-Flesh 5 & C. Ghobril 5 & G. Pourroy 5 & P. Simonet 1

Received: 1 September 2015 / Accepted: 13 October 2015 / Published online: 26 October 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Horizontal gene transfers are critical mechanisms of bacterial evolution and adaptation that are involved to a significant level in the degradation of toxic molecules such as xenobiotic pesticides. However, understanding how these mechanisms are regulated in situ and how they could be used by man to increase the degradation potential of soil microbes is compromised by conceptual and technical limitations. This includes the physical and chemical complexity and heterogeneity in such environments leading to an extreme bacterial taxonomical diversity and a strong redundancy of genes and functions. In addition, more than 99 % of soil bacteria fail to develop colonies in vitro, and even new DNA-based investigation methods (metagenomics) are not specific and sensitive enough to consider lysis recalcitrant bacteria and those belonging to the rare biosphere. The objective of the ANR Responsible editor: Philippe Garrigues * J. Pivetal [email protected] 1

Ecole Centrale de Lyon, CNRS UMR 5005, Laboratoire Ampère, F-69134 Écully, France and Université Lyon1, Laboratoire Ampère, F-69100 Villeurbanne, France

2

University Grenoble Alpes, Inst NEEL, F-38000 Grenoble, France

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CNRS, Inst NEEL, F-38042 Grenoble, France

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G2ELab, Grenoble-INP/UJF/CNRS UMR 5269, F-38402 Grenoble, France

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Institut de Physique et Chimie des Matériaux de Strasbourg (IPCMS), UMR CNRS/UdS 7504, 23 rue du Loess, F-67034 Strasbourg, France

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Univ Limoges, CNRS, SPCTS UMR 7315, Ctr Europeen Ceram, F-87068 Limoges, France

funded project BEmergent^ was to develop a new culture independent approach to monitor gene transfer among soil bacteria by labeling plasmid DNA with magnetic nanoparticles in order to specifically capture and isolate recombinant cells using magnetic microfluidic devices. We showed the feasibility of the approach by using electrotransformation to transform a suspension of Escherichia coli cells with biotinfunctionalized plasmid DNA molecules linked to streptavidin-coated superparamagnetic nanoparticles. Our results have demonstrated that magnetically labeled cells could be specifically retained on micromagnets integrated in a microfluidic channel and that an efficient selective separation can be achieved with the microfluidic device. Altogether, the project offers a promising alternative to traditional culturebased approaches for deciphering the extent of horizontal gene transfer events mediated by electro or natural genetic transformation mechanisms in complex environments such as soil. Keywords Horizontal gene transfer . Electrotransformation . Magnetic DNA labeling . Magnetic nanoparticles . Micromagnets . Magnetic cell sorting

Background, state of the art The remediation of polluted soil is a major concern for public health and environment. In France, for instance, over 4000 sites require urgent remediation in order to restore degraded environments and prevent risks for human health (Commissariat Général au developpement durable 2013). The relevance of soil remediation strategies has been proven since their emergence in the late 80s for the treatment of petroleum hydrocarbon-contaminated environments (Wilson and Jones

Environ Sci Pollut Res (2015) 22:20322–20327

1993). Today, however, although physical and biological processes for remediation treatments have been developed, their application is still not completely mastered (Scullion 2006). Over the past few decades, bioremediation, a process by which an environment is capable of natural self-remediation through the action of its indigenous microorganisms, has gained significant attention as an efficient and cost-effective method to supplement standard remediation strategies (Kumar et al. 2011). In bioremediation, contaminants are generally used as a source of energy or nutrients by genetically adapted microorganisms which, by increasing their fitness, also contribute to cleanup the environment (Eyers et al. 2004; Vogel 1994). Due to their considerable genetic diversity, soil bacterial communities represent a huge potential for bioremediation. Numerous studies have already shown the ability of soil bacteria to degrade a wide variety of pollutants, and today, many experiments have been conducted in order to use those microorganisms for bioremediation strategies (Lee et al. 2012; Pepi et al. 2011). Bacteria also possess a great adaptive capability. In a process called horizontal gene transfer (HGT), bacteria can exchange their genes within the microbial population resulting in a rapid and direct acquisition of new functions (Ochman et al. 2000). Usually considered to be an inconvenience when inducing antibiotic resistant gene dissemination, it becomes a powerful tool to further improve the degradative activities of targeted pollutants. Numerous examples confirm the fundamental role of HGT in bioremediation processes, and the interest to understand how this mechanism is regulated in situ in order to fully use its potential as a source of bioremediation (Ikuma and Gunsch 2012; Lyon et al. 2010). However, studying HGT in soil is particularly challenging due to the complexity and heterogeneity of this natural environment combined with the extremely high level of bacterial diversity, the very limited proportion of cultivable bacteria (Amann et al. 1995) and the lack of sensitivity and specificity of culturebased investigation methods (Sørensen et al. 2005). Most experimental approaches developed to study transfer events occurring in situ have involved inoculation of donor and/or recipient strains in samples of the studied environments and have relied on the expression of naturally or cloned marker gene(s) carried by donor DNA (Ray and Nielsen 2005). Most of the literature papers then compare HGT frequency based on the number of recombinant colonies growing on selective (or not) media when the marker gene(s) confer(s) resistance to toxic compounds such as antibiotics or contaminant molecules. Genes producing a fluorescent protein might also be used to allow a direct visualization of the labeled cells under a microscope unless the mixture is processed through fluorescent-activated cell sorting to specifically separate the untransformed bacteria from the fluorescent recombinants (Arends et al. 2012). Each separate bacteria can then be individually treated for DNA extraction, genome amplification, and sequencing. However, flaws related to unsatisfactory

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purification of the microbial suspension, in particular from naturally autofluorescent particles, hinder the process of cell sorting (Veal et al. 2000). Moreover, the low expression level of these genes in phylogenetically far remote heterologous hosts also contributes to strongly underestimate the detected transfer event frequency and, when based on the in vitro growth of recombinant clones from the indigenous microflora, the range of potential recipient bacteria (Terpe 2006). To overcome some of these limitations, the BEmergent^ project proposed to develop an alternative cultureindependent method with the aim to assess the full potential of HGT for bioremediation purpose in soils. The methodology developed in the framework of the Emergent project consisted in grafting magnetic nanoparticles onto DNA molecules before introducing them into bacterial cells by electroporation. Transformed cells are trapped using micromagnet arrays while magnetic nanoparticle free bacteria are eluted from the chamber. The transformed cells are then released by increasing the flow rate of the carrier liquid (Fig. 1).

Technical and scientific approach To develop a magnetic microfluidic manipulation tool applicable to the study of horizontal gene transfer between bacteria, we established a multidisciplinary consortium involving researchers specialized in the fields of chemistry, physics, microsystems, and environmental microbiology. The project was a collaboration between four French laboratories: Ampère (Lyon, project coordinator), G2Elab (Grenoble), Institut Néel (Grenoble), and IPCMS (Strasbourg). Bacteria transformed with magnetic DNA

Untransformed bacteria

Micro-magnet

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Attraction of magnetically labeled bacteria

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Elution of untransformed bacteria

Release and Analysis of transformed bacteria

Fig. 1 Schematic representation of the magnetic micromanipulation approach developed in the EMERGENT project

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Micropatterned magnetic film (zone of reversed magnetization) Unlabelled bacteria

Magnetically labeled bacteria (transformed bacteria)

Fig. 2 Schematic representation of a specific attraction of transformed bacteria onto a 100×100 μm2 micromagnet arrays

The project consisted of two main phases: firstly, partners had to collaborate in order to develop a micromanipulation system composed of high performance micromagnets integrated within a microfluidic device. The second part included the development of the magnetic labeling method for bacterial cell isolation. 1. The development of the micromanipulation system involved the following steps: (i) Analytical modeling was performed by G2Elab to predict, compare, and optimize forces exerted on magnetic particles for different microsystem configurations. (ii) The preparation and characterization of high performance micromagnets was performed by Institut Néel. (iii) Micromagnet integration within a microfluidic device was performed by Ampère Lab and/or Institut Néel. (a) Simulations were carried out to estimate the appropriate amount of magnetic material that has to be internalized by cells with respect to micromagnet size/geometry. The objective was to obtain significant forces to allow effective attraction of the Bmagnetized^ cells onto micromagnet arrays. These simulations took into account hydrodynamic forces in order to estimate the trajectory of superparamagnetic nanobeads captured on a micromagnet array localized at the bottom of a microfluidic channel (Zanini et al. 2011). The influence of the magnetic layer thickness, the size, and the shape of the magnetic patterns were quantified. Different geometries including chessboards and slanted strips have been proposed and studied (Zanini et al. 2012). (b) High rate triode sputtering was used to deposit highperformance neodymium iron boron (NdFeB) hard magnetic layers on Si wafers. Thermo-magnetic patterning (TMP) was used to produce arrays of oppositely magnetized regions in these flat films. This technique exploits the drop in coercivity achieved by locally heating the film by laser

irradiation through a mask, in the presence of an external magnetic field (Dumas-Bouchiat et al. 2010). The stray field and field gradient values at the surface of such structures, which are maximum at the interface between neighboring magnets, surpass those achieved with topographically patterned films (Kustov et al. 2010). The TMP structures used in this project consisted of arrays of out-of-plane magnetized 100×100 μm2 micromagnets (Fig. 2). (c) The micromagnet arrays were integrated into a microfluidic channel fabricated in polydimethylsiloxane (PDMS), a transparent polymer (Fig. 3). The PDMS channel obtained by micromolding was reversibly bound to the micromagnet arrays. For that purpose, the Ampère laboratory has developed a technique to deposit a thin PDMS membrane (250 nm) upon the micromagnet arrays acting as both a protective layer against corrosion and as an interface for bonding a PDMS microchannel with oxygen plasma (Vézy et al. 2011). While supporting up to one bar pressure, the resulting flexible polymer chamber could be easily removed from the micromagnet surface, allowing reuse of the latter. 2. The system application for bacteria micromanipulation involved the following steps: (i) The synthesis of very small size magnetic nanoparticles to enable their introduction into bacteria by electroporation and the functionalization of these particles to enable their grafting onto DNA and confer them fluorescent properties. (ii) The binding of DNA molecules to magnetic nanoparticles. a)

PDMS interface layer

NdFeB Film

100 100 µm 2 magnetic pattern

Mixed sample

b)

Microfluidic channel Micro-magnet arrays

Fig. 3 a Schematic illustration of bacterial cells flowing through the channel above the micromagnet arrays. b Photograph of the microfluidic chip


50 nm Fig. 4 Ten-nanometer iron oxide nanoparticles obtained by thermal decomposition

(iii) The internalization of nanoparticle-labeled DNA into bacteria and their purification. (iv) The isolation of magnetically labeled bacteria onto micromagnets. (a) IPCMS has succeeded in synthesizing iron oxide magnetic nanoparticles with an average diameter size of 11±1 nm covered with oleic acid, by the thermal decomposition method (Fig. 4) (Basly et al. 2013). A ligand exchange and a phase transfer were then realized in order to graft dendrons functionalized with peripheral carboxylate groups onto the nanoparticle surfaces (Ghobril et al. 2013). A dendritic approach for the decoration of nanoparticles appeared promising as the diversity of functionalization brought by the arborescent structure answers simultaneously all the criteria of biocompatibility, low toxicity, and specificity (Walter et al. 2014, 2015). Biotin and/or fluorophore molecules were then further introduced onto the dendritic nanoparticles through the postfunctionalization of carboxylate groups present at the periphery of the dendrons. (b) In parallel, Ampère has developed a method to graft commercial streptavidin-coated magnetic nanoparticles (Milteny Biotech®, 50 nm in diameter) on plasmid DNA molecules. The labeling was carried out on a plasmid carrying two antibiotic resistance genes (tetracycline and kanamycin) and the linA gene encoding the first step of the lindane pesticide (gamma-hexachlorocyclohexane) degradation. Plasmid molecules were labeled with 50-nm streptavidin-coated magnetic nanoparticles based on the biotin-streptavidin interaction. Plasmids were functionalized with biotin molecules, and functionalized plasmids were mixed with the magnetic nanoparticles to complete the labeling process of DNA. (c) DNA-nanoparticle internalization was then carried out by electroporation, a transformation process in which bacteria are subjected to high-voltage electrical pulses to create temporary pores in the bacterial envelopes for a passive entry of DNA. This process was used with a model bacteria (Escherichia coli).

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(d) To confirm nanoparticle-plasmid conjugation and internalization, we observed the organization of transformed bacteria upon micromagnet surfaces compared to different controls. Only bacteria electroporated in the presence of the conjugated nanoparticle plasmids were attracted to micromagnet surfaces, more specifically at the boundaries between micromagnets, where the stray field value is maximum.

Obtained results In this project, we used magnetism to attract superparamagnetic nanoparticles. Very small size magnetic nanoparticles were required in order to facilitate their introduction into bacteria (cell size ≈2 μm) and to limit steric hindrance when conjugated onto DNA molecules. However, the magnetic force exerted on an object is proportional to its volume, its magnetic moment, and the magnetic field gradient it experiences (Furlani 2002). Therefore, one solution to generate significant forces over cells carrying weak magnetic moments (such as bacterial cells labeled with ultra-small superparamagnetic particles) lies in the miniaturization of the magnetic flux sources, which leads to an increase of the magnetic field gradient according to scaling laws (Cugat et al. 2003). The micromagnets used in this project had field gradients as high as 106 T/m, compared to values in the range 10–103 T/m produced by bulk magnets of the same material. The effectiveness of using TMP micromagnets to attract small size particles was validated using magnetic micro- or nanoparticles, liposomes encapsulating magnetic nanoparticles (Pivetal et al. 2010), and magnetically labeled bacteria. Very small size fluorescent/magnetic hybrid nanoparticles developed by the

a)

b)

100 µm

100 µm

d)

c)

100 µm

100 µm

Fig. 5 Attraction of magnetic entities onto 100 × 100 μm 2 TMP micromagnets. a Fluorescent 50 nm sized nanoparticles. b Liposomes encapsulating 100 nm sized nanoparticles. c Magnetically labeled bacteria. d Twelve-nanometer fluorescent/magnetic hybrid nanoparticles developed by the IPCMS

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IPCMS (12 nm) could also be attracted onto the micromagnets (Fig. 5a, b, c, d). The micromagnets were integrated within a microfluidic channel to allow manipulation of bacteria, thanks to the combined use of magnetic and hydrodynamic forces. Our magnetic nanoparticle-DNA labeling process was successful. After the electroporation of bacteria in the presence of the magnetically labeled plasmid, a number of bacteria were organized upon the micromagnets surface, suggesting that magnetic plasmids had been internalized into bacteria (Pivetal et al. 2014a). Key issues were however identified as the presence of nanoparticles conjugated onto the plasmid DNA interfered with plasmid replications and gene expressions within the bacterial cells. This apparent limitation, however, might pave the way toward a totally new and innovative approach to assess horizontal gene transfer in microbiology as the selection will only occur on the recipient bacterial cells, excluding any other selection process. Magnetic selection could then offer the ability to establish an accurate transfer frequency of gene flow within bacterial communities. We also showed that tools and concepts developed for the study of horizontal gene transfer could be applied to the study of bacterial diversity (Pivetal et al. 2014b, c). In parallel to the horizontal gene study, we have evaluated the possibility of using the magnetic microfluidic system to isolate bacteria specifically in a mixture after labeling them magnetically using a magnetic in situ hybridization (Pivetal et al. 2014b). The sorting device led to the isolation of target cells present in very low abundance in a mixture (0.04 % of target cells against 99.96 % of non-target cells). In spite of the very strong initial imbalance between the two bacterial strain populations, the process enabled us to almost entirely eliminate contamination with non-target cells, to produce highly enriched target cell suspensions (98.46 % purity).

Conclusion The Emergent project has enabled the development of a new technology for the study of environmental bacterial diversity and horizontal gene transfer processes. Significant technological issues were addressed during this project, in particular concerning the fabrication of very small size hybrid nanoparticles, the magnetic labeling of DNA molecules, and the development of microfluidic systems with integrated miniaturized autonomous magnetic field sources. These technological advances have also been used for the development of a new bacterial cell isolation method using magnetic in situ hybridization. Based on these results, the objectives could now be to isolate bacterial cells specifically from complex environments such as soil to sequence their genome completely. Future work might concern both fundamental studies (e.g., study of the adaptive potential of bacteria), as well as applied research (e.g., industrial exploitation of the functional properties of


the vast majority of uncultured, and thus unknown, bacterial species). Acknowledgments The authors thank the Région Rhône-Alpes for the financial support provided including the PhD grant of J. Pivetal. This work also benefited from the financial support of the French National Research Agency (ANR 09-CESA-013), the CNRS, and Cemagref interdisciplinary Ecological engineering program 2009 (BNanogénomique^ project) which are gratefully acknowledged.

References Amann RI, Ludwig W, Schleifer KH (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59(1):143–69 Arends K, Schiwon K, Sakinc T, Hübner J, Grohmann E (2012) Green fluorescent protein-labeled monitoring tool to quantify conjugative plasmid transfer between gram-positive and gram-negative bacteria. Appl Environ Microbiol 78(3):895–9 Basly B, Popa G, Fleutot S, Pichon BP, Garofalo A, Ghobril C, BeginColin S (2013) Effect of the nanoparticle synthesis method on dendronized iron oxides as MRI contrast agents. Dalton Trans 42(6):2146–57 Commissariat Général au developpement durable (2013) Basol: un panorama des sites et sols pollués, ou potentiellement pollués, nécessitant une action des pouvoirs publics Cugat O, Delamare J, Reyne G (2003) Magnetic micro-actuators and systems (MAGMAS). IEEE Trans Magn 39(5):3607–3612 Dumas-Bouchiat F, Zanini LF, Kustov M, Dempsey NM, Grechishkin R, Hasselbach K, Givord D (2010) Thermomagnetically patterned micromagnets. Appl Phys Lett 96(10):102511 Eyers L, George I, Schuler L, Stenuit B, Agathos SN, El Fantroussi S (2004) Environmental genomics: exploring the unmined richness of microbes to degrade xenobiotics. Appl Microbiol Biotechnol 66(2):123–30 Furlani EP (2002) Particle transport in magnetophoretic microsystems. In Microfluidic devices in nanotechnology 3461–3473 Ghobril C, Popa G, Parat A, Billotey C, Taleb J, Bonazza P, Felder-Flesch D (2013) A bisphosphonate tweezers and clickable PEGylated PAMAM dendrons for the preparation of functional iron oxide nanoparticles displaying renal and Hepatobiliary elimination. Chem Commun 49(80):9158–60 Ikuma K, Gunsch CK (2012) Genetic bioaugmentation as an effective method for in situ bioremediation functionality. Bioengineered 3(4): 236–241 Kumar A, Bisht B, Joshi V, Dhewa T (2011) Review on bioremediation of polluted environment : a management tool. Int J Environ Sci 1(6): 1079–1093 Kustov M, Laczkowski P, Hykel D, Hasselbach K, Dumas-Bouchiat F, O’Brien D, Dempsey N (2010) Magnetic characterization of micropatterned Nd–Fe–B hard magnetic films using scanning hall probe microscopy. J Appl Phys 108(6):063914 Lee KY, Bosch J, Meckenstock RU (2012) Use of metal-reducing bacteria for bioremediation of soil contaminated with mixed organic and inorganic pollutants. Environ Geochem Health 34(1):135–142 Lyon DY, Pivetal J, Blanchard L, Vogel TM (2010) Bioremediation via in situ electrotransformation. Bioremediat J 14(2):109–119 Ochman H, Lawrence JG, Groisman E a (2000) Lateral gene transfer and the nature of bacterial innovation. Nature 405(6784):299–304 Pepi M, Gaggi C, Bernardini E, Focardi S, Lobianco A, Ruta M, Focardi SE (2011) Mercury-resistant bacterial strains pseudomonas and psychrobacter spp. Isolated from sediments of orbetello lagoon (Italy) and their possible use in bioremediation processes. Int Biodeterior Biodegrad 65(1):85–91

Environ Sci Pollut Res (2015) 22:20322–20327 Pivetal J, Osman O, Vezy C, Frénéa-robin M, Haddour N (2010) Trapping of magnetically-labelled liposomes on flat micropatterned hard magnetic films. In 8TH International conference on the scientific and clinical applications of magnetic carriers 192–197 Pivetal J, Ciuta G, Frenea-Robin M, Haddour N, Dempsey NM, DumasBouchiat F, Simonet P (2014a) Magnetic nanoparticle DNA labeling for individual bacterial cell detection and recovery. J Microbiol Methods 107:84–91 Pivetal J, Royet D, Ciuta G, Frenea-Robin M, Haddour N, Dempsey NM, Simonet P (2014b) Micro-magnet arrays for specific single bacterial cell positioning. J Magn Magn Mater 380:72–77 Pivetal J, Toru S, Frenea-Robin M, Haddour N, Cecillon S, Dempsey NM, Simonet P (2014c) Selective isolation of bacterial cells within a microfluidic device using magnetic probe-based cell fishing. Sensors Actuators B Chem 195:581–589 Ray JL, Nielsen KM (2005) Experimental methods for assaying natural transformation and inferring horizontal gene transfer. Methods Enzymol 395:491–520 Scullion J (2006) Remediating polluted soils. Die Naturwissenschaften 93(2):51–65 Sørensen SJ, Bailey M, Hansen LH, Kroer N, Wuertz S (2005) Studying plasmid horizontal transfer in situ: a critical review. Nat Rev Microbiol 3(9):700–10 Terpe K (2006) Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 72(2):211–22

20327 Veal DA, Deere D, Ferrari B, Piper J, Attfield PV (2000) Fluorescence staining and flow cytometry for monitoring microbial cells. J Immunol Methods 243(1–2):191–210 Vézy C, Haddour N, Dempsey NM, Dumas-Bouchiat F, Frénéa-Robin M (2011) Simple method for reversible bonding of a polydimethylsiloxane microchannel to a variety of substrates. Micro Nano Lett 6(10):871 Vogel TM (1994) Natural bioremediation of chlorinated compounds. In: Norris RD, Kerr RS (eds) Handbook of bioremediation. CRC Press Inc, Boca Raton Walter A, Billotey C, Garofalo A, Ulhaq-Bouillet C, Lefèvre C, Taleb J, Begin-Colin S (2014) Mastering the shape and composition of dendronized iron oxide nanoparticles to tailor magnetic resonance imaging and hyperthermia. Chem Mater 26(18):5252–5264 Walter A, Garofalo A, Parat A, Jouhannaud J, Pourroy G, Voirin E, Felder-Flesch D (2015) Validation of a dendron concept to tune colloidal stability, MRI relaxivity and bioelimination of functional nanoparticles. J Mater Chem B 3(8):1484–1494 Wilson SC, Jones KC (1993) Bioremediation of soil contaminated with polynuclear aromatic hydrocarbons (PAHs): a review. Environ Pollut 81(3):229–249 Zanini LF, Dempsey NM, Givord D, Reyne G, Dumas-Bouchiat F (2011) Autonomous micro-magnet based systems for highly efficient magnetic separation. Appl Phys Lett 99(23):232504 Zanini LF, Osman O, Frenea-Robin M, Haddour N, Dempsey NM, Reyne G, Dumas-Bouchiat F (2012) Micromagnet structures for magnetic positioning and alignment. J Appl Phys 111(7):07B312