CORDIS

Executive Summary:
The NanoMag project objectives were to standardize and harmonize ways to measure and analyse the data for magnetic nanoparticle (MNP) systems. NanoMag brought together Europe’s leading experts in; synthesis of magnetic single- and multi-core nanoparticles, characterizations of magnetic nanoparticles and national metrology institutes. In the consortium, we have gathered partners within companies, metrology institutes, universities and research institutes, all carrying out front end research and developing applications in the field of MNP systems. The NanoMag project was a for year project started in 2013 and ended in 2017. The objectives of the NanoMag project has been to improve and redefine existing analysing methods and in some cases, to develop new analyzing methods for MNPs. By using improved manufacturing technologies, we have synthesized MNPs with specific properties that have been analysed with a multitude of characterization techniques (focusing on both structural as well as magnetic properties) and the experimental results was correlated between different analysis techniques, and we obtained a self-consistent picture that describes how structural and magnetic properties are interrelated. In the NanoMag project we have used almost all existing analysis techniques to study MNPs. The project has defined standard measurements and techniques which are necessary for defining a magnetic nanostructure and quality control. The areas we have studied in the NanoMag project was focused on biomedical applications, for instance bio-sensing (detection of different biomarkers), contrast substance in tomography methods (Magnetic Resonance Imaging and Magnetic Particle Imaging) and magnetic hyperthermia (for cancer therapy). In the NanoMag project we have had an associated stakeholder committee group (17 members) of companies and universities in the fields of synthesis of MNPs and analysis techniques. These group have helped the NanoMag consortium in the different surveys we have sent out to understand the industrial needs and challenges in the fields of MNP synthesis, standardization and analysis methods. Four surveys were distributed to over 250 organizations and the answers were analysed to improve our standardization work and strategies. During the project NanoMag members have made substantial standardization contributions to ongoing ISO standardization work in the field of MNP systems. For most of the measurement methods standardization procedures (SOPs), uncertainty budgets and metrological descriptions has been developed. Over 50 new MNP systems (single- and multi-core particles) have been synthesized in the project and also SOPs for the synthesis of MNP systems have been developed. We have obtained improvements in reproducibility of MNP synthesis. In the project we have developed new MNP products and MNP analysis methods as a result of the work in the project. To reach out to the public with respect to basic MNP knowledge and MNP applications, four electronic-learning modules has been developed and will be available also after the NanoMag project. Over 80 new publications in international high impact journals and over 160 presentations and seminars have been carried out by NanoMag partners. During the project we have also developed a clearer nomenclature in order to describe structure and magnetic properties of MNPs and MNP ensembles. Surveys of measurement methods for MNPs and their pros and cons, classification of these methods including also classification of different types of MNP systems has been reported and published. In the project we have used Monte-Carlo simulations in order to explain the experimental results, which have led to improved modelling of MNP magnetic properties, especially the dynamic magnetic behaviour (for instance to be used in the magnetic hyperthermia field). Round Robin measurements (same analysis methods on the same MNP samples but in different labs utilizing the developed SOPs) using different types of MNP systems have been carried and the result was promising.
Project Context and Objectives:
The figures, tables, publications/conferences (over 80 publications and 170 attended conferences/seminars) are all listed in the attachment to this final report and also in table A1 and A2.

Activities and context of the NanoMag project can be seen below and which WP’s the activities belongs to.

• Synthesis of MNP with defined properties WP1/WP3
• Improvement of existing analysis methods WP2/WP4
• Development of new analysis methods WP2/WP4/WP6
• Understanding of structural and magnetic properties WP3/WP4/WP6
• Definition of standard measurements WP4/WP5
• Definition of magnetic nanostructures and quality Control WP3/WP4/WP5
• Dissemination and exploitation WP7
• Administration and management WP8

The workpackages (WP) in the NanoMag project were:

WP1: Definitions of magnetic nanoparticles
WP2: Definitions of analysis methods
WP3: Magnetic nanoparticle synthesis
WP4: Magnetic nanoparticle analysis
WP5: Standardization of magnetic nanoparticles
WP6: Application and benchmarking
WP7: Dissemination and exploitation
WP8: Adiministrative and financial management

The strategic objectives of the NanoMag project were:
• To identify analysis and characterization techniques that can be used as standardization measurements in the field of magnetic nanoparticle research and development and that will provide valuables tools to the manufacturing process of magnetic nanoparticles and the regulatory work on magnetic nanoparticles.
• Use new or improved analysis techniques to control the properties of magnetic nanoparticles that improve their specific application.
• Promote the standardization techniques so they can be used both in research as well in industry, SME or large companies.
• Provide/enable a traceable route for novel characterization techniques from a laboratory research towards the basis of new metrological standards, which do currently not exist in the area of magnetic nanoparticles.

The achievement of the strategic goals of the NanoMag concept requires the advancement in several complimentary characterization technologies.

The specific technical objectives of the project were:
• Correlate the magnetic and structural properties of magnetic nanoparticles.
• Develop new analysis techniques and models in the field of magnetic nanoparticles.
• Improve the traceability of the total magnetic nanoparticle “life time” from manufacturing to the specific application.
• To present standardized procedures for manufacturing magnetic nanoparticles with specific properties, for instance the size and size distribution and the aggregation state for a given material.

In the application work package (WP6) we tested the new synthesized optimized MNP systems for each application area.
Project Results:
The NanoMag project summarizing the actual results are listed below. Also, some recommendations for future work are also listed.

MNP synthesis
• Comprehensive description of synthesis routes
• Extensive synthesis of MNP covering different synthesis routes and MNP properties
• MNP series with gradually changing parameters
• Reproducibility of MNP synthesis in different labs

Improvement of existing analysis methods
• Overview and classification of MNP analysis methods
• SOPs and technical documentation
• Uncertainty budgets (in some cases)

Development of new analysis methods
• Numerical inversion techniques for reconstruction of distributed parameters
• Optical measurement of dynamic susceptibility

Understanding of structural and magnetic properties
• Comprehensive analysis of MNP series with gradually changing parameters
• Characterization of multi-core MNPs
• Neutron scattering measurements in combination with other characterization methods

Definition of standard measurements
• Metrological checklists
• SOPs and technical documentation
• Active participation in ISO/TC229 (ISO 19807 and PG14)

Definition of magnetic nanostructures and quality control
• Definition of structural concepts and terminology for single-core and multi-core particles
• Examples of comprehensive characterization of (complicated) MNPs, (e.g. nanoflowers)
• Quality control (Task 4.2 in the analysis work package WP4)

Dissemination and exploitation
• Stakeholder interaction (four online surveys, workshops)
• E-learning modules, four e-learning modules was developed in the project and available link from NanoMag website and hosted at the NPL website
• Scientific publications
• Commercial exploitation of project results
• Follow-up project EMPIR MagNaStand (start 2017, coordinated by PTB)
• PR activity (EuroNanoForum 2015, NanoMag was voted to be among the 10 best EU projects related to Nanoscience)

Main technical and scientific impacts in the project are listed below.
• Clear nomenclature for describing structure and magnetic properties of MNPs and MNP ensembles
• Improvements in reproducibility of MNP synthesis. Over 50 new MNP systems were synthesized in the project.
• Surveys of measurement methods for MNPs and their pros and cons, classification of these methods including classification of different types of MNP systems
• A stakeholder committee group was formed in the NanoMag project that helped with the MNP surveys performed
• Standard operation procedures, uncertainty budgets and metrological descriptions were developed
• Improved modelling of MNP magnetic properties, especially: dynamic magnetic behaviour
• Correlation analysis between MNP parameters determined by different analysis methods
• Over 80 new publications in international high impact journals and over 160 presentations and seminars
• E-learning modules (NPL-web site, linked from the NanoMag website). 132 people have already enrolled to the first 2 modules. The number is expected to increase significantly when all modules are launched after the end of the project.
• Substantial contribution to ongoing ISO MNP standards (ISO 19807 ” Nanotechnology — Liquid suspension of magnetic nanoparticles — Characteristics and measurements', PG14 "Superparamagnetic beads for free cell DNA extraction)
• Analysis service in the future (information will be collected at NanoMag homepage and PTB data server)
• Continuation with the MagnaStand EU project coordinated by Uwe Steinhoff (PTB).
• Round Robin measurements (same analysis methods but in different labs) using different types of MNP systems

A detailed description of the results in each work package (WP) are given in the following sections.

WP1: Definitions of magnetic nanoparticles
Work undertaken
In WP1 we defined the MNP systems that were synthesized in WP3 and analysed in WP4. We will also decide the additional commercial MNP systems that were analysed in WP2. The work was separated into three parts:
• Definitions of single-core particles
• Definitions of multi-core particles
• Definitions of commercial magnetic nanoparticles

Scientific and Technical Results

3.1.2.1 Definition of single-core particles
In the initialization phase of the project, we have defined relevant parameters to be considered for identifying particles as single-core MNPs. We have listed the main structural and magnetic characteristics of single-core MNPs in D1.1. Single-core MNPs can be produced by several chemical methods. The main routes are the aqueous phase and the organic phase synthesis techniques. An overview of the most commonly used approaches both synthesis techniques has been prepared. As a result of this task, we agreed on a list of single-core MNPs prepared by different synthesis routes with a variety of structural and magnetic characteristics, and different surface modifications. Responsibilities and roles of the project partners in synthesizing these MNPs were defined.

3.1.2.2 Definition of multi-core particles
The main structural and magnetic characteristic of multi-core MNPs were summarized in D1.2. An overview has been compiled of multi-core MNP systems and their synthesis routes being appropriate to produce MNPs for different applications such as magnetic hyperthermia, magnetic resonance imaging magnetic particle imaging, magnetic separation and MNPs for bio-sensing and lab-on-chip platforms. Based on this, we identified multi-core MNPs to be synthesized and characterized in WP3. The most promising MNPs were selected for comprehensive analysis in WP4. We agreed on a list of multi-core MNPs with magnetic cores from magnetite or maghemite, with small size distributions, iron oxide core sizes in the range of 5 nm to 100 nm and hydrodynamic diameters below 100 nm or between 100 nm and 200 nm. The coating materials were dextran or carboxy dextran, starch, silica, polyethylene glycol or corresponding polymer derivatives.

3.1.2.3 Definition of commercial magnetic nanoparticles
We established a comprehensive survey on available commercial single- and multi-core MNPs based on the information of the involved partners in WP1 (MICROMOD, NANOPET, SP, CSIC, ACREO, TUE, UCL, PTB). In report D1.3 we compiled information available from internet product descriptions published on companies’ websites. Four types of commercial players were identified: 1. Biomedical MNP manufacturer and companies placing MNP on the market for in-vitro applications, 2. Medical in-vivo injectable, 3. Technical engineering, and 4. Industrial chemicals supply. The project consortium decided to work with focus on biomedical commercial MNP according to Type 1. Six commercial MNP systems were defined for further analysis. Four of them are single- and multi-core MNPs with hydrodynamic diameter below 100 nm and different magnetic relaxation behaviour at room temperature, and two samples with larger particle size for magnetic separation and micro-sensing applications.

Impact on other Work Packages
The defined single- and multi-core MNPs were synthesised and initially characterized in WP3. The MNPs were utilized in our comprehensively studied in WP4 in order to improve and re-define the analysis methods. The defined MNPs were also used for our application and benchmark activities in WP6. The defined commercial MNPs were characterized in WP2 in order to decide which analysis technique and which models will be used in WP4 and which MNP parameters can be obtained.

WP2: Definitions of analysis methods
Work undertaken
In this work package, we have used the commercial single- and multi-core MNPs defined in WP1 for an initial characterization using various analysis technique. We have investigated the analysis techniques and decided which techniques we use in WP4 for the respective MNP system and which nanoparticle parameters were important for our standardization work in WP5. We have also studied which models are needed in order to determine relevant parameters for a deeper understanding of the MNPs. The work was divided into three parts:
• Analysis of the initial measurements
• Definition of characterization and analysis methods
• Definition of models

Scientific and Technical Results
3.2.2.1 Analysis of the initial measurement
A comprehensive overview (D2.1) has been prepared which comprises the structural and magnetic parameters of the commercial MNPs that we have defined in WP1. Here, we summarized the results obtained by the available analysis techniques in NanoMag. The parameters of the following commercial particle system were summarized:
• Micromod BNF multi-core particle with a hydrodynamic diameter of 80 nm.
• NanoPET FeraSpin-R multi-core particle system with a hydrodynamic diameter of 60 nm.
• Ocean Nanotech SHP25 and SHP20 single-core particles with hydrodynamic diameters of 25 nm and 20 nm respectively.

Along with the results of the characterization of the particle system, the overview D2.1 comprises for each analysis technique and based on the analysis results, a summary of the standard practice measurements including information on the sample preparation and amount, used concentrations, details on measurement systems and parameters, measurement time, calibrations and used models for data analysis. From these information, a measurement matrix has been generated. It gives a summary of the analysis techniques that are available in NanoMag and the accessible structural and magnetic MNP properties.

3.2.2.2 Definition of analysis methods and models
In the definition stage in WP2, a survey was compiled based on information on the data analysis, the structural and magnetic parameters that can be determined, assumptions or additional input parameters for the data analysis and a discussion on possible uncertainties and their magnitude. The survey further comprises information on interrelation with other analysis methods, proposals for measurement improvement and modifications for more reliable output parameters and modelling and simulations for the interpretation of measurement results.

Based on this survey the analysis methods were classified to:

Group 1: Structure, chemical composition and particle size distribution
• Conventional scattering techniques (XRD, ND)
• Small-angle scattering (SAXS, SANS)
• Electron microscopy (SEM, TEM)
• Dynamic light scattering (DLS), electrophoretic light scattering, zeta-potential
• Asymmetrical field flow fractionation (AF4)
• Inductively Coupled Plasma Mass Spectrometry (ICP-MS)
• Mössbauer spectroscopy

Group 2: Temperature and field dependent magnetization and resonance measurements
• Magnetization versus temperature (m vs. T)
• Isothermal magnetization measurements (m v H)
• AC susceptibility vs. temperature (AC v T)
• Ferromagnetic resonance (FMR)

Group 3: Frequency and time dependent magnetization/relaxation measurements
• Magnetorelaxometry (MRX)
• Frequency dependent AC-susceptibility (ACS)
• Magnetic particle spectroscopy (MPS)
• Rotating magnetic field method (RMF)

Group 4: Application oriented magnetic measurements
• NMR T1- and T2-Relaxivity
• Magnetic Separation
• Magnetic Hyperthermia
• Brownian relaxation measurements using on-chip relaxometry using a magnetoresistive sensor and optomagnetic sensing

Models were defined for those analysis techniques where modelling is needed to extract the MNP parameters from the measurement data.

Impact on other Work Packages
The results of the initial characterization were used in WP3 for the comparison of commercial MNPs and new synthesized single- and multi-core MNPs. The identified chemical, structural and magnetic characteristics that can be determined by our defined methods and models were studied in WP4 for the characterization of new synthesized MNPs. We continuously improved our analyses and assess our definitions within WP4 which provided the scientific background for our standardization work in WP5.

WP3: Magnetic nanoparticle synthesis
Work undertaken
In this WP we have synthesised magnetic single-core and multi-core particles with special magnetic, chemical and structural properties according to the definitions in WP1 and we used these MNPs for our analysis work in WP4 in order to define the analysis standard techniques in the standardization method work in WP5. We also used commercial magnetic nanoparticles for the comparison with the new synthesized MNPs.

Scientific and Technical Results
We have identified different synthesis routes susceptible for being standardized for the production of single- (task 3.1) and multi-core (task 3.2) magnetic nanoparticles suspensions for biomedical applications (Milestone MS3), according to the definitions in WP1.
Commercial nanoparticles were chosen from a broad range of manufacturers and suppliers marketing nanoparticles. The quantity and quality of information provided varies significantly between. The comparison of different commercial with new synthesized nanoparticles (WP2) was challenging and not feasible without their in-house characterization (task 3.3). For example, the Fe concentration provided by the manufacturer, which was critical for WP2, was not reliable and differed significantly from the actual measured values. Consequently, we elaborated a report describing the best way to determine the Fe concentration in those suspensions to assure uncertainty below 3%.
The resulting samples have been characterized by particle size, hydrodynamic size, Fe concentration, osmolality, pH, zeta potential and dispersion stability (task 3.4) and they have been compared with commercial samples selected in WP1 (task 3.3 D3.3). Only those having long-term colloidal stability (months) were distributed to WP4 and WP6 (D3.1 and D3.2) and a technical data sheets was designed.
We have developed standard operating procedures (SOP), describing the synthesis and surface modification of these iron oxide magnetic nanoparticles to obtain colloidal suspensions in water at pH 7 following recommendations from WP5. Second batches of single-core (D3.4) and multi-core particles (D3.5) prepared following these SOPs were delivered to WP4 and WP6.
We have analyzed the reproducibility of the selected synthesis methods and the inter-lab reproducibility based on the characterization results provided by WP4. The SOPs have been extended including more details on the experimental procedures and finally, non-expert labs have been invited to carry out the synthesis following the extended SOPs (work in progress).
We have analysed the key parameters controlling particle size, aggregation and colloidal stability. Analyses of the mechanism of particle formation and degradation have also been done in order to improve reproducibility and reaction yield.
A tight collaboration has been stablished between synthesis groups in WP3 for improving the synthesis of magnetic nanoparticles and its dispersion in water. Samples produced in this WP present high uniformity, good crystallinity, long term stability and good control of core, particle and hydrodynamic size, better than most of the commercial samples.

3.3.2.1 Selected MNP samples
Single-core particles
Magnetic nanoparticles with very low polydispersity (<10%) have been synthesized by high temperature decomposition of iron oleate and they have been coated with silica via microemulsions (Sample CSIC01) and dimercaptosuccinic acid, DMSA (Sample CSIC12) leading to single core particles (Fig. 1A). Silica and DMSA were chosen for the coating since they are well-studied materials, easily functionalizable and biocompatible. Silica and DMSA has been chosen for the coating since it is a well-studied material, easily functionalizable and biocompatible. The synthesis and coating methods were optimized to control the particle size and coating thickness.

Other method that was explored to obtain single core particles without size selection was this involving the synthesis of antiferromagnetic precursors which were subsequently coated with silica and reduced to magnetite without change in morphology. Cores with different morphology were obtained. First, this approach has the advantage of low inter-particle interactions of as-synthesized antiferromagnetic nanoparticles. Moreover, it covers different core size ranges and allows obtaining different morphologies such as rhombohedra (CSIC09), discs or needles below 200 nm [4].
The synthesis and coating methods were optimized to control the particle size and coating thickness. On the other hand, cores with different morphology have been synthesized from antiferromagnetic precursors which were subsequently coated and reduced to magnetite without change in morphology. Examples of different core size from 12 to 20 nm, same coating thickness, different coating thickness from 14 to 24 nm, same core size 16 nm and different morphology, same silica coating.

Multi-core particles
Controlling number of cores per aggregates, same core size
BNF-Starch particles with nominal diameters of 80 nm and 100 nm were selected as commercially available multi-core particles from Micromod in WP1. Within WP3 Micromod followed different strategies to modify the coating process of the iron oxide cores of BNF particles (around 20 nm core size) to change the number of cores per aggregate and to obtain particles with a more uniform number of iron oxide crystals per particle to improve the magnetic particle properties. Magnetic fractionation techniques and the peptization of iron oxide cores before coating led to a smaller and more uniform number of iron oxide cores per particle.

The commercial and biocompatible sample FeraSpin R, which is synthesized by aqueous coprecipitation of Fe(II) and Fe(III) in the presence of carboxydextran as stabilizing agent, consists of clustered 5 nm iron oxide nanoparticles forming aggregates of various sizes. Via magnetic fractionation different narrowly distributed size fractions of these aggregates were obtained by NanoPET, while all aggregates are composed of the same sized cores having a diameter of about 5 nm. From these the smallest size fraction FeraSpin XS, containing solely single-core particles, and a medium size fraction FeraSpin L were chosen for an extensive characterization in WP4.

Controlling core-core interaction
Different strategies to obtain flower-shaped iron oxide assemblies in the size range 25-100 nm were examined. The routes are based on the partial oxidation of Fe(OH)2, polyol mediated synthesis or the reduction of iron acetylacetonate. The nanoparticles are functionalized either with dextran or citric acid and their long-term stability is assessed. Key synthesis parameters driving the self-assembly process capable of organizing colloidal magnetic cores into highly regular and reproducible multi-core nanoparticles were determined. This is the first step towards standardized protocols of synthesis and characterization of flower-shaped nanoparticles.

Different core size, same particle size
The polyol-mediated synthesis has been explored and developed for the preparation of well-controlled magnetic nanoparticles with different core size and arrangement to form the final multicore particles. A prolonged heating of the flowers (nanoflower nanoparticles) leads to particles with larger cores with interesting magnetic and colloidal properties.

Different coating
In order to have a broad portfolio of different nanoparticles covering a wide range of physical and magnetic properties, several other synthesis techniques and coating materials were utilized [11-14]. NPG3310 for example was synthesized by a partial oxidation pathway and coated by the biocompatible polymer dextran, inducing a steric stabilization. By using citric acid and coprecipitation, charge stabilised nanoparticles have been synthesized. Nanoparticles, being stabilised sterically and by charge, have been synthesized by using poly(acrylic acid) as coating material.
Flower like particles of 100 nm core sizes have also been coated with dextran, citric acid, amino propyl silane (APS) and amino dextran with interesting colloidal properties and different surface charge, going from positive (+42 mV) with animodextran to negative (-30 mV) with citric acid and Z average sizes around 200 nm.

Different super-structure
SP synthesized nanoflowers (SP06) by means of sodium borohydride, which acts as reducing agent of iron (III) acetylacetonate (Fe(acac)3). Nanoflowers synthesized by this route were embedded on polystyrene spheres via the emulsion solvent evaporation (ESE) process or encapsulated forming super-structures. The differences in nature of aggregation of the magnetic cores could lead to different modes of inter-particle magnetic interactions. This would have an impact on application oriented properties such as NMR relaxivities, hyperthermia etc.
Interesting series from single- to multi-core MNPs
Interesting series of magnetic suspensions going form multicore to single core nanoparticles were obtained with particles made of 5 nm cores by NanoPET as mentioned before (FeraSpain samples) and for 30 and 60 nm cores prepared by ICMM [15] and Micromod by magnetic fractionation after dextran coating (CSIC04, 5, 6) and sedimentation fractionation after PAA-Na coatings with different molecular weights (CSIC08). We observed excellent reproducibility of CSIC05 and CSIC04.
Single-core and multi-core, including hollow spheres and nanoflowers, can be prepared by the polyol process. Sodium acetate (NaAc) can control the nucleation and assembly process to obtain the different particle morphologies that can be further stabilized in water by coating with citric acid. The particles are formed by burst nucleation and growth processes that determine the final nanostructure [2].
Finally, by controlling the internal structure of anisometric particles [4], we have developed single and multicore particles. The possibility of generating a discontinuous structure within a particle by forcing the pore formation may be an interesting strategy to develop new materials with tuned magnetic properties for biomedical applications.
Multi-core nanoparticles prepared in different polyol media (DEG/EG) and in the presence of different reactants such as NaAc and NMDEA that modify the viscosity and the boiling point of the media [5], [6].

Impact on other Work Packages
The single- and multi-core MNPs synthesized in WP3 were characterized in WP4 and WP6.
Synthesized particles with defined properties were used in WP4 to verify, improve and harmonize the analysis methods to be standardized in WP5.

WP4: Magnetic nanoparticle analysis
Work undertaken
In WP4 we carried out the characterization and analysis work and we used the analysis methods and models that were defined in WP2. We have classified the analysis methods according standard methods that were used for all MNP samples and more specialized methods used for selected MNP systems. We studied single- and multi-core MNPs that were synthesized in WP3 and we compared our results with commercial MNP systems. We correlated the results obtained by different analysis techniques in order to get a self-consistent picture of the MNP systems. The work was divided into four parts:
• Characterization and analysis results from the single-core nanoparticles
• Characterization and analysis results from the multi-core nanoparticles
• Finely tuned characterization and analysis methods for the single-core and multi-core nanoparticles
• Correlation between analysis techniques

Scientific and Technical Results

Terminology for magnetic nanoparticles
A clear terminology for describing the structural and magnetic properties of single- and multi-core MNPs and MNP ensembles has been developed and published in [7]. Here, we also considered those definitions for MNPs that are available in existing standard documents. Such a summary of definitions of the MNP components was essential for comparing the results among the project partners and among different measurement techniques, and it is indispensable for a coherent standardization structure MNP samples.

Classification of magnetic nanoparticles
The outcome of our characterization work in WP4 led to a classification of nanoparticles according to their number of cores inside a particle which leads to differentiation in single- and multi-core MNPs, and we reviewed different approaches for the synthesis of these MNP systems [8]. Single-core MNPs contain one magnetic core per particle and the core is covered by a matrix to prevent aggregation and agglomeration. In multi-core MNPs several cores exist within the matrix forming an individual unity.
The arrangement of cores within a MNP sample has significant influence on the magnetic properties. Whereas in single-core MNPs in suspension the magnetic moment of the particle cores can rotate via Néel and Brownian rotation, in multi-core MNP systems Brownian rotation is suppressed as the cores are anchored in the matrix. Only Brownian rotation of the entire particle is allowed in this system. In addition to that, for describing the static and dynamic magnetization behavior, magnetic interaction between the cores in multi-core structures may become relevant if the cores are closely packed. This led to the concept described in [9], where we classified MNP systems according to their magnetic relaxation properties and particle size parameters which are determined by utilizing magnetic analysis techniques such as ACS or MRX and techniques like TEM and DLS capable to probe the core and particle size and the hydrodynamic size.

The complex inner structure of multi-core MNPs lead to unique magnetic properties and can explain for example their high performance in magnetic particle imaging [10], [11] or other biomedical applications [12]. This shows that especially multi-core MNPs needed a comprehensive characterization and a careful interpretation of structural and magnetic parameters including the correlation of different MNP parameters.
The so-called flower-shaped MNPs [13] or “nanoflowers”, as a special type of multi-core MNPs, have been intensively studied within WP4. These multi-core particles consist of clusters of strongly coupled cores which exhibit unique magnetic properties and thus they are potentially relevant in for example magnetic hyperthermia. The focus of the characterization work was on the exploration of interaction between the nanocrystals inside an MNP. Thus, in [1] we classified the flower-shaped MNPs according their interaction between the cores and between the particles which can be adjusted by different synthesis routines. This enables us to tune the static and dynamic magnetic properties of MNPs for specific applications.

3.4.2.1 Classification of analysis methods
A classification of analysis methods into standard and advanced analysis techniques has been created and published in [14]. Standard techniques have large potential for standardization, whereas more advanced methods that provide additional information in order to gain a more detailed picture of an MNP system, however, due to their complexity and availability, these methods have limited potential to pose as standard methods for MNP characterization. Partners of the NanoMag consortium being experts in the field of nanoparticle research have assessed the analysis techniques considering different scientific, technological and economic aspects [14].
We furthermore developed a measurement matrix which provides information on the sample requirements (amount and sample forms) for each analysis technique and we gave an overview of the estimated cost of a single measurement using the technique and the types of sample, the method should be applied to.

The assessment of the analysis methods as a classification, resulted into standard methods, advanced methods and intermediate methods. The latter could potentially serve as standard methods; however, their results have to be correlated with those obtained by other techniques. Note that some methods belong to several categories (e.g. Mössbauer and ND providing both structural and magnetic information). Detailed information on assessment of analysis methods have been published in [14] and in our report D4.1.
The results of this task had consequences for our subsequent analysis work as we used the standard methods for the characterization of all NanoMag samples, whereas the advanced methods are used to gain a deeper understanding of selected MNP systems. Furthermore, we have identified for our standardization work in WP5 a selection of commonly used techniques, which should undergo a standardization process with respect to the properties of MNPs. The experience from the present analysis of characterization methods for MNP provided valuable arguments in ongoing formal ISO standardization processes.

3.4.2.2 Characterization and analysis results from the single-core MNPs
Our report D4.2 provided a detailed description of the structure and the magnetism of single-core MNPs and MNP ensembles and we pointed out that only a few measurement techniques are available which provide information on a single nanoparticle. The majority of techniques measure the properties of an MNP ensemble and in the simplest case, the MNPs can be considered to be identical. However, in practise, the MNPs exhibit some distribution of parameters, e.g. for MNP size, magnetic moment, magnetic anisotropy, etc. Here often a certain functional form for the probability density function in the model is assumed and is most cases a lognormal distribution is used. An alternative to that is the regularized inversion method which we have developed and published in [15]. The main advantage of the numerical inversion method is that no ad hoc assumptions regarding the line shape of the extracted distribution functions are required. This approach has been verified by comparing the results with the results obtained by standard model fits, i.e. where lognormal distribution was assumed.
Along with the basic description of single-core MNPs and ensembles our report D4.2 gave comprehensive summary of analysis methods and models for single-core MNP description. The information on each analysis techniques are summarized according the metrological checklist [16] which provides a guideline for the development of a standard. We furthermore presented an overview of the results of structural and magnetic characterization of single-core MNPs which have been synthesized within the project. As a consequence of the variety of analysis methods, there is some redundancy in parameters which helps one to verify and refine models. In [17] we have published the results of the comparison of single-core MNP sizes determined by different non-magnetic and magnetic analysis techniques. It was found that the mean core diameters determined from TEM, DCM, ACS and MRX measurements agree well although they are based on different models. We found good agreement among the results which proved the applicability of these techniques and their related models for the characterization of single-core nanoparticles. In a similar study, we found for single-core MNPs also very good agreement between core diameter obtained by TEM, DCM and MRX [18]. However, it turned out that in the investigation of multi-core MNPs large differences were present mainly caused by the applied models and the complex core structure.
In [4] we correlated structural and magnetic properties of large single-core MNPs using a variety of analysis methods was published in. We could demonstrate how the magnetic behavior is affected by the shape of the MNPs, their internal structure and the reduction process.
We could demonstrate in [19] that measurements of the complex susceptibility as a function of frequency on suspensions of thermally blocked MNP allow the simultaneous estimation of the hydrodynamic size and of the effective anisotropy constant.
The magnetic field dependence of the Brownian and Néel relaxation has been investigated using two single-core MNP systems [20]. Here we have shown that we are capable to model the dynamic magnetic behavior as a function of field amplitude and under application of an ac field superimposed by a static magnetic field.
In [21] the micro- and mesostructure of self-assembled mesocrystals composed of nanocubes with different edge lengths in the absence and presence of an applied magnetic field has been investigated. The results of this study are summarized in a qualitative phase diagram which outlines the preparation of mesocrystals and arrays with tunable micro- and mesostructure.

3.4.2.3 Characterization and analysis results from the multi-core MNPs
In our report D4.3 we presented a detailed description of the inner structure and the magnetism of multi-core MNPs. As in most cases interactions between the cores inside a nanoparticle must be considered for describing the magnetism of multi-core MNPs, we gave a detailed description of the magnetic interaction and relaxation properties on the basis of multi-core and flower-shaped MNPs. Report D4.3 also presented a comprehensive survey on the structural and magnetic properties of a selection of four multi-core MNPs that have been synthesized in our project. We summarized here information on each analysis techniques and models in accordance to the metrological checklist [16] and we considered particularities which arise from interactions in multi-core MNPs.
We were able to disclose the distribution of cores inside the MNPs and we could explain the magnetic behaviour which is significant affected by dipolar interactions between the cores [22]. Therefore, we studied the structural and magnetic properties of multi-core MNPs using a generalised numerical inversion technique.
In [23] we could show on the commercial FeraSpin series synthesized by nanoPET that FeraSpin XS can be described as individual nearly spherical single-core MNPs, while FeraSpin L consists of larger, slightly elongated clusters in which the interaction between the cores significantly alters the magnetic properties.
We comprehensively studied flower-shaped MNPs diverting core sizes and different packing densities inside a particle and thus variating interactions [1]. By comparing the results obtained from various analysis methods, we could disclose the inner structure of the core-clusters and link them to their magnetic properties. The heat generation of the flower-shaped MNPs in different stages of the synthesis was determined in [2] and compared with other particle types.
We explored the effect of the alignment of the magnetic easy axes on the dynamic magnetization of immobilized multi-core MNPs under an AC excitation field [24] and we obtained quantitative agreement between experiment and simulation. These results indicate that the dynamic magnetization of immobilized MNPs is significantly affected by the alignment of the easy axes.
Characterization of multi-core MNPs with application oriented methods
Large effort has been made in characterizing multi-core MNPs with application oriented methods with the aim to link the structural and magnetic characteristic and their performance in final application. We could show for example, how the amplitude and shape of the MPS spectra is affected by different physiological media [25]. Additionally, the observed linear correlation between MPS amplitude and shape alterations can be used to reduce the quantification uncertainty for MNP suspended in a biological environment.
In [26] we proposed a dual-frequency acquisition scheme to enhance sensitivity and contrast in the detection of different particle mobilities compared to a standard single-frequency MPI protocol. The method takes advantage of the fact, that the magnetization response of the tracer is strongly frequency-dependent, i.e. for low excitation frequencies a stronger Brownian contribution is observed.
In [27] we studied the structure and the magnetism of MNPs that were synthesized by a promising diffusion-controlled synthesis. From the characterization, we could demonstrate that a diffusion-controlled synthesis approach process allows to produce MNP suspensions with a large fraction of particles exhibiting a mean effective magnetic core size of about 28 nm which lies within the size range considered ideal for MPI.
We revealed how the particle size and concentration have influence on the separation of multi-core MNPs [28]. It was found that an increasing particle concentration leads to a reduction of the separation time for large nanoparticles due to the higher probability of building chains. For smaller MNPs the chain-formation is suppressed due to faster thermal fluctuation which led to concentration-independent separation times.
In [29] we explored the suitability of multi-core-MNPs in different polymer matrices to be used in MRI and we compared the MNPs with a MNP system approved for clinical use as contrast agent. Along with a comprehensive structural and magnetic analysis we could show that the synthesized MNPs exhibited higher R2 relaxivities and R2/R1 ratios compared to the commercial MNPs indicating their potential as new MRI contrast agents.
The effect of nanoclustering and dipolar interactions on the efficiency in magnetic hyperthermia was analyzed in [30]. We have shown that the magnetic hyperthermia performances of nanoclusters and single nanoparticles are distinctive and that nanoclustering of particles with randomly oriented easy axes is detrimental to the SAR. A decrease in ILP is observed when the nanocluster size and number of particles in the nanoclusters increase. This result is very interesting as in nanoflowers, where the cores inside the particles exhibit the same crystalline orientation, the SAR increases with increasing cluster size. For the individual MNPs, the SAR depends on particle concentration and thus increasing dipolar interaction.
We explored ellipsoidal MNPs subjected to an external AC magnetic field. First, the heat release is increased due to the additional shape anisotropy [31]. The rods can also dynamically reorientate perpendicular to the AC field direction. Importantly, the heating performance and the directional orientation can be controlled by changing the AC field treatment duration, thus opening the pathway to combined hyperthermic/mechanical nanoactuators for biomedicine.

3.4.2.4 Finely tuned characterization and analysis methods for the single-core and multi-core nanoparticles
The interaction among the NanoMag partners allowed to synthesize series of MNPs with varying structural and magnetic parameters. These model MNPs have been used to verify and to improve our analysis methods and the physical models.
Measurements on MNP series
As described in our report D4.4 a characterization strategy has been developed for MNPs which consist of the identical cores but different assembly size, with the aim to provide a consistent procedure for standardized analysis of multi-core nanoparticles. Along with a comprehensive structural and magnetic characterization we could show that the number of cores inside a MNP has significant influence on the heat generation in magnetic hyperthermia.Alongside other analysis techniques we utilized FMR and AF4 to derive a comprehensive understanding of the FeraSpin series synthesized by nanoPET [23]. We could disclose the coupling between the cores and we could show that FeraSpin R can be described as a superposition of the size fractions FeraSpin XS and L.

Verified and improved analysis methods
In [32] we demonstrated that improved SAXS in combination with SLS measurements delivered estimates of these morphological parameters and this information can then be transferred into a mean core distance inside the multi-core structure.
In [33] we could prove that ACS is capable to simulate the dynamic magnetic response of a sample possess a bimodal size distribution, proposed that the two particle sizes result in sufficiently deviating time constants. The numerical approach is more reliable when one particle fraction relaxes via Néel and the other via Brownian mechanism. Using Monte Carlo simulations for fitting the SAXS, we are also able to resolve the bimodality in the particle size. This result is important since only a few methods that can be used to dissolve a size distribution with more than one fraction.
The dependency of the Brownian and Néel relaxation times were studied by ACS as a function of frequency and field amplitude [20]. It was found that the Néel relaxation time decays much faster with increasing field amplitude than the Brownian one. Whereas the dependence of the Brownian relaxation time on the ac and dc field amplitude can be well explained with existing theoretical models, a proper model for the dependence of the Néel relaxation time on ac field amplitude for particles with random distribution of easy axes is still lacking. These findings are of great importance of applications where larger magnetic fields are used, e.g. MPI and magnetic hyperthermia.
In [34] we presented a new experimental approach to characterize an MNP system with respect to quantitative MRI. We could show that the hydrodynamic fractionation by AF4 and the subsequent structural and magnetic characterization of the size fractions by DLS, MALS, MPS and NMR enables us to evaluate the suitability of a MNP system for quantitative MRI and verify the theoretical predictions for the size dependence of relaxation rates at the same time. The approach could facilitate the choice of MNPs for quantitative MRI and helps clarifying the relationship between size, magnetism and relaxivity of MNPs in the future.
Along with the numeric inversion method presented above, we have developed a new method based on the iterative Kaczmarz algorithm that enables the reconstruction of the size distribution from magnetization measurements without a priori knowledge of the distribution form [35]. We concluded that this method is a powerful and intuitive tool for reconstructing particle size distributions from magnetization measurements. We have also used the Kaczmarz' algorithm for the determination of hydrodynamic size distribution from MRX measurements and we could show that this method is able to determine the hydrodynamic size distribution in agreement with either the known input distribution, in the case of simulated data, or other size estimates determined with different methods such as thermal magnetic noise spectroscopy and dynamic light scattering in the case of measured data [36].
In [37] we could identify the similarities and the differences in MRX and thermal magnetic noise spectroscopy. Both techniques are based on the same physical principle, i.e. the thermal fluctuations of the magnetic moment.
A new MPS setup was built which allows temperature dependent measurements in order to investigate the temperature dependence of the harmonics spectra [38].

Standard operation procedures
In preparation of our standardization work in WP5, Standard Operating Procedures (SOPs) for each analysis methods have been developed within WP4. For analysis methods being available at different facilities, a harmonized standard operation procedure has been compiled. The SOPs were published in our report D5.2. On the basis of the prepared SOPs, we have performed Round Robin tests, where we compared the MNP parameters measured by different partners. The results of these comparison studies were utilized to continuously improve the SOPs and to identify influence factors that were relevant for discrepancies among the results. However, within the project duration we were not able to finish our studies and to measure identical values at different institutes. This is still a challenge for future research projects.

Uncertainty budget calculation
In a Mössbauer study [39], we were able to identify independent influence factors that are relevant for the uncertainty of the mean isomer shift, used for the quantification of the magnetite content in MNPs, and we could derive a quantitative expression for the uncertainty budget. This concept has been applied at two different laboratories where the magnetite fraction values determined from the Mössbauer measurements agree within their respective uncertainties. This activity served as a model project for other analysis techniques in order to develop an uncertainty budget for all derived structural and magnetic parameters that are relevant for the reliable characterization of MNPs. Within the project we could identify the main influence factors for uncertainty for most of the analysis methods, however, we could not achieve the preparation of uncertainty budgets for all techniques. This should be solved in future research projects, for instance the newly started MagNaStand project in July 2017, coordinated by PTB where some of the NanoMag partners are participating (PTB, RISE Acreo, UCL, Micromod) as well as new partners from both industry and metrological institutes.

3.4.2.5 Correlation between analysis techniques
The comparison of MNP parameters is of utmost importance in order to get a self-consistent description of MNP systems. Consequently, we correlated our results in most of our activities. Our public report D4.5 gives an overview of the main results of a) correlation between the same MNP parameter determined by different techniques and b) the comparison of different MNP parameters. A consistent picture was found for the hydrodynamic particle size. However, quite some scatter between data was observed for core parameters, such as core size, effective anisotropy constant, and magnetic moment.
It was found that for single-core nanoparticles a number of methods consistently provide the same value for the core size and hydrodynamic size distribution. Slight differences can be attributed e.g. to the applied models. The situation is somewhat more complicated for multi-core MNP and nanoflowers.
Although there is a variety of analysis methods e.g. for determining the core size (TEM, DCM, MRX, ACS, SAXS, etc.), there is no best method which could be identified as standard one. However, the good agreement of size parameters, found when comparing techniques applied to the same sample, indicates that basically any of the methods can be applied.
The estimation of the effective anisotropy constant K remains an open task. Although a number of magnetic methods provide information on K, the physical background is manifold, ranging from the determination of the anisotropy energy via the Néel relaxation time to the intra-potential-well contribution in the dynamic susceptibility and the blocking temperature. A major problem for estimating the anisotropy constant applying the various methods is that all techniques require their very specific nanoparticles.
A remaining challenge for future research will be a better understanding of the effective parameters and their correlations for multi-core MNP and nanoflowers.

Impact on other Work Packages
The results and the finding in this work package have been utilized in WP3 for the improvement of MNPs and to synthesize MNPs with specific properties.
The scientific results of our work on the classification of MNP and the classification of analysis methods have been used in our standardization work in WP5.
The characterization of single- and multi-core MNPs has been utilized to harmonize and to improve the analysis methods. The correlation of results led to a better understanding of link between the structural and magnetic properties of MNPs. As a consequence, our achievements were relevant in the standardization work in WP5 and it provided the technological background for our cooperation in the ISO/TC 229.
The partners in WP4 have provided the technical and scientific content for the preparation of a SOP for each analysis technique considering the mandatory elements of the metrological checklist. This work was important for WP5 as standard operation procedures are an essential step toward a standardized MNP characterization. Furthermore, the development of uncertainty budgets was crucial for our standardization work
The extracted structural and magnetic properties were relevant in WP6 for the verification and improvement of application measurements.

WP5: Standardization of magnetic nanoparticles
Work undertaken
In WP5 we studied the results from the synthesis work in WP3 and the analysis result of WP4 obtained on new synthesized and commercial MNPs and we have defined standardization methods and parameters. The work was divided into three parts:
• Standardization strategies of magnetic nanoparticle systems
• Defined standardization methods and relevant parameters
• Standardization roadmap

Scientific and Technical Results

3.5.2.1 Standardization work strategies
Our report D5.1 presented an overview of the identified key physical parameters and the defined terminology to be used for classification and description of magnetic nanoparticle systems. This overview represented the state-of-the-art in science and technology which can be found in literature considering also existing standards and our knowledge we gained in WP4.
We further summarized commonly used analysis techniques for the characterization of MNPs. Here, we have drawn together the information contained within existing reports submitted by the NanoMag partners, and to structure it in a manner similar to the requirements listed within the ISO/IEC directives for the drafting of international standards.
We gave an overview of existing standardization work on sampling definitions, characteristics and measurements relating to MNPs, labelling, MNP manufacturing and on standard reference material for the MNP characterization. Finally, the standardization work strategies report D5.1 contained information on the current and future need for metrology of MNPs.
The main results of this task have been published in [7]. Along with a summary of the state-of-the-art in MNP science, the paper represents significant opportunities for future research projects, and are intended to provide an overview or roadmap of major topics which should be addressed within the coming years. This work is intended to act as a precursor to the future development of MNP standards. The standardization work and result are also reported in D5.2 (Defined standardization methods and relevant parameters) and D5.3 (Standardization road map).
A result of this activity is a mix-and-match approach as a combinatorial map showing the suggested structure around which standards relating to MNP suspensions may be built. Underlying all of the other document types are the vocabulary standards and materials specifications class, whose content feeds into the development of each of the other document types. The connections between the different classes, and the manner in which they build upon the content of others is depicted by the arrows.
The proposed structure is intended to allow the development and cross referencing of many interlinked standards with the least possible confusion or amendment. Each application standard can draw upon the specific documents which are relevant to it. Additional measurement and application standards may be developed as and when the need arises.

3.5.2.2 Defined standardization methods and relevant parameters
SOPs that have been developed in WP4 for all measurement techniques we used in NanoMag were published in D5.2. The SOPs have been prepared on the basis of a template [40] where the relevant points needed for a successfully preparation of a SOP are summarized. Furthermore, already existing standardization work was also considered in the SOPs. Furthermore, the SOPs have been established according the metrological checklist [16] providing guideline for a development of a standard. The SOPs have been used by the analysis partner for a standardized characterization of single- and multi-core MNPs.
The synthesis project partners in WP3 have also developed SOPs describing established and harmonized synthesis routine to produce MNP reference systems. These reference systems cover single- and multi-core MNPs that may be used to test and verify different harmonized characterization procedures. The developed SOPs (analysis and synthesis) has only been used by NanoMag partners during the time of the project but will be used outside the NanoMag consortium in the new started MagNaStand project (EMPIR project coordinated by PTB).
We classified the relevant parameters according to parameters needed to identify the material, structural and magnetic parameters and properties assessing the MNP performance in final application. Here, we took up the NanoMag achievements and we considered also the outcome of our third survey where project partners and stakeholders have been asked what are the most important MNP properties that could be defined for an international standard. In order to transfer all our results to the ISO/TC 229 “Nanotechnology” and to the currently developing standard ISO 19807 [41] and ISO TC/229 N 1421 [42], four NanoMag partners (SP, NPL, PTB, CSIC) sent technical experts to their national standardization organizations, with the exception of CSIC, we entered also ISO/TC229 WG4 “Nanotechnologies- Material description” as technical experts. There, we used the NanoMag knowledge for active participation in the development of new standards, which was also acknowledged by the convenor of the committee.
Furthermore, within the NanoMag project, we gave recommendations of entire MNP analyses utilizing a variety of characterization methods. Flow charts illustrate divergent approaches of a particle system analysis focusing different requirements to the characterization output.
We made a proposal for a unified technical specification sheet that contains the relevant MNP parameters.
Another important finding of the NanoMag work is that the decision which MNP parameter are relevant in application and thus are important for standardization, this decision strongly depends on the specific application. There is not even one parameter that is always needed in the description of MNPs.

3.5.2.3 Standardization road map
Our report D5.3 (standardization road map) summarizes the standardization work within the NanoMag project. After a short survey of the definitions and the relevant parameters of MNPs, we illustrated the current market trends and application of MNPs. We further showed our standardization strategy (see Figure 3) that has been developed in [7].
We highlight the status and the roadmaps of European standardization bodies (CEN/IEC) with respect to the standardization of nanoobjects and for ISO/TC 229 “Nanotechnology”, where we in detail present the current and future activities in working group (WG)1 “Terminology and Nomenclature”, WG2 “Metrology and Characterization”, WG3 “Health, Safety and Environment” and WG4 “Material Specifications”.
The standardization of MNP definitions and terminology has the highest hierarchy for standardization. We thus gave an overview of which existing standard documents already cover the characteristics of MNPs and we gave a recommendation for a roadmap for MNP terminology standardization.
For the most relevant MNPs parameters and relating characterization methods that have been identified during our work and which were revealed by surveys, we summarized the available standards.
We further noticed that for none of the relevant MNP biomedical applications standard documents exist so far and we thus recommended also a roadmap for MNP application standardization.
Impact on other Work Packages
We gave feedback to WP3 regarding the synthesis of MNP system with specific properties with the aim to verify and harmonize analysis methods to be used for standardization.We were in strong collaboration with WP4 concerning the preparation of SOPs and Round Robin tests. We supported the partners in WP3 and WP4 in case of metrological issues and we provided the basis for the preparation of uncertainty budgets in WP4. We have continuously informed all NanoMag partners about the current activities at ISO to that they had the opportunity to contribute in the development of work items.

WP6: Application and benchmarking
Work undertaken
This work package concerned the application and benchmarking of nanoparticles produced within the project in WP3 as well as commercially available nanoparticles with the goal of tailoring the particle properties to achieve improved performance in the chosen applications. Three application oriented techniques were selected for this investigation, where each technique had its own set of requirements:
(1) Magnetic particle rotation
(2) Brownian relaxation
(3) Magnetic particle imaging
Below, we report the main results and achievements separately for each of the techniques and list the publications resulting from the work.

Scientific and Technical Results

3.6.2.1 Magnetic particle rotation
The work was divided in three tasks: First we demonstrated the feasibility of measuring the nano-mechanical stiffness of single proteins with MMPs (Task 6.1). In the next phase (Task 6.2) we investigated whether the nano-mechanical stiffness could be related to the change in shape also denoted as the conformation of a protein, which determines its function. In this study, we focused on cardiac troponin, a protein released into the bloodstream upon heart failure and therefore an important biomarker for cardiovascular diseases. In the third phase (Task 6.3) we explored the possibility to probe the interaction between functionalized MMPs during their approach to functionalized substrates, an important requisite for creating sandwiched proteins in applications.
All experimental work was carried out using a home-built quadrupole electromagnet, which acts as a magnetic torque tweezer in combination with a microscope in order to monitor the rotation of the MMPs. In order to visualize the rotation of a single MMP with the microscope, we used fluorescent particle labels to break the rotational symmetry. Within the project we used both commercial particles (Dynal M-270, Thermo-Fisher) as well as particles synthesized in the NanoMag consortium by partner SP. The latter turned out to have a broad size distribution and the biofunctionalization turned out to be significantly more challenging than for the commercially available particles. As a result, we decided to continue using the commercially available Dynal M-270 particles. These particles were analysed within the consortium using standard techniques described in WP5 as well as non-standard techniques quantifying the torque described in the deliverables 6.1 and 6.2.
An impressive result, which illustrates the achieved goals of both task 6.1 and 6.2 is the angular deformation of a cardiac troponin protein complex as function of time in a rotating magnetic field (Task 6.1). This work showed a reversible change in twisting amplitude that can be related to the conformation of the troponin complex present in a flow cell when the local calcium concentration is altered. These conformations correspond to different stages of heart muscle contraction induced by the local calcium concentration (Task 6.2).
In biosensor applications, MMPs are magnetically pulled towards a surface to form the sandwiches that are described above. The interaction potential of functionalized MMPs with the antibody coated surfaces determines to a large extent the type of bond that will be formed and thereby the sensor performance. In task 6.3 we explored probing this interaction potential with rotating MMPs. We analysed both the magnetic field-induced particle rotation and the thermally induced random Brownian motion of the particle when it approaches the surface. We varied the distance between the particles and the surface by using a magnetic field to pull the particles towards the surface as well as the dilution of the buffer which influences the electrostatic repulsion between the particle and the surface.
The measurements successfully identify two regimes of interactions that can be described by the particle surface distance. These regimes can be explained by the particle-surface interaction potential (DLVO theory), which predicts two energy minima separated by an energy barrier.
Experimental parameters determine that particles are either within a few nanometers from the surface with a bond type that can be identified by the particle rotation behaviour (the primary minimum) or further away in a secondary minimum. In this secondary minimum MMPs rotate with a significant friction to the surface identified by the increasing phase lag to the rotating magnetic field. Here, the particles do not form a molecular bond indicated by the relatively large translational, Brownian, motion amplitudes.
These measurements prove that magnetic particle rotation can be used to probe the interaction between a functionalized MMP and a surface, which can be applied to optimize biosensor performance. The results obtained in this activity are documented in [43] and in the manuscripts [44], [45].

3.6.2.2 Brownian Relaxation
This activity utilizes nanosized particles with sizes in the range of 100 nm and below to detect and investigate biomolecules in a sample based on changes in the hydrodynamic size of the particles. The ability of a particle to rotate in response to a rotating or oscillating magnetic field (Brownian relaxation) depends on its size – smaller particles can rotate faster and follow the oscillating magnetic field up to higher frequencies than larger particles. Therefore, measurements of the ability of the particles to rotate as function of the frequency of the magnetic field can be used to estimate the size of the particles with attached molecules. Several ways to use such measurements for bio-detection have been investigated in the project.
The technique employed in the project is a newly developed and promising optomagnetic (OM) technique that measures the intensity of light transmitted through a suspension of nanoparticles in response to an oscillating magnetic field applied either along or perpendicular to the light path. The technique has been further developed in the NanoMag project and evaluated on all new synthesised MNP systems in the project. The technique takes advantage of the experimental fact that many particle systems are not spherical and have a remanent magnetic moment along their long axis. Therefore, an applied oscillating magnetic field produces a modulation of the intensity of transmitted light as the particles rotate to align their magnetic moments along the magnetic field.
In the project we have developed several setups for optomagnetic measurements (Task 6.4):

(1) a setup that combines centrifugal microfluidic disk with OM measurements [46]
(2) a setup that allows for simultaneous real-time measurements at a controllable temperature in four chips [47]
(3) a setup that allows for easy change of wavelength of the light

The principle of the technique has been described in detail by Fock et al [48].
We have developed a new quantitative method to determine the magnetic moment and the hydrodynamic size of particles using OM measurements [48]. The method was applied to all particle systems synthetized in WP3 and on some commercially available particle systems to identify the particle systems with the best performance and to gain knowledge on which of the parameters of the particle systems that are important for the quality of measurements on the particle suspensions (Task 6.5). We have also developed a method, which uses AC Susceptibility (ACS) and OM measurements vs. field and frequency to obtain directly the number-weighted magnetic moment and hydrodynamic size distribution, as well as the correlation between the distributions. The method utilizes the nonlinear response of the particle at high magnetic field strengths [49].
When used for bio-sensing, the optomagnetic method is used to measure changes in the hydrodynamic size distribution and/or changes in the extinction properties of the MNP dispersion upon binding of target bio-molecules to the MNPs. This binding could be detected as either an increase of the size of the individual nanoparticles or as a change of the signal due to clustering of the nanoparticles. Using commercial available magnetic nanoparticles (mainly multicore particles from partner Micromod with diameters of 80 nm or 100 nm) we have used this principle ourselves or in collaboration (Task 6.6) to detect:

• Coils of DNA formed by a rolling circle amplification (RCA) reaction [46]
• Monomerized (chopped up) rolling circle amplified DNA [50]
• Products formed from synthetic Dengue DNA by loop-mediated isothermal amplification (LAMP) and detected in real-time during the reaction [51]
• C-Reactive Protein (CRP) and comparing/benchmarking to a readout on the same samples using AC susceptibility [52]
• NS1 protein dengue biomarker in serum [53]
• prostate- specific antigen (PSA) using shape anisotropy enhanced optomagnetic measurement [54]
• Thrombin [55]
• the pH dependence of DNA triplex nanoswitches [47]

From the vast amount of particle systems synthesized in WP3, a limited set of candidates for bio-detection was identified, and one particle system (prepared by Micromod) was selected and optimized for bio-detection (Task 6.6). We obtained six different batches of this particle system, and optimized the probe density and the buffer used in the experiment. We were able to obtain slightly better performance with this system compared to our conventional commercially available magnetic nanoparticles (also from Micromod). We learned that the transition from a system showing reproducible physical properties to one that also has reproducible performance after surface functionalization and exposure to bioassay conditions should not be underestimated and is at least as demanding as defining the optimum physical properties.
During this activity, we have investigated and developed a new optomagnetic technique for characterization and bio-detection. The technique has been applied on the characterization of the hydrodynamic size and magnetic moment on essentially all particle systems in the project and the information obtained (when possible) has been compared to alternative determinations. A variety of setups have been developed and applied for the detection of a range of biomolecules using a range of different detection strategies (see references for more information). Most of these experiments were performed using commercially available nanoparticles produced by one of the project partners (Micromod), which were also subject to characterization in WP4. A particle system – also prepared by Micromod – that showed promising properties in physical measurements was selected and functionalized. However, the performance of this system when used for bio-detection, under the conditions tested, was found to be only marginally better than that used previously.

3.6.2.3 Magnetic particle imaging
The new tomographic imaging modality magnetic particle imaging (MPI) is able to combine high spatial and temporal resolution with excellent sensitivity, opening new opportunities in clinical applications. The principle of MPI is based on the nonlinear magnetization curve of superparamagnetic iron oxide nanoparticles and does not stress the patient with harmful radiation. An overview of current scanner topologies and tracer materials can be found in Panagiotopoulus et al. [56]. With optimized scanner topologies and advanced tracers, a spatial submillimeter resolution is possible. In 2015, Bringout et al. [57] introduced the first concept for a rabbit sized field free line MPI scanner. Also in 2015, Graeser et al. [58] introduced a magnet particle spectroscopy (MPS) device which is able to generate field free point and field free line field sequences while applying different possible offset fields. The flexibility of this measurement device allows the comparison of different particle responses of different field sequences with the same setup. A sensitivity study by Bente et al. [59] verified the linearity of the reconstructed signal of a permanent magnet field free line MPI scanner with respect to the particle concentration of Resovist (Bayer Schering Pharma AG, Berlin, Germany), which is used as a gold standard in MPI, and found a lower detection limit of 15 μg iron in magnetite. In 2017, Dieckhoff et al. [60] were able to demonstrate an improved MPI visualization of the liver applying Resovist and an enhanced system function approach and multiple patches reconstructions.
In task 6.7 of WP6, we investigated the suitability of particles that were synthesized, analyzed and standardized in the NanoMag consortium. The resolution study was performed using a magnetic particle scanning device (preclinical MPI scanner, Bruker BioSpin GmbH, Ettlingen Germany) and three MPS devices [Biederer et al., J. Phys. D Appl. Phys., 42(20), 2009; Graeser et al., Phys. Med. Biol., 62(9), 2017; Chen et al., IWMPI, 2017] to compare the particles regarding their sensitivity and spatial resolution. Over all, three commercial particles selected in WP1, 28 particles synthesized in WP3 by CSIC, nanoPET, micromod, DTU, and SP, as well as nine particles reproduced by nanoPET and micromod regarding to the acquired standard operating procedures of WP5 were available. For reconstruction model-based, hybrid-based as well as measurement-based reconstruction techniques were used as introduced amongst others in Schmidt et al. [61] and von Gladiss et al. (Phys. Med. Biol., 62(9), 2017). All measurement results were compared to the results of Resovist,
Using the results of MPS measurements [62], [63], all the above particles were compared in terms of signal strength in MPI both with and without the presence of magnetic offset fields. Twenty-five particles (two commercial, 14 synthesized, eight reproduced) were selected for further investigation considering their performance.
The evaluation of the measurement results identified some particles with a possible suitability for use in MPI. The spatial resolution as well as the sensitivity of the particles showed results comparable to those of Resovist.
In order to match some of the chemical and structural parameters of the particles with a possible MPI suitability, parameters, such as the iron content, the particle shape and the core size as defined in the technical data sheets or characterized by consortium members, were investigated. The comparison of those parameters does not allow for the definition of a sharp quality criterion for the analysis and the synthesis in respect to MPI devices. Neither the different synthesis methods nor the different particle coatings used for the synthesis of the particles within the consortium seem to have a noticeable impact on the MPI suitability. It can be stated though that particles with an iron core below 30 nm and an irregular or flower-shaped core seem to be more suitable for MPI than particles with a spherical or rhombohedral core and a core size above 30 nm. The results of this activity will be documented in [64].

Impact on other Work Packages
The partners in WP6 were in strong collaboration with WP4 and have contributed in the standardization activities in WP5.

WP7: Dissemination and exploitation
The main results in WP7 (dissemination and exploitation) are listed under “Potential Impact and Main Dissemination Activities” section.

References

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Potential Impact:
In the NanoMag project we see the following main impacts during the project:

• Clear nomenclature for describing structure and magnetic properties of MNPs and MNP ensembles
• Improvements in reproducibility of MNP synthesis. Over 50 new MNP systems synthesized in the project.
• Excellent surveys of many common measurement methods for MNPs and their pros and cons, classification of these methods including also classification of different types of MNP systems
• Stakeholder committee group connected to the NanoMag project.
• For some measurement methods: SOPs, uncertainty budgets, metrological descriptions
• Improved modelling of MNP magnetic properties, especially: dynamic behavior
• Correlation analysis between MNP parameters determined by different analysis methods
• Over 80 new publications in international high impact journals and over 170 presentations and seminars carried out by NanoMag partners
• E-learning modules (NPL-web site, linked from the NanoMag homepage)
• Substantial contribution to ongoing ISO MNP standards (ISO 19807 ” Nanotechnology — Liquid suspension of magnetic nanoparticles — Characteristics and measurements', PG14 "Superparamagnetic beads for free cell DNA extraction)
• Analysis service in the future (information will be collected at NanoMag homepage and PTB data server)
• Continuation with the MagnaStand EU project coordinated by Uwe Steinhoff (PTB).
• Round Robin measurements (same analysis methods but in different labs) using different types of MNP systems
• Our industrial partners in the project have commercialized some of the most promising MNP systems that was synthesized during the project

The dissemination after the NanoMag project ended are described in the NanoMag exploitation plan. Main results from the exploitation plan are described in the following text: “Within NanoMag project we have identified three main types of exploitable outcomes. These are areas where the work that is currently being done within the project can be used to generate impact, either directly, through the creation of best practice and standards, or indirectly, through the upskilling of researchers and industry in the use of MNPs.”

1. Standardisation practices and procedures. As with any emerging field of research that is finding applications in industry, there is a need to ensure that users are supplied with the correct information and supporting guidelines detailing what they are dealing with and how it should be used. This information is currently lacking in the field of MNPs and is a necessary part of building confidence in new products, from both users and investors, as well as ensuring that these products are used correctly, which is especially important in the many medical applications that exist.
2. Services/measurement services. As this project represents the formation of a measurement community for MNPs, it is important to be clear about the expertise and potential services that exist and can be offered to both industry and academia.
3. Teaching aids such as e-learning products, best practice guides, and reports on standard operation procedures (SOPs) that will make results accessible to users. Aimed more at students and new researchers moving into the field, the production of teaching and learning resources will help to train the next generation of researchers and provide established researchers with the capabilities and understanding to pursue further research into the use of MNPs in biomedical science.

These three outcomes are further described in the following pages.

Initiation of new standards
In 2015 ISO/TC229 WG4 “Nanotechnologies – Material specification” started independently of NanoMag the development of a material specification for MNPs (ISO 19807). Immediately, NanoMag reacted to this development by sending technical experts to the national standard developing organisations (SDOs) in the UK, Spain, Sweden and Germany, which are all countries with considerable industrial involvement in the MNP sector. This was necessary, because NaoMag was the dominant pre-normative activity focusing on MNPs in Europe. In addition, these SDOs have now documented plans to adopt ISO 19807 as a national standard. Except Spain, the NanoMag partners delegated their technical experts also to ISO/TC229 WG4, where they subsequently contributed to the development of ISO 19807. Currently ISO 19807 is in draft form with completion anticipated by the end of 2018. NanoMag partners PTB, NPL, RISE Acreo, MICROMOD, and UCL will continue to work on ISO 19807 within the follow-up project MagNaStand. A specific task in MagNaStand is to secure the public results of NanoMag and make it available for further MNP standardisation at ISO and CEN level.
In September 2016, ISO/TC229 WG4 started to prepare a Preliminary Work Item “Specification for superparamagnetic beads composed of nanoparticles for circulating tumour DNA extraction”. This proposal did not come from NanoMag members. It has now been accepted and ISO has started the development of the next standard involving MNPs under active contribution and involvement of the NanoMag members. This new standard will directly affect a number of European SMEs and larger companies working in the field. One European company, Roche-Diagnostics, has published a market turnover in 2014 of 2.6 bln € with their immunodiagnostics (including reagents and instruments), which are based on the use of MNPs .
The NanoMag project has brought immense input into the development of these new standards, which has been recognized several times by the ISO TC229/WG4 working group. In addition, an agreement has been reached with the respective European standardisation committee CEN/TC352 “Nanotechnologies” that the committee will support the developments at ISO and that, after finalization, it will adopt the ISO standards as European normative documents.

Participation in other relevant MNP projects
Five members of the NanoMag consortium (NPL, RISE Acreo, DTU, PTB, UCL) are also active in the TD COST network 1402 “RADIOMAG: Multifunctional Nanoparticles for Magnetic Hyperthermia and Indirect Radiation Therapy”. This research network with over 140 participants covers several aspects of a new cancer therapy based on MNPs. One workpackage is specifically dedicated to the standardisation of MNP characterisation, where the NanoMag partners are actively involved. Participation of NanoMag partners in a ring comparison of MNP heating characteristics was of a great importance, where the NanoMag consortium supported the data analysis and interpretation for a larger number of participants. Another goal within this is the setup of calibration samples for magnetic hyperthermia. The RADIOMAG network will continue running until late 2018, well after the end of NanoMag.

Follow-up projects of MNP analysis standardisation (MagNaStand)
Acknowledging the situation that NanoMag does not have the necessary funding for a continuous support of standardisation and it does not last long enough to finalise an ISO standards development, it was necessary to create a follow-up project achieving these goals. For this reason, some members of the NanoMag consortium initiated a new co-normative EMPIR project on MNPs, that in contrast to NanoMag, which is a pre-normative project. In June 2017, the new project MagNaStand went into operation. Participating NanoMag partners are PTB as the coordinator, NPL, UCL, RISE Acreo and Micromod.
From the beginning, a strong collaboration and transmission of the results between NanoMag and MagNaStand projects was envisaged. Specifically, in the MagNaStand working plan, a Task 3.1 is defined under the title;” Knowledge transfer from the pre-normative EU FP7 project NanoMag”. The aim of this task is to gather the metrological knowledge gained in the FP7 project NanoMag (2013-2017) and make it available for the international standardisation of MNPs. The information collected from NanoMag will be used within MagNaStand in the development of magnetic measurement methods, in the preparation of metrological checklists and in the development of new standards at a national and an international level.

Measurement Services
One of the most direct ways for this project to generate impact is through ensuring that companies and researchers are aware of the services and expertise that exist in MNP metrology. In order to grow this awareness NanoMag is currently carrying out the following actions:
A reference sheet for methodological standards will be made free and publicly available and will be able to be downloaded from the NanoMag website (that still will be available and updated after the NanoMag project). This reference sheet will be designed for the customers that buy MNP systems to be used in specific applications (such as bio-separation, hyperthermia, or bio-sensing). Each of the application areas are interested in different specifications of the MNP systems, however knowledge of the particle size and particle morphology (single- or multi-core), and how to treat each of these, is required for each application. Additionally, specific magnetic saturation value and initial magnetic susceptibility are two important parameters that customers find valuable. This sheet will be updated on the NanoMag site up to two years after the end of the project. After this point, one suggestion would be that the synthesis partners will keep the NanoMag specifications for their MNP systems at their own websites. This would be preferable to leaving an out-of-date copy on the NanoMag website for later reference, however, it would require continued coordination.
The development of new samples or reproducing of old formulations and their characterisation – at cost – may be possible after the project’s completion. It is desirable to maintain communication between the 17 partners of the NanoMag consortium after the project. This will create a post-NanoMag community and allow external companies to contact this community (through project coordinator Christer Johansson RISE Acreo as primary contact), if they require guidance on synthesis, analysis and standardisation of MNP systems. In this case the NanoMag partners might charge such companies individually for each specific consultancy or action.
In order to facilitate this interaction between companies and the NanoMag community ‘Yellow Pages’ of NanoMag techniques and contacts is being generated and will be able to download this from the NanoMag website. This will be a directory stating what services each partner is capable of, which can be used be industry to find relevant points of contact and increase awareness of what the consortium as a whole can deliver. One example is an operation of the Core Facility “Metrology of Ultra-Low Magnetic Fields” at PTB since June 2017. PTB grants external scientists from universities, international metrology institutes and companies access to its know-how and its equipment for the measurement of extremely small magnetic fields, explicitly including the thorough characterization of magnetic nanoparticles.
This directory will be held on the NanoMag website. It would be efficient to have this stored with the reference sheet and linked across partner sites after updates of the NanoMag site cease. This would need to be well organised with defined leads and a main site to hold the curated document. This would then be shared with organisers of conferences and meetings on MNP systems to ensure that attendees are aware of the extent of the community that has been established to assist in the standardisation and assurance of MNP characterisation. The NanoMag website and the NanoMag data at the PTB server will be active after the NanoMag project has ended.

Teaching/Learning
We understand that the setting of standards and the provision of services alone will not produce impact if the requisite understanding does not exist in both the research and industrial communities. In order to grow this understanding, NanoMag is producing tools and guidance for education in MNPs.
The main thrust of this outcome is being pursued through production of a series of e-Learning modules under the common title Magnetic Nanoparticles: Standardisation and Biomedical Applications. The course comprises 4 modules, linked to the knowledge and skills generated by the NanoMag project, each of which covers a number of lessons on different subject areas and which will be freely available indefinitely. The content and structure of the course is summarised in the course map below.
Learners can take the modules individually as a stand-alone learning resource, or can undertake all four together as a full e-Learning course. Information on these modules, including links to the e-Learning course, can be found on the NanoMag website (www.nanomag-project.eu) and will remain live after the website curation ends, i.e. two years after the project completion. The modules themselves are held on the NPL e-learning website and will be accessible indefinitely. Up to now 132 people have already enrolled to the first 2 modules. The number is expected to increase significantly when all modules are launched after the end of the project.
For information on more routine processes, the project is developing best practice guides for specific MNP measurement/characterisation that will be easily contextualised to relate to product information. This will be led by PTB.

Stakeholder Surveys, Stakeholder meeting and Dissemination Conference
In order to maximise the output from the stakeholder participation, surveys were carried out in four main areas. The surveys were implemented online using SurveyMonkey. These questions were typically formatted in a binary or point based manner with the option to leave additional comments. The surveys were carried out over the course of 36 months and concentrated on four main areas:

• Initial assessment of stakeholder/consortium interests and views.
• Characterisation techniques of MNP’s.
• Standards and role of standards in MNP industry.
• Performance of MNP’s in applications.

The objective of the first survey was to form an initial assessment of the stakeholder interests and gauge the general views of the stakeholder/consortium. The second survey concerned specific areas of characterisation techniques of MNPs. The third survey concerned specific areas of standardisation and the role of standards in the MNP industry. The fourth, and final, NanoMag survey looked at applications of MNPs and whether available particles limit performance of these applications. It also included questions on the importance of MNPs' characteristics (such as single or multiple magnetic cores, hydrodynamic size and colloidal stability) to their purpose.
During the project, we have had two major meetings, in connection to the ordinary project meetings, with the NanoMag stakeholder committee (PTB Berlin M18 and at SP Stockholm M36). During these meetings, we have discussed the standardization parameters that are important in industry and how these are applied for instance in MNP synthesis process and analysis. We have also discussed the project result uptake and how the results can be further be used in the future.
The final dissemination conference (deliverable D7.8) took place at Waterfront Hotel in Göteborg, Sweden on 25-26 September 2017. The presentations were chosen to show the most scientific impacts in the project related to; I) synthesis, II) analysis and II) applications. In total 52 attended the conference. The NanoMag stakeholder committee was represented by Barry Moskowitz and Joachim Schwender (Ferrotec), Lluis Martinez (Sepmag), Filiz Ibraimi (Lifeassays), Grete Irene Modahl (Thermofisher), Jose Maria Abad (Nano Immunotech) and Gunnar Schütz (Bayer). Also, external participants invited by NanoMag partners attended the conference. We got a very good response from both the stakeholders and the NanoMag partners that the conference was very much appreciated.

During the project, over 80 publications submitted to international journals and over 170 presentations at international conferences and seminars disseminating the NanoMag results (listed in the attachment to this final report)
List of Websites:
The NanoMag website can be found at http://www.nanomag-project.eu/home.html.

Contact:
Christer Johansson, Professor
Senior Expert
RISE Research Institutes of Sweden
Division ICT – RISE Acreo AB
+46 72 723 33 21
Arvid Hedvalls Backe 4, 400 14 Göteborg, Sweden
christer.johansson@ri.se