Magnetophoretic velocities of superparamagnetic particles, agglomerates and complexes

https://doi.org/10.1016/j.jmmm.2015.02.031Get rights and content

Highlights

  • Analysis of the magnetically induced drift velocity of superparamagnetic particles.

  • Zeta potential of superparamagnetic particles and non-magnetic particles found.

  • Drift velocity of single particles, chains and complexes determined experimentally.

  • Magnetic drift velocities of chains and complexes predicted by simple models.

Abstract

A study into the magnetically induced mobility of four types of superparamagnetic particles (SMPs) was conducted using a video camera, an inverted light microscope and ImageJ tracking software. The objective is to improve the understanding of how SMP-capture assays perform by measuring mobilities of SMPs, when aggregated together or attached to non-magnetic beads (NMB). The magnetically induced velocities of self-assembled SMP chains were measured and found to meet the proposed models. A study into the zeta potential of the SMPs was completed to determine a scenario for maximal electrostatic interactions and efficient capture of the SMPs to a target. SMPs were bound to biotinylated NMBs, representing attachment to a disease biomarker. The drift velocity of SMP chains and SMP–NMB complexes in a gradient magnetic field was compared. It is expected that the observable changes to the magnetophoretic mobility of SMPs attached to a disease biomarker will lead to new biosensor technology.

Introduction

Due to the commercial availability, low cost and easy functionalization of superparamagnetic particles (SMPs), their use in the separation and detection at the micro-scale is ever increasing. SMPs have been described in many batch applications, which include but are not limited to: the detection of pathogenic bacteria in the environment [1], [2] and in food [3], [4].

In clinical settings [5], the use of SMPs for the recognition of biomarkers from blood [6], [7], including nucleic acids, microvesicles [8] and proteins [9], have been documented along with applications in biomedical imaging [10]. Environmental contaminants have been removed through adsorption to SMPs [11], and algae have been harvested using the same technique [12].

A simple and reliable method is required to determine the magnetic characteristics of SMPs in order to enhance the predictability for in vivo applications. A better understanding of the properties of SMPs will allow for their use in more advanced, continuous-flow systems [13] for the separation of disease biomarkers from heterogeneous samples.

A study was conducted into the magnetic properties of four commercially available SMPs, using light microscopy. The aim was to develop a better understanding of the movement of the particles within a magnetic field, as single particles, self-assembled chains and as complexes bound to a non-magnetic bead (NMB). A diameter of 1 µm was chosen for the NMB as this is representative of the approximate size of certain biomarkers such as circulating endothelial microparticles associated with coronary artery disease [14], osteocyte apoptotic bodies associated with bone resorption [15] and pathogenic bacteria such as Haemophilus influenza and Streptococcus pneumonia causative agents of pneumonia [16]. The study will enable the accurate prediction of particle velocities and trajectories and can mimic the case of a bacterium or cell bound to an SMP.

Superparamagnetism refers to the random magnetisation of magnetic crystallites in the absence of an external magnetic field [17]. The unique properties of superparamagnetism enable the free dispersion of SMPs throughout a sample matrix. The later application of an external magnetic field allows SMPs to be focused, separated or concentrated from a sample, at a specific location. When required, removal of the magnetic field results in magnetic relaxation of the SMPs, i.e. they “switch off” [18], allowing the SMPs to be re-dispersed and removed from the location.

There are a variety of commercially available SMPs with different manufacturing procedures and therefore differing characteristics. The loading of iron oxide nanoparticles into a silica shell, for example, occurs either as dispersed particles within the silica shell or as a group forming the core [19]. It can be expected that each brand of SMPs will behave differently when subjected to a magnetic field.

The response of a magnetic material to an applied magnetic field is termed the magnetic susceptibility [20]. The susceptibility is an important parameter, but may vary greatly [17]. Given this variation, it is not sufficient to determine only the magnetic susceptibility. The susceptibility provides an approximation of magnetic responsiveness in in vivo applications [21], the SMPs trajectory and induced magnetic velocity can be better calculated from experimentally determined magnetic mobility.

The ability to coat several superparamagnetic nanoparticles with a silica or polystyrene shell allows the magnetic content to be maximised, increasing the magnetic mobility of the particle, while maintaining the superparamagnetism (which is dependent upon the nanoparticle size) [22]. The coating encases the toxic iron oxide content; as such the conjugate becomes biocompatible [10].

Functional SMPs are used in a variety of applications for the capture, transport and detection of magnetically labelled cells [23]. The functionalization of a microparticle is achieved through the attachment of, for example, a ligand, amino acid or protein to the surface [24]. A specific capture molecule may be immobilised upon the surface of an SMPs [25]. When a particle comes into contact with a target cell, a binding event occurs. The target cell inherits the magnetic properties of the SMPs [26] and will thus be influenced by a magnetic field. The most widely used capture method is the immunological antibody–antigen interaction [1], or a protein–ligand interaction (as was used in this study). Immuno-magnetic separation is generally an efficient and rapid method to perform and can be achieved in almost any sample matrix [17].

Biotin–streptavidin binding has one of the highest association constants in biochemistry [27]. This ligand–protein interaction acts as a good model for binding due to its low cost and the considerable enthalpy generated (approximately 107 kJ/mol) [28]. Biotinylation is a popular method for attaching proteins, carbohydrates and nucleic acids to particles. Due to the small size of biotin, it is unlikely to change the natural functions of the molecule to which it is attached [29].

Given the strong binding properties to biotin and the ease with which proteins may be biotinylated, many manufacturers offer SMPs coated with streptavidin. This binding interface was used in this study to demonstrate the potential for using immuno-labelling of SMPs for target detection through attachment of a biotinylated antibody to a streptavidin coated SMPs. Tu et al. [2] describe a streptavidin-coated immunomagnetic bead assay to detect E. coli O157:H7 with the associated cost being just 0.50 USD/assay.

There are many conjugation methods for coating particles with binding molecules. The linkers that are used to attach the molecule to the particle surface vary in length. Two types of biotinylated beads were used as mock biomarkers in this study. The first bead had biotin attached via a “short” linking arm (SLB); the second with biotin attached by a bespoke “long” linking arm (LLB) as supplied by Chemicell (Germany) with four additional methylene bridges in the linking arm attaching biotin to the bead.

All suspended particles carry a charge; the components of the SMPs and interaction with surrounding media give the particle an electrical charge. A double layer is formed between a particle and the suspending medium, the first layer, named the Stern-layer, contains immobilised ions surrounded by the diffuse layer [30]. The potential between the diffuse layer and the suspending medium is measurable and is termed the zeta potential [31]. The zeta potential is measurable by commercially available instruments such as Malvern Instruments' Zetasizer Pro. Electrophoretic light scattering is used to measure the electrophoretic mobility of a particle towards either the cathode or anode, and the Helmholtz–Smoluchowski equation [32] used to extrapolate a value for the zeta potential.

To establish efficient binding of SMPs to a target, it is important to know the charge of the binding particle and the charge of the target particle. If both particle types carry the same charge when suspended in the solution, binding of the SMP to the target will be in adverse conditions, i.e. particles will be repelled from each other and will remain isolated.

Information regarding the charge of Dynabeads, as used in this study, is available from the manufacturer [33]. However, charge information for the Chemicell particles was not available to the authors. A study into the zeta potential of the Chemicell particles was conducted in order to gain an approximation of the isoelectric point and charge of the particles across a pH range. Ideally, the solution should have a pH such that the SMP and NMB have opposite charges to promote binding.

In order to maximise the potential for SMPs in ultrasensitive detection of disease biomarkers, there is a need to improve their extraction from a continuous flow of assay solution. The continuous separation from a sample flow will allow the use of SMPs in even more diverse and complex systems. This will have advantages; for example within environmental applications for the removal of contaminants from wastewater, and in a medical context, for the extraction of bacteria from a patient with septicaemia [34].

Continuous methods have been described [35] however, these methods meet certain difficulties. The agglomeration of SMPs is a particular problem. The greater the number of SMPs within close proximity of each other, the greater the magnetic force acting upon them. Under high concentrations scenarios, particle agglomeration is unavoidable [33], [34]. An understanding of the magnetic properties of particle agglomerates is required to allow for predictability when designing a continuous flow system.

Section snippets

Transport model

There are two dominant forces which act upon SMPs when in a magnetic field. Primarily, the magnetic force imposed by a gradient field and secondly the Stokes Drag which is generated in the opposite direction to movement.

Materials and methods

The magnetophoretic mobility of five types of SMPs was determined using light microscopy, and tracking of individual SMPs using the ImageJ software package (http://rsb.info.nih.gov/). The magnetically induced velocity was determined for a single SMP, chains and SMP–NMB complexes. The zeta potential of particles was determined using a Malvern Zetasizer Pro, as was the isoelectric point of the Chemicell particles. In total, this study extends to six types of particles.

Results and discussion

A study into the magnetophoretically induced velocity upon SMPs was conducted. Single particles, chains and SMP–NMB complexes were analysed. Velocity data was compared to predictions derived from a simple drag force model to determine if relative drift velocity of SMP–NMB complexes could be a useful parameter in a bioassay. SMPs bound to a non-magnetic biomarker, for example, are predicted to have a lower drift velocity (i.e. mobility) compared to a single SMP; detection of the disease

Conclusions

A method has been developed that allows the analysis of the magnetically induced drift velocity of SMPs in a known magnetic gradient. The trajectories of the SMPs within a magnetic field, as single particles, chains and complexes, were observed. The Dynabead M270 had the greatest magnetophoretic mobility. Chains of SMPs aligned with the field lines of the magnet (Fig. 2).

This study shows that the magnetic drift velocity of SMPs formed into chains or attached to NMBs can be predicted using

Acknowledgements

The authors would like to thank the Oxford University Materials Department for the use of the Malvern Zetasizer Pro and Chemicell, Germany, for the supply of the long-linker biotinylated beads used within this study. In addition, the authors are thankful of the financial assistance received from Trinity College, University of Oxford and Silver Star Society (Oxford Radcliffe Hospitals Charitable Fund 0347).

References (41)

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