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Sensitive and quantitative pEPR detection system for SPIO nanoparticles


Abstract and Figures

A new system for the quantification of super paramagnetic iron oxide nanoparticles (SPIONs) is presented. The proposed system relies on the particle electron paramagnetic resonance (pEPR) technique and utilises the linear response of the SPIONs to generate a pEPR signal under a static field. By extracting the pEPR signals, whose intensity is proportional to the SPIONs concentration, the quantitative information of the SPIONs is derived. To evaluate the system performance, sensitivity measurements have been conducted with two commercial SPIONs, FeraSpin M and Resovist, and reveal a measurement sensitivity of 300 ng.
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A Sensitive and Quantitative pEPR
Detection System for SPIO Nanoparticles
X. Li, G. Torfs, J. Vandewege, J. Bauwelinck,
and J. R. Verbiest
This letter presents a new system for super paramagnetic iron oxide
nanoparticles (SPIONs) quantification. The proposed system design
relies on the particle electron paramagnetic resonance (pEPR) technique
and it utilizes the linear response of SPIONs to generate a pEPR signal
under a static field. The pEPR signal intensity is proportional to the
SPIONs concentration, by extracting the pEPR signals, the quantitative
information of SPIONs is derived. To evaluate the system performance,
sensitivity measurements have been conducted with two commercial
SPIONs, FeraSpin M and Resovist, and reveal a measurement
sensitivity of 300 ng..
Introduction: Super paramagnetic iron oxide nanoparticles (SPIONs)
have been recently implemented in medical and biomedical research
applications, such as disease diagnosis, hyperthermia and drug delivery
[1]. Accurate quantification of SPIONs is crucial in these applications
[2-3]. Several methods to quantification of iron ions in aqueous solution
have been described in the past, among these are inductively coupled
plasma spectrometry (ICP), Perls' Prussian blue reaction spectroscopy,
fluorometry and continuous wave electron paramagnetic resonance
(CW EPR) spectrometry [3]. ICP offers extremely high sensitivity,
although comes from high cost, and it is neither suitable for ex vivo
analysis [4]. In contrast to Perls' reaction-based colorimetry technology
and the fluorometry, the CW EPR technology had been shown to be a
more sensitive technique for SPION quantification [4]. CW EPR
spectrometer allows indirect EPR response measurements by recording
microwave power absorption during spin resonance, and a sweep of
static field is required .
In this letter, we present a new quantitative measurement system for
SPIONs based on the proposed particle electron paramagnetic response
(pEPR) technique [5]. Compared to a typical EPR spectrometric process,
pEPR technology directly detects EPR radiated from SPIONs, and the
pEPR signal intensity is proportional to the magnetization of the
nanoparticles. Due to the high magnetic susceptibility of SPIONs, a
high magnetization can be achieved at a relatively low magnetic field
(<50 mT) configuration, the low static magnetic field results in a low
resonant frequency, which is particularly interesting for biological and
medical applications, because the dielectrics losses in biological units
are significantly lower at low frequencies [5]. Moreover, pEPR
technology only requires fix static magnetic field configuration, hereby
the sweep of static field is no longer indispensable.
A b
Fig. 1 pEPR measurement fields configuration
a Positive B0 field configuration;
b Positive and negative B0 field configuration.
Measurement principle: In pEPR technology, the signal intensity of the
pERP wave is directly proportional to the concentration of the SPIONs.
By precisely detecting the active pEPR wave amplitude, the quantity of
SPIONs can be derived subsequently. To generate the pEPR signal, a
static magnetic field, B0, is required to orient the electron spins and an
alternating electromagnetic (EM) field, B1, at the corresponding Larmor
frequency is required to resonate the spins, as shown in Fig. 1 (a). M
represents the magnetization of spin, which precesses about B0 field
direction. The two fields, B0 and B1, are perpendicularly imposed on the
SPIONs. When resonating, the spin absorbs RF energy from the EM
field and emits a pEPR wave, My, at the Larmor frequency [5].
However, in pEPR measurements, the signal intensity of nanoparticle
sample is also determined by the static magnetic field strength, the EM
field strength and local temperature [6]. Hence, all these parameters are
expected to be highly stable during quantification measurement.
In the presented system, the relative orientation of the magnetic fields
is critical. The static field, B0, and the EM field, B1, are perpendicular
and the pEPR detection is set along y axis to avoid disturbance from the
static field and the EM field, as shown in Fig. 1. Nevertheless, in
practical, an EM field feed through signal is still presented together with
the pEPR signal because of strong coupling. Additionally, based on the
fact that the direction of SPIONs magnetization, M, will respond to a
direction change of B0 field, as shown in Fig. 1(b), a positive and
negative B0 field configuration is introduced in the proposed system. By
subtracting the received signals under positive and negative B0 field, the
feed through signal of EM field is eliminated as shown in (1) [7], SPositive
and SNegative represent the received signals under positive and negative
B0 field respectively, and SpEPR represents the pEPR signal, which is
proportional to the SPIONs quantity.
noiseSSS pEPRNegativePositive 2
Fig. 2 Conceptual block diagram of pEPR system
System architecture: The high level system architecture is shown in Fig.
2. Four main blocks can be distinguished: an RF transmitter (TX), an
RF receiver (RX), the coils and a control and data processing unit. The
coils are the central part of the pEPR system, which include a
Helmholtz coil, RX- and TX-coil. The Helmholtz coil builds the B0
field, and a low jitter synthesizer followed by a power amplifier (PA)
drives the TX coil which establishes the EM field, B1, that excites the
nanoparticles to resonance. The emitted pEPR signal is captured by the
RX coil, amplified by the low noise amplifier (LNA) and sampled with
an analog to digital converter (ADC). The digital processing unit
analyzes the digitalized pEPR signal and processes the collected data to
yield the quantification result of SPIONs. To achieve high measurement
sensitivity, a high quality factor RX coil is utilized, and a high isolation
between the TX coil and the RX coil is achieved by careful positioning
the two coils in the proposed system. Besides high isolation, precise
control of the measurement timing is also important, low jitter ADC
clock is highly applied. Furthermore, to further reduce the system noise
level, each measurement result would be repeated and averaged over a
million times.
Results and discussion: The proposed system was evaluated by
performing pEPR measurements on two different SPIONs, namely
FeraSpin M (Miltenyi Biotec, Leiden, Netherlands) and Resovist
(Schering AG, Berlin, Germany). Samples with different SPION
concentrations were prepared in 150 μL demineralized water. During
the measurements, a magnetic field of 10 mT and a RF frequency that
a b
Fig. 3 pEPR measurement results
a Five Resovist samples between 0.1 μg and 2 μg were quantified by
pEPR, an error bar of one SD was added on each sample measurement;
b Ten FeraSpin samples between 0.08 μg to 41 μg were quantified by
pEPR, an error bar of one SD was added on each measurement.
corresponds to the Larmor frequency were employed. Each
concentration was quantified 10 times and the mean value and the
standard deviation (SD) of the pEPR signal intensity were calculated.
The measurement results were plotted in Fig. 3. Both measurement
results exhibited the pERP signal intensity was proportional to the
SPION quantities of sample solutions. The pEPR signal intensity of
Resovist was 1.87 mV/μmol, in Fig. 3 (a), which is almost two times
smaller than 3.49 mV/μmol of FeraSpin M, in Fig. 3 (b), caused by the
different characteristics of the two SPIONs. By comparing the deviation
with the SD of each measurement result, the detective sensitivity of the
proposed system was estimated as 300 ng.
Fig. 4 FeraSpin M measurement with both pEPR and ICP-OES and an
error bar of one SD was added on each measurement. Two wavelengths
of iron ion, 238.204 nm and 259.940 nm, were chosen for FeraSpin M
under ICP-OES measurements.
Subsequently, a reference measurement was performed to validate
previous measurement results. 8 FeraSpin M samples ranging from 0.3
μg to 41 μg were quantified by inductively coupled plasma optical
emission spectrometry (ICP-OES). The iron ions quantity results
derived from ICP-OES measurement were plotted together with the
pEPR results in Fig. 4. The measurement results of the pEPR technique
showed agreement with ICP-OES results, proving the viability of the
presented pEPR technique, and verifying the previous measurement
Conclusion: This letter demonstrates a sensitive quantification system
for SPIONs based on the pEPR technique, which allows direct
measurements of the emitted signal from SPIONs under specific
magnetic fields configurations. The designed system was evaluated by
quantifying two types of commercial nanoparticle, and the experiment
results were also validated by ICP-OES measurements. The
measurements prove that the presented system has a sensitivity down to
300 ng.
X. Li, G. Torfs, J. Vandewege, J. Bauwelinck (INTEC_Design, UGent,
Gent 9000, Belgium)
J. R. Verbiest (Pepric NV, Kapeldreef 75, Leuven 3001, Belgium)
1 Q. A. Pankhurst, J. Connolly, S. K. Jones, and J. Dobson,
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technical note. Contrast Media & Molecular Imaging. Vol. 4, Issue 6,
pp. 299304, Nov./Dec. 2009.
4 P. Danhier, G. De Preter.Electron paramagnetic resonance as a
sensitive tool to assess the iron oxide content in cells for MRI cell
labeling studies. Contrast Media & Molecular Imaging. Vol. 7, Issue 3,
pp. 302307, May/June 2012.
5 V. Peter, T. Stephanie. " Isolating Active Electron Spin Signals in
EPR" WO/2012/126968, September 27, 2012.
6 Gareth R. Eaton, Sandra S. Eaton, David P. Barr, Ralph Thomas
Weber. Quantitative EPR, New York: Springer, 2010, ch. 2.
... The pickup coil measures the component of the magnetization that is perpendicular to the applied magnetic field and radio frequency wave. This component is dependent on the amount of MNP present in the sample [52]. This measurement is performed for anti-parallel directions of the applied magnetic field, so a high signal-to-noise ratio (SNR) can be achieved and therefore the obtained MNP amount is very accurate. ...
... These principles are also described in Refs. [51,52,305]. Section 2.1.1 mentioned that existing EPR setups use a resonator to amplify the weak absorption signal and measure the absorption or derivative of the absorption by the sample. ...
... M y is the magnetization component of the MNP measured by the pickup coil. M y is proportional to the MNP amount of the sample [52]. Even though the excitation and pickup coil are placed orthogonally, in practice some RF feedthrough is still measured by the pickup coil: ...
... Aforementioned applications require the knowledge of the spatial distribution of the MNP because only malignant tissue should be targeted. In this study, two techniques are considered for retrieving this spatial distribution, namely magnetorelaxometry (MRX) imaging [9] and electron paramagnetic resonance (EPR) [10]. In the first approach, a volume containing MNP is placed in a homogeneous magnetic field. ...
... In EPR, the sample is again placed in a homogenous magnetic field but additionally excited by a radio frequency wave that brings the MNP's magnetic moments into resonance. The magnetization of the resonant MNP is then measured by a pick-up coil [10]. EPR is a very sensitive, direct and selective technique for MNP quantification in vitro and ex vivo in small samples without the need of extensive sample preparation [22]. ...
... The frequency is chosen with the intention to bring the MNP's magnetic moments into resonance such that the resulting total magnetic moment precesses around the direction of the external magnetic field at a fixed angle. The component orthogonal to the direction of the applied field, measured by a pick-up coil, is proportional to the amount of MNP [10]. The EPR signal in the pick-up coil originating from a MNP amount c v at a certain position (labeled as index m) depends on its distance with respect to the excitation coil and pick-up coil and the local magnetic field amplitude B at this position m [25,26]. ...
Full-text available
Magnetorelaxometry (MRX) imaging and electron paramagnetic resonance (EPR) are two non-invasive techniques capable of recovering the magnetic nanoparticle (MNP) distribution. Both techniques solve an ill-posed inverse problem in order to find the spatial MNP distribution. A lot of research has been done on increasing the stability of these inverse problems with the main objective to improve the quality of MNP imaging. In this paper a proof of concept is presented in which the sensor data of both techniques is fused into EPR-MRX, with the intention to stabilize the inverse problem. First, both techniques are compared by reconstructing several phantoms with different sizes for various noise levels and calculating stability, sensitivity and reconstruction quality parameters for these cases. This study reveals that both techniques are sensitive to different information from the MNP distributions and generate complementary measurement data. As such, their merging might stabilize the inverse problem. In a next step we investigated how both techniques need to be combined to reduce their respective drawbacks, such as a high number of required measurements and reduced stability, and to improve MNP reconstructions. We were able to stabilize both techniques, increase reconstruction quality by an average of 5% and reduce measurement times by 88%. These improvements could make EPR-MRX a valuable and accurate technique in a clinical environment.
... In this study we present a newly introduced alternative analytical method [15] for detecting SPIONs in vitro and ex vivo that does not require complex sample preparation. Sampling errors often induced by the selection, manipulation and preparation, such as homogenization, dehydration or dilution, of tissue and blood samples can be avoided, resulting in a higher quality dataset. ...
... A radio frequency excitation is applied at the resonant frequency and the received signal is a measure of the amount of SPIONs present in the sample. The Pepric-developed instrument for particle electron paramagnetic resonance (pEPR) detection [15] constitutes a spectrometer (pepric particle spectrometer [PPS]) which was developed to measure the particle content in blood and tissue in a reliable and straightforward manner. Applicability of such instrumentation ranges from the laboratory analytical activity to the clinical environment. ...
... The PPS is an instrument for detection and quantification of magnetic iron oxide nanoparticles and is based on a direct and selective detection method pEPR [15]. In this study, all pEPR measurements were carried out with Pepric PPS2 instrument (Pepric, Leuven, Belgium). ...
Full-text available
Superparamagnetic iron oxide nanoparticles (SPIONs) may play an important role in nanomedicine by serving as drug carriers and imaging agents. In this study, we present the biodistribution and pharmacokinetic properties of SPIONs using a new detection method, particle electron paramagnetic resonance (pEPR). The pEPR technique is based on a low-field and low-frequency electron paramagnetic resonance. pEPR was compared with inductively coupled plasma mass spectrometry and MRI, in in vitro and in vivo. The pEPR, inductively coupled plasma mass spectrometry and MRI results showed a good correlation between the techniques. The results indicate that pEPR can be used to detect SPIONs in both preclinical and clinical studies.
... During the measurements, a radiofrequency (RF) field of 300 MHz and a magnetic field of 10 mT were employed. The pEPR measurement voltage was directly proportional to the particles in resonance with the RF-field [30]. Hence, there was no contamination from iron in tissue, blood, and intraplaque hemorrhages, as would be the case for a more conventional inductively coupled plasma mass spectrometry (ICP-MS) determination of iron content. ...
Full-text available
The purpose of our study was to monitor the iron oxide contrast agent uptake in mouse brachiocephalic artery (BCA) atherosclerotic plaques in vivo by quantitative T2-mapping magnetic resonance imaging (MRI). Female ApoE-/- mice (n = 32) on a 15-week Western-type diet developed advanced plaques in the BCA and were injected with ultra-small superparamagnetic iron oxides (USPIOs). Quantitative in vivo MRI at 9.4 T was performed with a Malcolm-Levitt (MLEV) prepared T2-mapping sequence to monitor the nanoparticle uptake in the atherosclerotic plaque. Ex vivo histology and particle electron paramagnetic resonance (pEPR) were used for validation. Longitudinal high-resolution in vivo T2-value maps were acquired with consistent quality. Average T2 values in the plaque decreased from a baseline value of 34.5 ± 0.6 ms to 24.0 ± 0.4 ms one day after injection and partially recovered to an average T2 of 27 ± 0.5 ms after two days. T2 values were inversely related to iron levels in the plaque as determined by ex vivo particle electron paramagnetic resonance (pEPR). We concluded that MRI T2 mapping facilitates a robust quantitative readout for USPIO uptake in atherosclerotic plaques in arteries near the mouse heart.
... This novel technique can take the advantage of the high magnetic susceptibility of SPIONs, so that the excited pEPR signal can be extracted under a low static magnetic field and at room temperature. The pEPR technology has proved to be able to quantify the concentration of SPION solutions with a high sensitivity under an RF continuous wave excitation [6]. In this Letter, a proof-of-concept system based on the pulsed pEPR technology is presented. ...
The first demonstration of a pulsed electron paramagnetic resonance (EPR) detection system based on the recently announced particle EPR (pEPR) technology for superparamagnetic iron oxide nanoparticles (SPIONs) applications is presented. The SPIONs have large magnetisation, which allows one to detect the electron resonance under a low magnetic field and at room temperature. However, the broad linewidth of the superparamagnetic particles leads to a short relaxation time which requires a quick damping of the transmit signal. The presented experiments on a commercial SPION prove the feasibility to detect a pulsed electron response from broad linewidth particles using the pulsed pEPR technology.
With the help of iron oxide nanoparticles, electron spin resonance spectroscopy (ESR) was applied to immunoassay. Iron oxide nanoparticles were used as the ESR probe in order to achieve an amplification of the signal resulting from the large amount of Fe³⁺ ion enclosed in each nanoparticle. Rabbit IgG was used as antigen to test this method. Polyclonal antibody of rabbit IgG was used as antibody to detect the antigen. Iron oxide nanoparticle with a diameter of either 10 or 30 nm was labeled to the antibody, and Fe³⁺ in the nanoparticle was probed for ESR signal. The sepharose beads were used as solid phase to which rabbit IgG was conjugated. The nanoparticle-labeled antibody was first added in the sample containing antigen, and the antigen-conjugated sepharose beads were then added into the sample. The nanoparticle-labeled antibody bound to the antigen on sepharose beads was separated from the sample by centrifugation and measured. We found that the detection ranges of the antigen obtained with nanoparticles of different sizes were different because the amount of antibody on nanoparticles of 10 nm was about one order of magnitude higher than that on nanoparticles of 30 nm. When 10 nm nanoparticle was used as probe, the upper limit of detection was 40.00 μg mL⁻¹, and the analytical sensitivity was 1.81 μg mL⁻¹. When 30 nm nanoparticle was used, the upper limit of detection was 3.00 μg mL⁻¹, and the sensitivity was 0.014 and 0.13 μg mL⁻¹ depending on the ratio of nanoparticle to antibody.
Purpose: Magnetic nanoparticles (MNPs) are an important asset in many biomedical applications. An effective working of these applications requires an accurate knowledge of the spatial MNP distribution. A promising, noninvasive, and sensitive technique to visualize MNP distributions in vivo is electron paramagnetic resonance (EPR). Currently only 1D MNP distributions can be reconstructed. In this paper, the authors propose extending 1D EPR toward 2D and 3D using computer simulations to allow accurate imaging of MNP distributions.
Magnetic nanoparticles play an important role in several biomedical applications such as hyperthermia, drug targeting, and disease detection. To realize an effective working of these applications, the spatial distribution of the particles needs to be accurately known, in a non-invasive way. Electron Paramagnetic Resonance (EPR) is a promising and sensitive measurement technique for recovering these distributions. In the conventional approach, EPR is applied with a homogeneous magnetic field. In this paper, we employ different heterogeneous magnetic fields that allow to stabilize the solution of the associated inverse problem and to obtain localized spatial information. A comparison is made between the two approaches and our novel adaptation shows an average increase in reconstruction quality by 5% and is 12 times more robust towards noise. Furthermore, our approach allows to speed up the EPR measurements while still obtaining reconstructions with an improved accuracy and noise robustness compared to homogeneous EPR.
Electron Paramagnetic Resonance (EPR) is a sensitive measurement technique which can be used to recover the 1-dimensional spatial distribution of magnetic nanoparticles (MNP) non-invasively. This can be achieved by solving an inverse problem that requires a numerical model for interpreting the EPR measurement data. This paper assesses the robustness of this technique by including different types of errors such as setup errors, measurement errors and sample positioning errors in the numerical model. The impact of each error is estimated for different spatial MNP distributions. Additionally, our error models are validated by comparing the simulated impact of errors to the impact on lab EPR measurements. Furthermore, we improve the solution of the inverse problem by introducing a combination of Truncated Singular Value Decomposition (TSVD) and Non-Negative Least Squares (NNLS). This combination enables to recover both smooth and discontinuous MNP distributions. From this analysis, conclusions are drawn to improve MNP reconstructions with EPR and to state requirements for using EPR as a 2-dimensional and 3-dimensional imaging technique for MNP.
Full-text available
The physical principles underlying some current biomedical applications of magnetic nanoparticles are reviewed. Starting from well-known basic concepts, and drawing on examples from biology and biomedicine, the relevant physics of magnetic materials and their responses to applied magnetic fields are surveyed. The way these properties are controlled and used is illustrated with reference to (i) magnetic separation of labelled cells and other biological entities; (ii) therapeutic drug, gene and radionuclide delivery; (iii) radio frequency methods for the catabolism of tumours via hyperthermia; and (iv) contrast enhancement agents for magnetic resonance imaging applications. Future prospects are also discussed.
Even if the question is simply “is there a radical present?” it is important to know, e.g., whether <1 or 100% of the species are in the radical form or in a particular metal oxidation state. There are many examples in the literature in which an impurity or a slight dissociation resulted in the EPR signal observed. A goal of this book is to provide guidance on issues that the EPR spectroscopist should consider when designing experiments to obtain quantitative results. The focus of this book is on radicals in condensed phases.
MRI cell tracking is a promising technique to track various cell types (stem cells, tumor cells, etc.) in living animals. Usually, cells are incubated with iron oxides (T(2) contrast agent) in order to take up the particles before being injected in vivo. Iron oxide quantification is important in such studies for validating the labeling protocols and assessing the dilution of the particles with cell proliferation. We here propose to implement electron paramagnetic resonance (EPR) as a very sensitive method to quantify iron oxide concentration in cells. Iron oxide particles exhibit a unique EPR spectrum, which directly reflects the number of particles in a sample. In order to compare EPR with existing methods (Perls's Prussian blue reaction, ICP-MS and fluorimetry), we labeled tumor cells (melanoma and renal adenocarcinoma cell lines) and fibroblasts with fluorescent iron oxide particles, and determined the limits of detection of the different techniques. We show that EPR is a very sensitive technique and is specific for iron oxide quantification as measurements are not affected by endogenous iron. As a consequence, EPR is well adapted to perform ex vivo analysis of tissues after cell tracking experiments in order to confirm MRI results.
Iron oxide (nano)particles are powerful contrast agents for MRI and tags for magnetic cellular labeling. The need for quantitative methods to evaluate the iron content of contrast media solutions and biological matrixes is thus obvious. Several convenient methods aiming at the quantification of iron from iron oxide nanoparticle-containing samples are presented.
Nanotechnology is a multidisciplinary field, which covers a vast and diverse array of devices derived from engineering, biology, physics and chemistry. These devices include nanovectors for the targeted delivery of anticancer drugs and imaging contrast agents. Nanowires and nanocantilever arrays are among the leading approaches under development for the early detection of precancerous and malignant lesions from biological fluids. These and other nanodevices can provide essential breakthroughs in the fight against cancer.
Isolating Active Electron Spin Signals in EPR
  • V Peter
  • T Stephanie
V. Peter, T. Stephanie. " Isolating Active Electron Spin Signals in EPR" WO/2012/126968, September 27, 2012.