Distinguishing Nanoparticle Aggregation from Viscosity Changes in MPS/MSB Detection of Biomarkers.

Magnetic particle spectroscopy (MPS) in the Brownian relaxation regime, also termed magnetic spectroscopy of Brownian motion (MSB), can detect and quantitate very low, sub-nanomolar concentrations of molecular biomarkers. MPS/MSB uses the harmonics of the magnetization induced by a small, low-frequency oscillating magnetic field to provide quantitative information about the magnetic nanoparticles' (mNPs') microenvironment. A key application uses antibody-coated mNPs to produce biomarker-mediated aggregation that can be detected using MPS/MSB. However, relaxation changes can also be caused by viscosity changes. To address this challenge, we propose a metric that can distinguish between aggregation and viscosity. Viscosity changes scale the MPS/MSB harmonic ratios with a constant multiplier across all applied field frequencies. The change in viscosity is exactly equal to the multiplier with generality, avoiding the need to understand the signal explicitly. This simple scaling relationship is violated when particles aggregate. Instead, a separate multiplier must be used for each frequency. The standard deviation of the multipliers over frequency defines a metric isolating viscosity (zero standard deviation) from aggregation (non-zero standard deviation). It increases monotonically with biomarker concentration. We modeled aggregation and simulated the MPS/MSB signal changes resulting from aggregation and viscosity changes. MPS/MSB signal changes were also measured experimentally using 100 nm iron-oxide mNPs in solutions with different viscosities (modulated by glycerol concentration) and with different levels of aggregation (modulated by concanavalin A linker concentrations). Experimental and simulation results confirmed that viscosity changes produced small changes in the standard deviation and aggregation produced larger values of standard deviation. This work overcomes a key barrier to using MPS/MSB to detect biomarkers in vivo with variable tissue viscosity.

One-Sided Multidimensional Statistical Significance Testing: A New Method of Calculating the Statistical Significance of Spectra Used to Demonstrate Magnetic Nanoparticle Sensitivity.

Estimating statistical significance of the difference between two spectra or series is a fundamental statistical problem. Multivariate significance tests exist but the limitations preclude their use in many common cases; e.g., one-sided testing, unequal variance and when few repetitions are acquired all of which are required in magnetic spectroscopy of nanoparticle Brownian motion (MSB). We introduce a test, termed the T-S test, that is powerful and exact (exact type I error). It is flexible enough to be one- or two-sided and the one-sided version can specify arbitrary regions where each spectrum should be larger. The T-S test takes the-one or two-sided p-value at each frequency and combines them using Stouffer's method. We evaluated it using simulated spectra and measured MSB spectra. For the single-sided version, mean of the spectrum, A-T, was used as a reference; the T-S test is as powerful when the variance at each frequency is uniform and outperforms when the noise power is not uniform. For the two-sided version, the Hotelling T2 two-sided multivariate test was used as a reference; the two-sided T-S test is only slightly less powerful for large numbers of repetitions and outperforms rather dramatically for small numbers of repetitions. The T-S test was used to estimate the sensitivity of our current MSB spectrometer showing 1 nanogram sensitivity. Using eight repetitions the T-S test allowed 15 pM concentrations of mouse IL-6 to be identified while the mean of the spectra only identified 76 pM.

Identifying in vivo inflammation using magnetic nanoparticle spectra.

We are developing magnetic nanoparticle (NP) methods to characterize inflammation and infection in vivo. Peritoneal infection in C57BL/6 mice was used as a biological model. An intraperitoneal NP injection was followed by measurement of magnetic nanoparticle spectroscopy of Brownian rotation (MSB) spectra taken over time. MSB measures the magnetization of NPs in a low frequency alternating magnetic field. Two groups of three mice were studied; each group had two infected mice and one control with no infection. The raw MSB signal was compared with two derived metrics: the NP relaxation time and number of NPs present in the sensitive volume of the receive coil. A four compartment dynamic model was used to relate those physical properties to the relevant biological processes including phagocytic activity and migration. The relaxation time increased over time for all of the mice as the NPs were absorbed. The NP number decreased over time as the NPs were cleared from the sensitive volume of the receive coil. The composite p-values for all three rate constants were significant: raw signal, 0.0002, relaxation, <10-16 and local NP clearance, <10-16. However, not all the individual mice had significant changes: Only half the infected mice had significantly different rate constants for raw signal reduction. All infected mice had significantly smaller relaxation time constants. All but one of the infected mice had significantly lower rate constants for local clearance. Relaxation is affected by both phagocytic activity, edema and temperature changes and it should be possible to better isolate those effects to more completely characterize inflammation using more advanced MSB methods. The MSB NP signal can be used to identify inflammation in vivo because it has the unique ability to monitor phagocytic absorption through relaxation measurements.

Measuring protein biomarker concentrations using antibody tagged magnetic nanoparticles.

Under physiological conditions biomarker concentrations tend to rise and fall over time e.g. for inflammation. Ex vivo measurements provide a snapshot in time of biomarker concentrations, which is useful, but limited. Approaching real time monitoring of biomarker concentration(s) using a wearable, implantable or injectable in vivo sensor is therefore an appealing target. As an early step towards developing an in vivo biomarker sensor, antibody (AB) tagged magnetic nanoparticles (NPs) are used here to demonstrate the in vitro measurement of ~5 distinct biomarkers with high specificity and sensitivity. In previous work, aptamers were used to target a given biomarker in vitro and generate magnetic clusters that exhibit a characteristic rotational signature quite different from free NPs. Here the method is expanded to detect a much wider range of biomarkers using polyclonal ABs attached to the surface of the NPs. Commercial ABs exist for a wide range of targets allowing accurate and specific concentration measurements for most significant biomarkers. We show sufficient detection sensitivity, using an in-house spectrometer to measure the rotational signatures of the NPs, to assess physiological concentrations of hormones, cytokines and other signaling molecules. Detection limits for biomarkers drawn mainly from pain and inflammation targets were: 10 pM for mouse Granzyme B (mGZM-B), 40 pM for mouse interferon-gamma (mIFN-γ), 7 pM for mouse interleukin-6 (mIL-6), 40 pM for rat interleukin-6 (rIL-6), 40 pM for mouse vascular endothelial growth factor (mVEGF) and 250 pM for rat calcitonin gene related peptide (rCGRP). Much lower detection limits are certainly possible using improved spectrometers and nanoparticles.

Quantification of magnetic nanoparticles by compensating for multiple environment changes simultaneously.

The quantification of magnetic nanoparticles is important for many applications, especially for in vivo biosensing. The magnetization harmonics used in spectroscopy of magnetic nanoparticles can be used to estimate nanoparticle number or weight. However, other effects such as temperature or relaxation time change can also influence the nanoparticle magnetization. Therefore, it is necessary to compensate for these factors when estimating the amount of magnetic nanoparticles. This paper shows through simulation that a two-dimensional scaling method can be used to improve the accuracy of nanoparticle quantification, especially when multiple effects are present which can influence the nanoparticle magnetization. Finally, an experiment was performed on a Magnetic Spectroscopy of Brownian motion (MSB) apparatus to demonstrate this method, and nanoparticle weight was determined with a mean error of 1.3 ng (1.81%).

Nonlinear Inversion MR Elastography With Low-Frequency Actuation.

Magnetic resonance elastography (MRE) has been developed to noninvasively reconstruct mechanical properties for tissue and tissue-like materials over a frequency range of 10 ~200 Hz. In this work, low frequency (1~1.5 Hz) MRE activations were employed to estimate mechanical property distributions of simulated data and experimental phantoms. Nonlinear inversion (NLI) MRE algorithms based on viscoelastic and poroelastic material models were used to solve the inverse problems and recover images of the shear modulus and hydraulic conductivity. Data from a simulated phantom containing an inclusion with property contrast was carried out to study the feasibility of our low frequency actuated approach. To verify the stability of NLI algorithms for low frequency actuation, different levels of synthetic noise were added to the displacement data. Spatial distributions and property values were recovered well for noise level less than 5%. For the presented experimental phantom reconstructions with regularizations, the computed storage moduli from viscoelastic and poroelastic MRE gave similar results. Contrast was detected between inclusions and background in recovered hydraulic conductivity images. Results and findings confirm the feasibility of future in vivo neuroimaging examinations using natural cerebrovascular pulsations at cardiac frequencies, which can eliminate specialized equipment for high frequency actuation.

Phantom evaluations of low frequency MR elastography.

Intrinsic activation MR elastography (IA-MRE) is a novel technique which seeks to estimate brain mechanical properties non-invasively and without external mechanical drivers. The method eliminates actuation hardware and patient discomfort while capitalizing on the brain's intrinsic low frequency motion. This study explores low frequency actuation (1 Hz) MR elastography in phantoms and analyzes performance of non-linear inversion (NLI) of viscoelastic and poroelastic mechanical models as a framework for assessing clinical results from IA-MRE. We present results from four gelatin phantoms and report stiffness resolution of 6 mm (two measurement voxels) with a stiffness contrast ratio of 4.21 relative to the background and 9 mm (three measurement voxels) with a lower stiffness contrast ratio of near 1.77. Stiffness edge resolution was also evaluated using edge spread and line spread functions and yielded a stiffness edge response distance of 9 mm. The intraclass correlation coefficient was high (0.93) between mechanical testing and poroelastic estimates, although quantitative agreement was affected by model-data mismatch. Viscoelastic MRE at low frequencies has issues with non-uniqueness due to small inertial forces, and performed worse than poroelastic MRE in terms of inclusion detection and consistency with mechanical testing. These results present the first evaluation of MR elastography using displacement measurements from an actuation frequency less than 5 Hz and support the validity of brain IA-MRE to recover spatially resolved stiffness changes. They provide a baseline of performance in terms of standard metrics for future animal and human brain stiffness studies and analyses based on intrinsic motion.

MR elastography at 1 Hz of gelatin phantoms using 3D or 4D acquisition.

Magnetic Resonance Elastography (MRE) detects induced periodic motions in biological tissues allowing maps of tissue mechanical properties to be derived. In-vivo MRE is commonly performed at frequencies of 30-100 Hz using external actuation, however, using cerebro-vascular pulsation at 1 Hz as a form of intrinsic actuation (IA-MRE) eliminates the need for external motion sources and simplifies data acquisition. In this study a hydraulic actuation system was developed to drive 1 Hz motions in gelatin as a tool for investigating the performance limits of IA-MRE image reconstruction under controlled conditions. Quantitative flow (QFLOW) MR techniques were used to phase encode 1 Hz motions as a function of gradient direction using 3D or 4D acquisition; 4D acquisition was twice as fast and yielded comparable motion field and concomitant image reconstruction results provided the motion signal was sufficiently strong. Per voxel motion noise floor corresponded to a displacement amplitude of about 20-30 µm. Signal to noise ratio (SNR) was 94 ±â€¯17 for 3D and dropped to 69 ±â€¯10 for the faster 4D acquisition, but yielded octahedral shear stress and shear modulus maps of high quality that differed by only about 20% on average. QFLOW measurements in gel phantoms were improved significantly by adding Mn(II) to mimic relaxation rates found in brain. Overall, the hydraulic 1 Hz actuation system when coupled with 4D sequence acquisition produced a fast reliable approach for future IA-MRE phantom evaluation and contrast detail studies needed to benchmark imaging performance.

7,7-Dimethyl-5H-dibenzo[e,i][1,4,8]triazacycloundecine-6,9,15(7H,8H,14H)-trione pyridine solvate.

The X-ray crystal structure and hydrogen-bonding patterns of the title compound, C18H17N3O3.C5H5N, a non-N-alkylated cyclotripeptide containing one alpha- and two beta-amino acids, are reported. The amides in the 11-membered ring have an unprecedented all-transoid configuration. The torsion angles and Dunitz parameters describing non-planarity of the amides contained in the cyclotripeptide are discussed.

XANES evidence against a manganyl species in the S3 state of the oxygen-evolving complex.

Manganyl Mn=O species have been suggested as possible intermediates in photosynthetic water oxidation and as the reactive species in asymmetric olefin epoxidation. The first X-ray absorption spectrum for a MnV=O complex is reported. Comparison of the EXAFS data for Na[MnV=O(HMPAB) with those for lower-valent Mn complexes suggests that EXAFS measurements and edge-energy measurements are unlikely to have sufficient sensitivity to reliably reflect the presence of Mn=O species. In contrast, the preedge transition for Na[MnV=O(HMPAB) is 4-fold more intense than the most intense preedge transition observed for nonmanganyl complexes. This increase in intensity is shown to be sufficiently sensitive to allow detection of a manganyl species if it is formed. These data together with published data for the photosynthetic oxygen-evolving complex provide strong evidence against the presence of manganyl Mn=O species in the oxygen-evolving complex through the S3 state.