Measuring protein biomarker concentrations using antibody tagged magnetic nanoparticles

Abstract

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.

Investigation of Commercial Iron Oxide Nanoparticles: Structural and Magnetic Property Characterization

“…For homogeneous bioassays that are based on a conjugation-mediated change in Brownian relaxation time, MNPs should be thermally blocked. ,− Thus, an in-depth study on the Brownian and Néel relaxation times of these MNPs under different driving fields should be carried out. , For magnetic hyperthermia therapy, the dissipated energy or specific absorption rate (SAR) is directly proportional to the imaginary component of AC susceptibility and saturation magnetization ( M s ) of MNPs, the applied field frequency, and the amplitude squared. − Thus, a high M s of MNPs does not guarantee a high SAR, and practical measurements on the hyperthermia performance are required to find out which SHA series MNPs are better suited for hyperthermia applications.…”

One-Step, Wash-free, Nanoparticle Clustering-Based Magnetic Particle Spectroscopy Bioassay Method for Detection of SARS-CoV-2 Spike and Nucleocapsid Proteins in the Liquid Phase

“… 23 − 26 By applying AC magnetic fields to MNPs, the magnetic moments of MNPs follow the time-varying external field directions through a combined Néel and Brownian relaxation mechanism. 16 , 18 , 27 − 29 The dominant relaxation of these two processes is dependent on the magnetic core sizes of MNPs (assuming unconstrained MNPs in liquid without surface binding of any chemical substances). 30 − 32 The Brownian relaxation process is dominant for single-core iron oxide MNPs with core sizes above 20 nm (this critical size may vary for different magnetic materials of different magnetic properties).…”

“…Conjugation of any chemical substances including protein molecules, aptamers, and other nonmagnetic materials onto MNPs can hinder or even block this Brownian relaxation. 23 , 27 , 38 − 45 As a result, this binding event causes a phase lag between magnetic moments and external AC fields (except at very low frequencies), and thus, weaker dynamic magnetic responses are observed from clustered MNPs. By exploiting this unique property of Brownian relaxation-dominant MNPs, researchers have applied it along with the MPS platform for the detection of H1N1 virus, 39 thrombin, 38 staphylococcal toxins, 20 SARS-CoV-2, 23 , 46 botulinum neurotoxins A, B, and E, 21 and so forth.…”

“…Magnetic particle spectroscopy (MPS) is one of the most promising candidates for rapid, inexpensive, and high-accuracy bioassays. − It is a homogeneous biosensing tool that monitors the dynamic magnetic responses of magnetic nanoparticles (MNPs) in the liquid phase. − By applying AC magnetic fields to MNPs, the magnetic moments of MNPs follow the time-varying external field directions through a combined Néel and Brownian relaxation mechanism. ,,− The dominant relaxation of these two processes is dependent on the magnetic core sizes of MNPs (assuming unconstrained MNPs in liquid without surface binding of any chemical substances). − The Brownian relaxation process is dominant for single-core iron oxide MNPs with core sizes above 20 nm (this critical size may vary for different magnetic materials of different magnetic properties). − This relaxation process reflects the degree of freedom of physical rotational motion of MNPs. Conjugation of any chemical substances including protein molecules, aptamers, and other nonmagnetic materials onto MNPs can hinder or even block this Brownian relaxation. ,,− As a result, this binding event causes a phase lag between magnetic moments and external AC fields (except at very low frequencies), and thus, weaker dynamic magnetic responses are observed from clustered MNPs. By exploiting this unique property of Brownian relaxation-dominant MNPs, researchers have applied it along with the MPS platform for the detection of H1N1 virus, thrombin, staphylococcal toxins, SARS-CoV-2, , botulinum neurotoxins A, B, and E, and so forth.…”

A Portable Magnetic Particle Spectrometer for Future Rapid and Wash-Free Bioassays

“…Furthermore, by surface functionalizing MNPs with different ligands (e.g., carboxylic acid and amine), nucleic acids (i.e., DNA and RNA), and proteins (e.g., antibodies, streptavidin, protein A, etc. ), the MNPs can be specifically modified for detecting different target analytes as well as diseases. ,,,,− Recently, it has been reported that a custom-built scanning MPS is used for biomolecule imaging, showing great potential for disease diagnostics and imaging …”