Five-Minute Magnetic Nanoparticle Spectroscopy-Based Bioassay for Ultrafast Detection of SARS-CoV-2 Spike Protein.

In this work, we report a 5-min magnetic particle spectroscopy (MPS)-based bioassay strategy. In our approach, surface-functionalized magnetic nanoparticles are incubated with target analytes at 37 °C with agitation for 3 min, and the MPS reading is then taken at the fifth minute. We prove the feasibility of 5 min ultrafast detection of SARS-CoV-2 spike protein with a detection limit below 5 nM (0.2 pmol). Our proposed 5-min bioassay strategy may be applied to reduce the assay time for other liquid-phase, volumetric biosensors such as NMR, quantum dots, fluorescent biosensors, etc.

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.

With the ongoing global pandemic of coronavirus disease 2019 (COVID-19), there is an increasing quest for more accessible, easy-to-use, rapid, inexpensive, and high-accuracy diagnostic tools. Traditional disease diagnostic methods such as qRT-PCR (quantitative reverse transcription-PCR) and ELISA (enzyme-linked immunosorbent assay) require multiple steps, trained technicians, and long turnaround time that may worsen the disease surveillance and pandemic control. In sight of this situation, a rapid, one-step, easy-to-use, and high-accuracy diagnostic platform will be valuable for future epidemic control, especially for regions with scarce medical resources. Herein, we report a magnetic particle spectroscopy (MPS) platform for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) biomarkers: spike and nucleocapsid proteins. This technique monitors the dynamic magnetic responses of magnetic nanoparticles (MNPs) and uses their higher harmonics as a measure of the nanoparticles' binding states. By anchoring polyclonal antibodies (pAbs) onto MNP surfaces, these nanoparticles function as nanoprobes to specifically bind to target analytes (SARS-CoV-2 spike and nucleocapsid proteins in this work) and form nanoparticle clusters. This binding event causes detectable changes in higher harmonics and allows for quantitative and qualitative detection of target analytes in the liquid phase. We have achieved detection limits of 1.56 nM (equivalent to 125 fmole) and 12.5 nM (equivalent to 1 pmole) for detecting SARS-CoV-2 spike and nucleocapsid proteins, respectively. This MPS platform combined with the one-step, wash-free, nanoparticle clustering-based assay method is intrinsically versatile and allows for the detection of a variety of other disease biomarkers by simply changing the surface functional groups on MNPs.

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

Magnetic nanoparticles (MNPs) have been extensively used as tiny heating sources in magnetic hyperthermia therapy, contrast agents in magnetic resonance imaging, tracers in magnetic particle imaging, carriers for drug/gene delivery, etc. There have emerged many MNP/microbead suppliers since the past decade, such as Ocean NanoTech, Nanoprobes, US Research Nanomaterials, Miltenyi Biotec, micromod Partikeltechnologie GmbH, nanoComposix, and so forth. In this paper, we report the physical and magnetic characterizations on iron oxide nanoparticle products from Ocean NanoTech. Standard characterization tools such as vibrating-sample magnetometry, X-ray diffraction, dynamic light scattering, transmission electron microscopy, and zeta potential analysis are used to provide MNP customers and researchers with an overview of these iron oxide nanoparticle products. In addition, the dynamic magnetic responses of these iron oxide nanoparticles in aqueous solutions are investigated under low- and high-frequency alternating magnetic fields, giving a standardized operating procedure for characterizing the MNPs from Ocean NanoTech, thereby yielding the best of MNPs for different applications.

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

Nowadays, there is an increasing demand for more accessible routine diagnostics for patients with respect to high accuracy, ease of use, and low cost. However, the quantitative and high accuracy bioassays in large hospitals and laboratories usually require trained technicians and equipment that is both bulky and expensive. In addition, the multistep bioassays and long turnaround time could severely affect the disease surveillance and control especially in pandemics such as influenza and COVID-19. In view of this, a portable, quantitative bioassay device will be valuable in regions with scarce medical resources and help relieve burden on local healthcare systems. Herein, we introduce the MagiCoil diagnostic device, an inexpensive, portable, quantitative, and rapid bioassay platform based on the magnetic particle spectrometer (MPS) technique. MPS detects the dynamic magnetic responses of magnetic nanoparticles (MNPs) and uses the harmonics from oscillating MNPs as metrics for sensitive and quantitative bioassays. This device does not require trained technicians to operate and employs a fully automatic, one-step, and wash-free assay with a user friendly smartphone interface. Using a streptavidin-biotin binding system as a model, we show that the detection limit of the current portable device for streptavidin is 64 nM (equal to 5.12 pmole). In addition, this MPS technique is very versatile and allows for the detection of different diseases just by changing the surface modifications on MNPs. Although MPS-based bioassays show high sensitivities as reported in many literatures, at the current stage, this portable device faces insufficient sensitivity and needs further improvements. It is foreseen that this kind of portable device can transform the multistep, laboratory-based bioassays to one-step field testing in nonclinical settings such as schools, homes, offices, etc.

Magnetic Particle Spectroscopy with One-Stage Lock-In Implementation for Magnetic Bioassays with Improved Sensitivities.

In recent years, magnetic particle spectroscopy (MPS) has become a highly sensitive and versatile sensing technique for quantitative bioassays. It relies on the dynamic magnetic responses of magnetic nanoparticles (MNPs) for the detection of target analytes in the liquid phase. There are many research studies reporting the application of MPS for detecting a variety of analytes including viruses, toxins, nucleic acids, and so forth. Herein, we report a modified version of the MPS platform with the addition of a one-stage lock-in design to remove the feedthrough signals induced by external driving magnetic fields, thus capturing only MNP responses for improved system sensitivity. This one-stage lock-in MPS system is able to detect as low as 781 ng multi-core Nanomag50 iron oxide MNPs (micromod Partikeltechnologie GmbH) and 78 ng single-core SHB30 iron oxide MNPs (Ocean NanoTech). We first demonstrated the performance of this MPS system for bioassay-related applications. Using the SARS-CoV-2 spike protein as a model, we have achieved a detection limit of 125 nM (equal to 5 pmole) for detecting spike protein molecules in the liquid phase. In addition, using a streptavidin-biotin binding system as a proof-of-concept, we show that these single-core SHB30 MNPs can be used for Brownian relaxation-based bioassays while the multi-core Nanomag50 cannot be used. The effects of MNP amount on the concentration-dependent response profiles for detecting streptavidin were also investigated. Results show that by using a lower concentration/ amount of MNPs, concentration-response curves shift to a lower concentration/amount of target analytes. This lower concentration-response indicates the possibility of improved bioassay sensitivities by using lower amounts of MNPs.

Folic Acid Targeting for Efficient Isolation and Detection of Ovarian Cancer CTCs from Human Whole Blood Based on Two-Step Binding Strategy.

Studies regarding circulating tumor cells (CTCs) have great significance for cancer prognosis, treatment monitoring, and metastasis diagnosis. However, due to their extremely low concentration in peripheral blood, isolation and enrichment of CTCs are the key steps for early detection. To this end, targeting the folic acid receptors (FRs) on the CTC surface for capture with folic acid (FA) using bovine serum albumin (BSA)-tether for multibiotin enhancement in combination with streptavidin-coated magnetic nanoparticles (MNPs-SA) was developed for ovarian cancer CTC isolation. The streptavidin-biotin-system-mediated two-step binding strategy was shown to capture CTCs from whole blood efficiently without the need for a pretreatment process. The optimized parameters for this system exhibited an average capture efficiency of 80%, which was 25% higher than that of FA-decorated magnetic nanoparticles based on the one-step CTC separation method. Moreover, the isolated cells remained highly viable and were cultured directly without detachment from the MNPs-SA-biotin-CTC complex. Furthermore, when the system was applied for the isolation and detection of CTCs in ovarian cancer patients' peripheral blood samples, it exhibited an 80% correlation with clinical diagnostic criteria. The results indicated that FA targeting, in combination with BSA-based multibiotin enhancement magnetic nanoparticle separation, is a promising tool for CTC enrichment and detection of early-stage ovarian cancer.

Preclinical evaluation of a urokinase plasminogen activator receptor-targeted nanoprobe in rhesus monkeys.

PURPOSE: To translate a recombinant peptide containing the amino-terminal fragment (ATF) of urokinase plasminogen activator receptor-targeted magnetic iron oxide (IO) nanoparticles (uPAR-targeted human ATF-IONPs) into clinical applications, we conducted a pilot study to evaluate the toxicity and pharmacokinetics of this nanoparticle in normal rhesus monkeys. METHODS: We assessed the changes in the following: magnetic resonance imaging (MRI) signals from pretreatment stage to 14 days posttreatment, serum iron concentrations from 5 minutes posttreatment to 12 weeks posttreatment, routine blood examination and serum chemistry analysis results from pretreatment stage to 12 weeks after administration, and results of staining of the liver with Perls' Prussian Blue and hematoxylin-eosin at 24 hours and 3 months posttreatment in two rhesus monkeys following an intravenous administration of the targeted nanoparticles either with a polyethylene glycol (ATF-PEG-IONP) or without a PEG (ATF-IONP) coating. RESULTS: The levels of alkaline phosphatase, alanine transaminase, and direct bilirubin in the two monkeys increased immediately after the administration of the IONPs but returned to normal within 20 days and stayed within the normal reference range 3 months after the injection. The creatinine levels of the two monkeys stayed within the normal range during the study. In addition, red blood cells, white blood cells, hemoglobin level, and platelets remained normal during the 3 months of the study. CONCLUSION: All of the results suggest that a transient injury in terms of normal organ functions, but no microscopic necrotic lesions, was observed at a systemic delivery dose of 5 mg/kg of iron equivalent concentration in the acute phase, and that no chronic toxicity was found 3 months after the injection. Therefore, we conclude that uPAR-targeted IONPs have the potential to be used as receptor-targeted MRI contrasts as well as theranostic agents for the detection and treatment of human cancers in future studies.

Targeted iron-oxide nanoparticle for photodynamic therapy and imaging of head and neck cancer.

Photodynamic therapy (PDT) is a highly specific anticancer treatment modality for various cancers, particularly for recurrent cancers that no longer respond to conventional anticancer therapies. PDT has been under development for decades, but light-associated toxicity limits its clinical applications. To reduce the toxicity of PDT, we recently developed a targeted nanoparticle (NP) platform that combines a second-generation PDT drug, Pc 4, with a cancer targeting ligand, and iron oxide (IO) NPs. Carboxyl functionalized IO NPs were first conjugated with a fibronectin-mimetic peptide (Fmp), which binds integrin ß1. Then the PDT drug Pc 4 was successfully encapsulated into the ligand-conjugated IO NPs to generate Fmp-IO-Pc 4. Our study indicated that both nontargeted IO-Pc 4 and targeted Fmp-IO-Pc 4 NPs accumulated in xenograft tumors with higher concentrations than nonformulated Pc 4. As expected, both IO-Pc 4 and Fmp-IO-Pc 4 reduced the size of HNSCC xenograft tumors more effectively than free Pc 4. Using a 10-fold lower dose of Pc 4 than that reported in the literature, the targeted Fmp-IO-Pc 4 NPs demonstrated significantly greater inhibition of tumor growth than nontargeted IO-Pc 4 NPs. These results suggest that the delivery of a PDT agent Pc 4 by IO NPs can enhance treatment efficacy and reduce PDT drug dose. The targeted IO-Pc 4 NPs have great potential to serve as both a magnetic resonance imaging (MRI) agent and PDT drug in the clinic.

Theranostic nanoparticles with controlled release of gemcitabine for targeted therapy and MRI of pancreatic cancer.

The tumor stroma in human cancers significantly limits the delivery of therapeutic agents into cancer cells. To develop an effective therapeutic approach overcoming the physical barrier of the stroma, we engineered urokinase plasminogen activator receptor (uPAR)-targeted magnetic iron oxide nanoparticles (IONPs) carrying chemotherapy drug gemcitabine (Gem) for targeted delivery into uPAR-expressing tumor and stromal cells. The uPAR-targeted nanoparticle construct, ATF-IONP-Gem, was prepared by conjugating IONPs with the amino-terminal fragment (ATF) peptide of the receptor-binding domain of uPA, a natural ligand of uPAR, and Gem via a lysosomally cleavable tetrapeptide linker. These theranostic nanoparticles enable intracellular release of Gem following receptor-mediated endocytosis of ATF-IONP-Gem into tumor cells and also provide contrast enhancement in magnetic resonance imaging (MRI) of tumors. Our results demonstrated the pH- and lysosomal enzyme-dependent release of gemcitabine, preventing the drug from enzymatic degradation. Systemic administrations of ATF-IONP-Gem significantly inhibited the growth of orthotopic human pancreatic cancer xenografts in nude mice. With MRI contrast enhancement by IONPs, we detected the presence of IONPs in the residual tumors following the treatment, suggesting the possibility of monitoring drug delivery and assessing drug-resistant tumors by MRI. The theranostic ATF-IONP-Gem nanoparticle has great potential for the development of targeted therapeutic and imaging approaches that are capable of overcoming the tumor stromal barrier, thus enhancing the therapeutic effect of nanoparticle drugs on pancreatic cancers.