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This paper describes a magnetic levitation-based method that can specifically detect the presence of antigens, either soluble or membrane-bound, by quantifying changes in the levitation height of capture beads with fixed densities.
The described method was developed based on the principles of magnetic levitation, which separates cells and particles based on their density and magnetic properties. Density is a cell type identifying property, directly related to its metabolic rate, differentiation, and activation status. Magnetic levitation allows a one-step approach to successfully separate, image and characterize circulating blood cells, and to detect anemia, sickle cell disease, and circulating tumor cells based on density and magnetic properties. This approach is also amenable to detecting soluble antigens present in a solution by using sets of low- and high-density beads coated with capture and detection antibodies, respectively. If the antigen is present in solution, it will bridge the two setsĀ of beads, generating a new bead-bead complex, which will levitate in between the rows of antibody-coated beads. Increased concentration of the target antigen in solution will generate a larger number of bead-bead complexes when compared to lower concentrations of antigen, thus allowing for quantitative measurements of the target antigen. Magnetic levitation is advantageous to other methods due to its decreased sample preparation time and lack of dependance on classical readout methods. The image generated is easily captured and analyzed using a standard microscope or mobile device, such as a smartphone or a tablet.
Magnetic levitation is a technique developed to separate, analyze, and identify cellĀ types1,2,3, proteins4,5 and opioids6 based solely on their specific density and paramagnetic properties. Cell density is a unique, intrinsic property of each cell type directly related to its metabolic rate and differentiation status7,8,9,10,11<....
The experimental protocol used in this study was approved by the Beth Israel Deaconess Medical Center Institutional Review Board (IRB).
1. Instrument setup
NOTE: Imaging levitating cells requires two rare earth neodymium magnets magnetized on the z-axis to be placed with the same pole facing each other to generate a magnetic field. The distance between the magnets can be customized depending on the intensity of the magnetic field and the density of the targets. In thi.......
Magnetic levitation focuses objects of different densities at different levitation heights depending on the object's density, its magnetic signature, the concentration of paramagnetic solution, and the strength of the magnetic field created by two strong, rare-earth magnets. As the two magnets are placed on top of each other, levitating samples can only be viewed, while maintaining Kƶhler illumination, by using a microscope turned on its side (Figure 1). The final levitation height .......
Gradient centrifugation is currently the standard technique for isolating subcellular components based on their unique densities. This approach, however, requires the use of specialized gradient media as well as centrifuge equipment. The magnetic levitation approach presented hereāÆallows detailed investigation of the morphological and functional properties of circulating cells, with minimum, if any manipulation of the cells, providing a near in vivo access to circulating cells.
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The authors would like to thank Dr. Getulio Pereira for his help with extracellular vesicle work.
This work was supported by the following National Institute of Health grants to ICG: RO1CA218500, UG3HL147353, and UG3TR002881.
....Name | Company | Catalog Number | Comments |
2-(N-Morpholino)ethanesulfonic acid hydrate | Sigma Aldrich | M-2933 | (MES); component of activation buffer |
50x2.5x1 mm magnets, Nickel (Ni-Cu-Ni) plated, grade N52, magnetized through 5mm (0.197") thickness | K&J Magnetics | Custom | Magnets used for the magnetic levitation device |
Capillary Tube Sealant (Critoseal) | Leica Microsystems | 267620 | Used to cap the ends of the capillary tubes |
Centrifuge tube filters (Corning Costar Spin-X) | Sigma Aldrich | CLS8163 | Used to wash beads |
Compact Lab Jack | Thorlabs | LJ750 | Used for adjusting the magnetic levitation device |
DPBS, no calcium, no magnesium | Gibco | 14190-144 | Solution for bead suspensions |
Ethanolamine | Sigma Aldrich | E9508-100ML | Used during a wash step for beads |
Fluorescent Plasma Membrane Stain (CellMask Green) | Invitrogen | C37608 | Used to stain Rh+ cells |
Gadoteridol Injection | ProHance | NDC 0270-1111-03 | Gadolinium (Gd3+); magnetic solution used to suspend cells |
HBSS++ | Gibco | 14025-092 | Solution for sample preparation |
Human C5b,6 complex | Complement Technology, Inc | A122 | Used to generate RBC Evs |
Human C7 protein | Complement Technology, Inc | A124 | Used to generate RBC Evs |
Human C8 protein | Complement Technology, Inc | A125 | Used to generate RBC Evs |
Human C9 protein | Complement Technology, Inc | A126 | Used to generate RBC Evs |
Mini Series Post Collar | Thorlabs | MSR2 | Used to secure magnetic levitation device to lab jacks |
N-(3-Dimethylaminopropyl)-Nā²-ethylcarbodiimide hydrochloride | Sigma Aldrich | E1769-10G | (EDC); used in antibody coupling reaction |
Normal Rabbit IgG Control | R&D Systems | AB-105-C | Used to coat beads as a control condition |
Phosphate Buffered Saline (10X Solution, pH 7.4) | Boston Bioproducts | BM-220 | Component of coupling buffer, used for washing steps |
Polysorbate 20 (Tween 20) | Sigma Aldrich | P7949-500ML | Component of activation buffer |
Polystyrene Carboxyl Polymer | Bangs Laboratories | PC06004 | Top density beads (1.05 g/mL), used for antibody coupling |
Rabbit RhD Polyclonal Antibody | Invitrogen | PA5-112694 | Used to coat beads for the dectection of Rh factor in red blood cells |
Research Grade Microscope | Olympus | Provis AX-70 | Microscoped used to mount magnetic levitation device and view levitating cells |
Rubber Dampening Feet | Thorlabs | RDF1 | Used to support the breadboard table |
Square Boro Tubing | VitroTubes | 8100-050 | Capillary tube used for loading sample into Maglev |
Sulfo-NHS | Thermoscientific | 24510 | Used in antibody coupling reaction |
Translational Stage | Thorlabs | PT1 | Used for focusing and for scanning capillary tube |
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