DENSITY AS A TOOL FOR CHEMISTRY AND BIOLOGY
Density, defined as the ratio of the mass and volume of an object, is a fundamental characteristic of all matter. In our lab, we have developed two density-based platforms—aqueous multiphase systems (AMPS) and magnetic levitation (MagLev)—that can be used to separate valuable materials, perform quality control, make medical diagnoses, enable self-assembly, and probe chemical and biological binding events.
AQUEOUS MULTIPHASE SYSTEMS (AMPS)
Aqueous multiphase systems (AMPS) are aqueous mixtures of polymers, salts, and surfactants that spontaneously separate, like oil and water, into discrete phases having distinct densities (Figure 1).1 AMPS provide seven distinct advantages: 1) AMPS can be designed to have two to six phases (we have demonstrated over 300 different systems), 2) the density difference between the phases of an AMPS can be tuned to be very small (Δρ ~ 0.0003 g cm-3), 3) the molecularly sharp interfaces between the phases of an AMPS provide bins to separate and extract objects based on their density, 4) they can be made to be biocompatible and, because they have extremely low interfacial surface energy between the phases (nJ m-2 to mJ m-2), can be used to separate fragile objects, such as cells, 5) unlike other density gradient systems, AMPS are stable over time: since the phases are immiscible, diffusional mixing of phases does not occur, and the steps in density reform after agitation, 6) they are scalable—they can be used in a capillary tube for low-cost diagnostics, or in a Falcon tube for the large scale separation of whole blood, and 7) the use of AMPS requires minimal training and only a benchtop centrifuge is necessary to perform an assay or separation.
Separations with AMPS
Many applications that exploit the unique properties of nanoparticles require particles (rods, spheres, etc.) having high purity. We have used AMPS as a means to separate nanoparticles based on their shape and size.2 When a particle is centrifuged in a viscous media (called rate-zonal centrifugation), the hydrodynamic behavior of the particle depends on its shape and size. We exploit this behavior to enrich gold nanorods from 48% to 99% purity in less than ten minutes using a benchtop centrifuge (Figure 2).
Using AMPS, we have created a simple method to enrich reticulocytes (immature red blood cells) that can be used to grow malaria species better than conventional reticulocyte purification methods.3
Diagnostics with AMPS
In certain cases, the ability to separate cells by density can provide a means to diagnose disease. Sickle cell disease is a genetic disease that approximately 300,000 children are born with each year. Most of these children are undiagnosed and at high risk for childhood mortality even though simple and effective interventions exist to manage the disease. The morphological changes in sickle cell disease lead to the formation of very dense erythrocytes that can be separated using AMPS. Using capillary tubes, AMPS, and portable centrifuges, we have created a low-cost, visual test for sickle cell disease that can be performed in ~15 minutes.4 After initial validation in the U.S., we worked with partners in Zambia to test the device in a larger population and to assess feasibility of the design in rural health centers.5
MAGNETIC LEVITATION (MagLev)
Another method to analyze density with minimal equipment is magnetic levitation.6,7 Using two opposing magnets, we create a magnetic field gradient. When a diamagnetic object is placed in a paramagnetic solution within the magnetic field gradient, the paramagnetic fluid will be attracted to the highest field (closer to the magnets) and will push diamagnetic object towards the point of lowest field (in the middle of the two magnets). When the object has a different density than the solution, an additional buoyant force pushes the object away from the center, and towards the top (float) or bottom (sink) of the solution. The balance of the magnetic and buoyant forces results in a stable levitation height within the paramagnetic fluid that depends on the density (Figure 3). The paramagnetic fluid used in MagLev can consist of paramagnetic salts (e.g., MnCl2 or GdCl3) in water,6 organic solutions of hydrophobic chelates (e.g., gadolinium(III) diethylenetriamine triacetic acid didecyldiacetamide),8 or paramagnetic ionic liquids.9
SEPARATIONS WITH MagLev
Over a billion pounds of plastics are recycled each year. The value of recycled plastics is related to its final purity. MagLev is able to levitate several objects simultaneously, based on their density. We have demonstrated that MagLev can be used to separate different grades of polymers (e.g., Nylon 6 and Nylon 6/6) or polymers having different stereochemistry (e.g., syndiotactic and atactic polystyrene).7
When organic molecules crystallize, they can produce a variety of different macroscopic structures (polymorphs), each having unique properties. Many small-molecule pharmaceuticals, for example, form several polymorphs and oftentimes only one structure possesses the desired properties. No convenient method for separating polymorphs exists. We have applied MagLev as a non-destructive tool to separate a variety of crystal polymorphs based on their density.10
QUALITY CONTROL USING MagLev
Food and Water
Density is commonly used in the food and beverage industries to obtain information about a substance. The alcohol content of wine, for example, can be inferred by measuring its density. Many tools exist to probe density of solids and liquids, but each has trade-offs between cost, ease of operation, and accuracy. MagLev provides a low-cost, simple, and rapid method to precisely assess the density of foods and water.8 Figure 4 shows several samples of milk levitating in a MagLev device—the height of levitation is dependent on the fat content of the milk. MagLev can also be used to assess the density of small pieces of solids (string cheese) and pastes (peanut butter).
Quality control is often the largest single cost in the manufacturing of small plastic parts. Visual evaluation, the most common form of quality control for plastic parts, cannot detect internal defects in opaque objects (i.e., the majority of plastic parts). Methods such as x-ray computed tomography and ultrasonic testing provide exquisite information on the structural characteristics of opaque parts, but they are generally slow and cannot be used to test a large number of parts without a prohibitive increase in cost and time for manufacturing. Many defects found in plastic parts (due to voids, cracks, unpolymerized material) have a different density than the bulk of the part. We have demonstrated the use of MagLev to rapidly and nondestructively assess injection-molded plastic parts for defects by monitoring, by eye, the orientation that a part adopts when levitating in the MagLev device (Figure 5).11
DIAGNOSTICS WITH MagLev
Density-based ELIA with MagLev
Using antigens immobilized onto the surface of polymeric beads, we have created a density-based ELISA. When the beads are exposed to a sample, antibodies specifically bind the antigens. Anti-human antibodies conjugated with gold particles then bind to the captured antibody. We then chemically deposit silver or gold onto the beads. The deposition of metal leads to a change in density of the bead that indicates the amount of antigen that was captured. Using parallel vials, we can perform a multiplexed density-based ELISA.12
NONCONTACT ORIENTATION AND SELF-ASSEMBLY USING MagLev
Noncontact Orientation in MagLev
The manipulation and orientation of objects having irregular shape is a major challenge in advanced manufacturing; materials that are soft, sticky, or brittle are hard to manipulate no matter their shape. We have developed mathematical models that allow us to predict the height and orientation of an object levitating in MagLev.13 From this understanding, we have shown the ability of MagLev to manipulate objects by 1) rotating the MagLev device around an object suspended in paramagnetic solution, or 2) using an external magnet to perturb the local magnetic field (Figure 6). We use MagLev to manipulate hard plastics, soft elastomers, stick hydrogels, and fragile droplets of jammed particles.
Self-Assembly using MagLev
Self-assembling objects on the mesoscale (mm to cm scale) is an unsolved challenge: structure tends to be dominated by gravitational forces rather than interactions between two assembling components. MagLev provides a buoyant force to counteract gravity, enabling objects to assemble based on size, shape, and density.14,15 Figure 7 displays a variety of self-assembled objects.
MEASURING ATOMIC LEVEL DIFFERENCES USING MagLev
Bioassays frequently involving use of proteins bound to beads to identify molecules of interest based on specific binding events. Most assays use labels (typically fluorescent) to detect and quantify binding events. While this approach is well studied and reliable, we sought to develop a new approach that may be useful in certain applications. We use MagLev to measure the binding between proteins and ligands by functionalizing small molecules (ligands) to diamagnetic beads.16,17 As the protein-ligand binding progresses, the density of the beads increases; the process can be monitored, by eye, with high accuracy.
Monitoring Chemical Reactions
MagLev can also be used to monitor non-biological chemical reactions.18 We functionalize beads with ten derivatives of 4-benzyloxybenzaldehyde polystyrene and perform a polymerization reaction inside of the MagLev device. By monitoring the change in levitation height, we can monitor the extent and rate of reaction.
1. Mace, C.R., Akbulut.O., Kumar.A.A., Shapiro.N.D., Derda.R., and Whitesides.G.M., "Aqueous multiphase systems of polymers and surfactants provide self-assembling step-gradients in density", J. Am. Chem. Soc. 134, 9094–9097 (2012).
2. Akbulut, O., Mace.C.F., Martinez.A.W., Kumar.A.A., Nie.Z., Patton.M.R., and Whitesides.G.M., "Separation of Nanoparticles in Aqueous Multiphase Systems through Centrifugation", Nano Lett. 12, 4060–4064 (2012).
3. Kumar, A.A, Lim.C., Moreno.Y., Mace.C.R, Syed.A., Tyne.D.V., Wirth.D.F, Duraisingh.D.T, and Whitesides.G.M,"Enrichment of reticulocytes from whole blood using aqueous multiphase systems of polymers", Am. J. Hematol. 90, 31–36 (2015).
4. Kumar, A.A, Patton.M.R, Hennek.J.W, Lee.S.Y.R, Alesio-Spina.D.D., Yang.X., Kanter.J., Shevkoplyas.S.S, Brugnara.C., and Whitesides.G.M, "Density-based separation in multiphase systems provides a simple method to identify sickle cell disease", Proc. Natl. Acad. Sci. U. S. A. 111, 14864–14869 (2014).
5. Kumar, A.A, Chunda-Liyoka.C., Hennek.J.W, Mantina.H., Lee.S.Y.R, Patton.M.R, Sambo.S., Sinyangwe.S., Kankasa.C., Chintu.C., Stossel.T.P, and Whitesides.G.M, "Evaluation of a Density-Based Rapid Diagnostic Test for Sickle Cell Disease in a Clinical Setting in Zambia", PLoS One 9, e114540 (2014).
6. Mirica, K. A., Shevkoplyas, S. S., Phillips, S. T., Gupta, M. & Whitesides, G. M. "Measuring Densities of Solids and Liquids Using Magnetic Levitation: Fundamentals", J. Am. Chem. Soc. 131, 10049–10058 (2009).
7. Winkleman, A., Perez-Castillejos, R., Gudiksen, K.L., Phillips, S.T., Prentiss, M., and Whitesides, G.M., "Density-based diamagnetic separation: devices for detecting binding events and for collecting unlabeled diamagnetic particles in paramagnetic solutions", Anal. Chem. 79, 6542–6550 (2007).
8. Mirica, K. A., Phillips, S. T., Mace, C. R. & Whitesides, G. M. "Magnetic Levitation in the Analysis of Foods and Water", J. Agric. Food Chem. 58, 6565–6569 (2010).
9. Bwambok, D.K., Thou.M.M., Atkinson.M.B.J., Mirica.K.A., Shaprio.N.D., and Whitesides.G.M. "Paramagnetic ionic liquids for measurements of density using magnetic levitation", Anal. Chem. 85, 8442–8447 (2013).
10. Atkinson, M.B.J., Bwambok.D.K., Chen.J., Chopode.P.D., Thuo.M.M., Mace.C.R., Mirica.K.A., Kumar.A.A., Myerson.A.S., and Whitesides.G.M., "Using magnetic levitation to separate mixtures of crystal polymorphs", Angew. Chem. Int. Ed. Engl. 125, 10398–10401 (2013).
11. Hennek, J.W., Nemiroski.A., Subramaniam.A.B., Bwambok.D.K., Yang.D., Harburg.D.V., Tricard.S., Ellerbee.A.K., and Whitesides.G.M., "Using Magnetic Levitation for Non-Destructive Quality Control of Plastic Parts", Adv. Mater. 27, 1587–1592 (2015).
12. Subramaniam, A. B., Gonidec, M., Shapiro, N. D., Kresse, K. M. & Whitesides, G. M. "Metal-amplified Density Assays, (MADAs), including a Density-Linked Immunosorbent Assay (DeLISA)", Lab Chip 15, 1009–1022 (2015).
13. Subramaniam, A.B, Yang.D., Yu.H., Nemiroski.A., Tricard.S., Ellerbee.A.K, Soh.S., and Whitesides.G.M., "Noncontact orientation of objects in three-dimensional space using magnetic levitation", Proc. Natl. Acad. Sci. U. S. A. 111, 12980–12985 (2014).
14. Mirica, K. A., Ilievski, F., Ellerbee, A. K., Shevkoplyas, S. S. & Whitesides, G. M. "Using Magnetic Levitation for Three Dimensional Self-Assembly", Adv. Mater. 23, 4134–4140 (2011).
15. Ilievski, F., Mirica, K. A., Ellerbee, A. K. & Whitesides, G. M. "Templated self-assembly in three dimensions using magnetic levitation", Soft Matter 7, 9113 (2011).
16. Shapiro, N.D., Mirica.K.A., Soh.S., Phillips.S.T., Taran.O., Mace.C.R., Shevkoplyas.S.S., and Whitesides.G.M.,"Measuring Binding of Protein to Gel-Bound Ligands Using Magnetic Levitation", J. Am. Chem. Soc. 134, 5637–5646 (2012).
17. Shapiro, N. D., Soh, S., Mirica, K. A. & Whitesides, G. M. "Magnetic levitation as a platform for competitive protein-ligand binding assays", Anal. Chem. 84, 6166–6172 (2012).
18. Mirica, K. A., Phillips, S. T., Shevkoplyas, S. S. & Whitesides, G. M. "Using magnetic levitation to distinguish atomic-level differences in chemical composition of polymers, and to monitor chemical reactions on solid supports", J. Am. Chem. Soc. 130, 17678–17680 (2008).