Paper works: Paper‐based indirect ELISA (see picture) has been demonstrated through the detection of rabbit IgG and the HIV‐1 envelope antigen gp41. This technique combines the sensitivity and specificity of ELISA with the low cost and ease‐of‐use of paper‐based platforms.

This communication demonstrates the generation of over 300 phase-separated systems—ranging from two to six phases—from mixtures of aqueous solutions of polymers and surfactants. These aqueous multiphase systems (MuPSs) form self-assembling, thermodynamically stable, step-gradients in density using a common solvent (water). The steps in density between phases of a MuPS can be very small (Δρ ~ 0.001 g/cm3), do not change over time, and can be tuned by the addition of co-solutes. We use two sets of similar objects—glass beads and pellets of different formulations of Nylon—to demonstrate the ability of MuPSs to separate mixtures of objects by differences in density. The stable interfaces between phases facilitate the convenient collection of species after separation. These results suggest that the stable, sharp, step-gradients in density provided by MuPSs can enable new classes of fractionations and separations based on density.

# These authors contributed equally to this work.1 This paper describes enzyme-linked immunosorbent assays (ELISA) performed in a 96-microzone plate made out of paper (paper-based ELISA, or P-ELISA). ELISA is widely used in biochemical analyses, including immunoassays, food industry assays for food allergens, and toxicological assays. These assays are typically carried out in microtiter plates or small vials.1,2 ELISA combines the specificity of antibodies and the high-turnover catalysis by enzymes, to provide specificity and sensitivity. 1,2 We have recently described a 96-microzone paper plate-fabricated by patterning hydrophobic polymer in hydrophilic paper-as a platform for biochemical analysis. 3,4 Although microfluidic paper-based analytical devices (µPADs) were designed primarily to provide analytical capability at low cost in developing countries, [5][6][7] we expect that they will also be useful in applications such as point-of-care clinical analysis, military field operations, and others where high throughput, low volumes of sample, low cost, and robustness are important. 6,7 These devices have so far been prototyped using analyses of simple metabolites: glucose, protein, and certain enzymes. 8-10 P-ELISA combines the sensitivity and specificity of ELISA with the convenience, low cost and ease-of-use of paper-based platforms.Porous membranes, including nitrocellulose and filter paper, have been used for decades in dot-immunobinding assays (DIA). [8][9][10][11][12][13] Though DIAs are the simplest form of immunoassays on paper, they typically require one piece of nitrocellulose for each assay, the pieces of nitrocellulose have to be processed individually in Petri dishes, and the assays take several hours to complete. 9 Quantitative DIAs have been reported, 14 but DIAs are typically qualitative, and provide only "yes/no" results. 15 Traditional ELISA, usually performed in 96-well plates (fabricated by injection molding in plastic), is quantitative 2 and well-suited for high-throughput assays, but each assay requires large volumes (~20-200 µL) of analyte and reagents, the incubation and blocking steps are long (≥1 h per step, because the reagents must diffuse to the surface of the wells), and the results are quantified using a plate reader, typically an ~$20,000 instrument. 9,16Paper microzone plates for ELISA can have the same layout as plastic 96-wellplates, but each test zone requires only ~3 µL of sample, and the results can be measured using a desktop scanner, typically a ~$100 instrument. In addition, an entire P-ELISA can be completed in less than one hour. The ease of fabrication of paper microzone plates also opens opportunities for a wide range of non-standard formats, and customized connections to carry reagents between zones. To evaluate the feasibility of P-ELISA, and the potential advantages and disadvantages of P-ELISA and 96-well-plate-based ELISA,we developed a three-step procedure that i) immobilizes targeted antigens and then incubates them with their primary antibodies on a 96-microzo...

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This paper demonstrates the use of aqueous multiphase systems (MuPSs) as media for rate-zonal centrifugation to separate nanoparticles of different shapes and sizes. The properties of MuPSs do not change with time or during centrifugation; this stability facilitates sample collection after separation. A three-phase system demonstrates the separation of the reaction products (nanorods, nanospheres, and large particles) of a synthesis of gold nanorods, and enriches the nanorods from 48 to 99% in less than ten minutes using a benchtop centrifuge.

This paper describes the use of magnetic levitation (MagLev) to measure the association of proteins and ligands. The method starts with diamagnetic gel beads that are functionalized covalently with small molecules (putative ligands). Binding of protein to the ligands within the bead causes a change in the density of the bead. When these beads are suspended in a paramagnetic aqueous buffer and placed between the poles of two NbFeB magnets with like poles facing, the changes in the density of the bead on binding of protein result in changes in the levitation height of the bead that can be used to quantify the amount of protein bound. This paper uses a reaction-diffusion model to examine the physical principles that determine the values of rate and equilibrium constants measured by this system, using the well-defined model system of carbonic anhydrase and aryl sulfonamides. By tuning the experimental protocol, the method is capable of quantifying either the concentration of protein in a solution, or the binding affinities of a protein to several resin-bound small molecules simultaneously. Since this method requires no electricity and only a single piece of inexpensive equipment, it may find use in situations where portability and low cost are important, such as in bioanalysis in resource-limited settings, point-of-care diagnosis, veterinary medicine, and plant pathology. It still has several practical disadvantages. Most notably, the method requires relatively long assay times and cannot be applied to large proteins (> 70 kDa), including antibodies. The design and synthesis of beads with improved characteristics (e.g., larger pore size) has the potential to resolve these problems.

All matter has density. The recorded uses of density to characterize matter date back to as early as ca. 250 BC, when Archimedes was believed to have solved “The Puzzle of The King's Crown” using density.[1] Today, measurements of density are used to separate and characterize a range of materials (including cells and organisms), and their chemical and/or physical changes in time and space. This Review describes a density‐based technique—magnetic levitation (which we call “MagLev” for simplicity)—developed and used to solve problems in the fields of chemistry, materials science, and biochemistry. MagLev has two principal characteristics—simplicity, and applicability to a wide range of materials—that make it useful for a number of applications (for example, characterization of materials, quality control of manufactured plastic parts, self‐assembly of objects in 3D, separation of different types of biological cells, and bioanalyses). Its simplicity and breadth of applications also enable its use in low‐resource settings (for example—in economically developing regions—in evaluating water/food quality, and in diagnosing disease).

Diagnostic assays can provide valuable information about the health status of a patient, which include detection of biomarkers that indicate the presence of an infection, the progression or regression of a disease, and the efficacy of a course of treatment. Critical healthcare decisions must often be made at the point-of-care, far from the infrastructure and diagnostic capabilities of centralized laboratories. There exists an obvious need for diagnostic tools that are designed to address the unique challenges encountered by healthcare workers in limited-resource settings. Paper, a readily-available and inexpensive commodity, is an attractive medium with which to develop diagnostic assays for use in limited-resource settings. In this article, we describe a device architecture to perform immunoassays in patterned paper. These paper-based devices use a combination of lateral and vertical flow to control the wicking of fluid in three-dimensions. We provide guidelines to aid in the design of these devices and we illustrate how patterning can be used to tune the duration and performance of the assay. We demonstrate the use of these paper-based devices by developing a sandwich immunoassay for human chorionic gonadotropin (hCG) in urine, a biomarker of pregnancy. We then directly compare the qualitative and quantitative results of these paper-based immunoassays to commercially available lateral flow tests (i.e., the home pregnancy test). Our results suggest paper-based devices may find broad utility in the development of immunoassays for use at the point-of-care.