Katherine A. Mirica

Paper-Based ELISA†

This paper describes enzyme-linked immunosorbent assays (ELISA) performed in a 96-microzone plate fabricated in paper (paper-based ELISA, or P-ELISA). ELISA is widely used in biochemical analyses; these assays are typically carried out in microtiter plates or small vials. 2] ELISA combines the specificity of antibodies with high-turnover catalysis by enzymes to provide specificity and sensitivity. We have recently described a 96-microzone paper plate— fabricated by patterning hydrophobic polymer in hydrophilic paper—as a platform for biochemical analysis. Although microfluidic paper-based analytical devices (mPADs) were designed primarily to provide analytical capability at low cost in developing countries, we expect that they will also be useful in applications such as point-of-care clinical analysis, military and humanitarian aid field operations, and others where high throughput, low volumes of sample, low cost, and robustness are important. These devices have so far been prototyped using analyses of simple analytes: glucose, total protein, and certain enzymes. P-ELISA combines the sensitivity and specificity of ELISA with the convenience, low cost and ease-of-use of paper-based platforms; P-ELISA (at it current state of development) is faster and less expensive than conventional ELISA, but somewhat less sensitive. Porous membranes, including nitrocellulose and filter paper, have been used for decades in dot-immunobinding assays (DIA). 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. Quantitative DIAs have been reported, but DIAs are typically qualitative, and provide only “yes/no” results. Conventional ELISA, usually performed in 96-well plates (fabricated by injection molding in plastic), is quantitative and well-suited for highthroughput assays, but each assay requires large volumes (ca. 20–200 mL) of analyte and reagents, the time required for 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 usually quantified using a plate reader, typically a

Quantifying Colorimetric Assays in Paper-Based Microfluidic Devices by Measuring the Transmission of Light through Paper

20 000 instrument. Paper microzone plates for ELISA can have the same layout as plastic 96-well plates, but each test zone requires only about 3 mL of sample, and the results can be measured using a desktop scanner, typically a

Measuring Markers of Liver Function Using a Micropatterned Paper Device Designed for Blood from a Fingerstick

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 adapted a standard procedure to our format and then demonstrated an indirect P-ELISA using rabbit IgG as a model analyte. We also established that P-ELISA can be used to detect and quantify antibodies to the HIV-1 envelope antigen gp41 in human serum using an anti-human IgG antibody conjugated to alkaline phosphatase (ALP) to produce a colorimetric readout. We used a 96-microzone paper plate with an array (12 8) of circular test zones for running multiple P-ELISAs in parallel (Figure 1A); the Supporting Information describes the details. The array was designed to have the same layout and dimensions as a standard plastic 96-well plate, so that it would be compatible with existing microanalytical infrastructure (eightor twelve-channel pipettes and plate readers). Each test zone was 5 mm in diameter and required 3 mL of solution to fill (e.g., to wet completely with fluid); this design was a good compromise between convenience and conservation of reagents, as it reduced the amount of reagents and sample required for the assay but ensured accurate distribution of fluids when using a manual pipette. We also examined smaller test zones, with the smallest test zone requiring 0.5 mL of solution to fill (e.g., to wet completely). This size is similar to that required in a 384-well plate format. The top and bottom faces of the test zones in papermicrozone plates are open to atmosphere. The advantage of this configuration is that the zones can be washed by adding a washing buffer to the top of the zone while pressing the bottom of the zone against a piece of blotting paper. The washing buffer goes through the test zone vertically and into [*] Dr. C.-M. Cheng, Dr. A. W. Martinez, Dr. J. Gong, Dr. C. R. Mace, Prof. S. T. Phillips, Prof. E. Carrilho, K. A. Mirica, Prof. G. M. Whitesides Department of Chemistry and Chemical Biology Harvard University Cambridge, MA 02138 (USA) E-mail: gwhitesides@gmwgroup.harvard.edu Homepage: http://gmwgroup.harvard.edu

Thread as a Matrix for Biomedical Assays

This article describes a point-of-care (POC) system--comprising a microfluidic, paper-based analytical device (micro-PAD) and a hand-held optical colorimeter--for quantifying the concentration of analytes in biological fluids. The micro-PAD runs colorimetric assays, and consists of paper that has been (i) patterned to expose isolated regions of hydrophilic zones and (ii) wet with an index-matching fluid (e.g., vegetable oil) that is applied using a disposable, plastic sleeve encasement. Measuring transmittance through paper represents a new method of quantitative detection that expands the potential functionality of micro-PADs. This prototype transmittance colorimeter is inexpensive, rugged, and fully self-contained, and thus potentially attractive for use in resource-limited environments and developing countries.

Rapid prototyping of carbon-based chemiresistive gas sensors on paper

This paper describes a paper-based microfluidic device that measures two enzymatic markers of liver function (alkaline phosphatase, ALP, and aspartate aminotransferase, AST) and total serum protein. A device consists of four components: (i) a top plastic sheet, (ii) a filter membrane, (iii) a patterned paper chip containing the reagents necessary for analysis, and (iv) a bottom plastic sheet. The device performs both the sample preparation (separating blood plasma from erythrocytes) and the assays; it also enables both qualitative and quantitative analysis of data. The data obtained from the paper-microfluidic devices show standard deviations in calibration runs and spiked standards that are acceptable for routine clinical use. This device illustrates a type of test useable for a range of assays in resource-poor settings.

Measuring Densities of Solids and Liquids Using Magnetic Levitation: Fundamentals

This paper describes the use of thread as a matrix for the fabrication of diagnostic assay systems. The kinds of thread used for this study are inexpensive, broadly available, and lightweight; some of them are already familiar materials in healthcare. Fluids wick along these threads by capillary action; no external power source is necessary for pumping. This paper demonstrates three designs for diagnostic assays that use different characteristics of the thread. The first two designs-the woven array and the branching design-take advantage of the ease with which thread can be woven on a loom to generate fluidic pathways that enable multiple assays to be performed in parallel. The third design-the sewn array-takes advantage of the ease with which thread can be sewn through a hydrophobic polymer sheet to incorporate assays into bandages, diapers and similar systems. These designs lead to microfluidic devices that may be useful in performing simple colorimetric assays that require qualitative results. We demonstrate the function of thread-based microfluidic devices in the context of five different colorimetric assays: detection of ketones, nitrite, protein, and glucose in artificial urine, and detection of alkaline phosphatase in artificial plasma.

Using Magnetic Levitation for Three Dimensional Self-Assembly

Significance This paper describes a rapid, solvent-free, two-step procedure for the fabrication of selective gas and vapor sensors from carbon nanotubes and graphite on the surface of paper that overcomes challenges associated with solvent-assisted chemical functionalization and integration of these materials into devices. The first step generates solid composites from carbon nanotubes (or graphite) and small molecules (chosen to interact with specific types of gases and vapors) by mechanical mixing and subsequent compression into a form similar to a conventional pencil “lead”. The second step uses mechanical abrasion (“drawing”) of these solid composites on the surface of paper to generate functional devices. The use of diverse composites yields sensing arrays capable of detecting and differentiating gases and vapors and part-per-million concentrations. Chemically functionalized carbon nanotubes (CNTs) are promising materials for sensing of gases and volatile organic compounds. However, the poor solubility of carbon nanotubes hinders their chemical functionalization and the subsequent integration of these materials into devices. This manuscript describes a solvent-free procedure for rapid prototyping of selective chemiresistors from CNTs and graphite on the surface of paper. This procedure enables fabrication of functional gas sensors from commercially available starting materials in less than 15 min. The first step of this procedure involves the generation of solid composites of CNTs or graphite with small molecule selectors—designed to interact with specific classes of gaseous analytes—by solvent-free mechanical mixing in a ball mill and subsequent compression. The second step involves deposition of chemiresistive sensors by mechanical abrasion of these solid composites onto the surface of paper. Parallel fabrication of multiple chemiresistors from diverse composites rapidly generates cross-reactive arrays capable of sensing and differentiating gases and volatile organic compounds at part-per-million and part-per-thousand concentrations.

Magnetic Levitation in the Analysis of Foods and Water

This paper describes an analytical system that uses magnetic levitation to measure densities of solids and water-immiscible organic liquids with accuracies ranging from +/-0.0002 to +/-0.02 g/cm(3), depending on the type of experiment. The technique is compatible with densities of 0.8-3 g/cm(3) and is applicable to samples with volumes of 1 pL to 1 mL; the samples can be either spherical or irregular in shape. The method employs two permanent NdFeB magnets positioned with like poles facing one another--with the axis between the poles aligned with the gravitational field--and a container filled with paramagnetic medium (e.g., MnCl(2) dissolved in water) placed between these magnets. Density measurements are obtained by placing the sample into the container and measuring the position of the sample relative to the bottom magnet. The balance of magnetic and gravitational forces determines the vertical position of the sample within the device; knowing this position makes it possible to calculate the density of the sample.

Fluoroalkyl and Alkyl Chains Have Similar Hydrophobicities in Binding to the “Hydrophobic Wall” of Carbonic Anhydrase

The development of practical strategies for the assembly of objects into 3D arrays is an unsolved problem. This paper describes the use of magnetic levitation (MagLev) to guide the self-assembly of millimeterto centimeter-scale dia magnetic objects, which we call “components”, each programmed by shape and distribution of density, into 3D assemblies and structures in a paramagnetic fl uid medium positioned in the magnetic fi eld gradient generated by NdFeB magnets. When the components are not in contact, their equilibrium confi guration depends on the balance of magnetic and gravitational forces they experience. This technique provides a convenient method to position components in 3D without mechanical contact; we demonstrate its unique capabilities using components with optical functions, and with components that form ordered, assembled structures when transferred into contact with solid supports. Most functional devices are assembled from components (by either humans or machines) and connected mechanically. The ability to carry out any part of these processes automatically, or even to preposition or preorient the components reliably, would simplify them. Self-assembly is a useful technique for generating ordered assemblies, [ 1–3 ] and although there are exceptions, it has been most highly developed when the components are all the same, and when there is a (quasi) 2D template (e.g., surface) to guide the process. [ 3–5 ] We and others have demonstrated self-assembly across a range of sizes, [ 6–14 ] and have also generated self-assembled functional structures, [ 3 , 15–25 ]

Wireless gas detection with a smartphone via rf communication

This paper describes a method and a sensor that use magnetic levitation (MagLev) to characterize samples of food and water on the basis of measurements of density. The sensor comprises two permanent NdFeB magnets positioned on top of each other in a configuration with like poles facing and a container filled with a solution of paramagnetic ions. Measurements of density are obtained by suspending a diamagnetic object in the container filled with the paramagnetic fluid, placing the container between the magnets, and measuring the vertical position of the suspended object. MagLev was used to estimate the salinity of water, to compare a variety of vegetable oils on the basis of the ratio of polyunsaturated fat to monounsaturated fat, to compare the contents of fat in milk, cheese, and peanut butter, and to determine the density of grains.