Andres W. Martinez

Diagnostics for the Developing World: Microfluidic Paper-Based Analytical Devices

Microfluidic paper-based analytical devices (microPADs) are a new class of point-of-care diagnostic devices that are inexpensive, easy to use, and designed specifically for use in developing countries. (To listen to a podcast about this feature, please go to the Analytical Chemistry multimedia page at pubs.acs.org/page/ancham/audio/index.html.).

Simple Telemedicine for Developing Regions: Camera Phones and Paper-Based Microfluidic Devices for Real-Time, Off-Site Diagnosis

This article describes a prototype system for quantifying bioassays and for exchanging the results of the assays digitally with physicians located off-site. The system uses paper-based microfluidic devices for running multiple assays simultaneously, camera phones or portable scanners for digitizing the intensity of color associated with each colorimetric assay, and established communications infrastructure for transferring the digital information from the assay site to an off-site laboratory for analysis by a trained medical professional; the diagnosis then can be returned directly to the healthcare provider in the field. The microfluidic devices were fabricated in paper using photolithography and were functionalized with reagents for colorimetric assays. The results of the assays were quantified by comparing the intensities of the color developed in each assay with those of calibration curves. An example of this system quantified clinically relevant concentrations of glucose and protein in artificial urine. The combination of patterned paper, a portable method for obtaining digital images, and a method for exchanging results of the assays with off-site diagnosticians offers new opportunities for inexpensive monitoring of health, especially in situations that require physicians to travel to patients (e.g., in the developing world, in emergency management, and during field operations by the military) to obtain diagnostic information that might be obtained more effectively by less valuable personnel.

Understanding Wax Printing: A Simple Micropatterning Process for Paper-Based Microfluidics

This technical note describes a detailed study on wax printing, a simple and inexpensive method for fabricating microfluidic devices in paper using a commercially available printer and hot plate. The printer prints patterns of solid wax on the surface of the paper, and the hot plate melts the wax so that it penetrates the full thickness of the paper. This process creates complete hydrophobic barriers in paper that define hydrophilic channels, fluid reservoirs, and reaction zones. The design of each device was based on a simple equation that accounts for the spreading of molten wax in paper.

Three-dimensional microfluidic devices fabricated in layered paper and tape

This article describes a method for fabricating 3D microfluidic devices by stacking layers of patterned paper and double-sided adhesive tape. Paper-based 3D microfluidic devices have capabilities in microfluidics that are difficult to achieve using conventional open-channel microsystems made from glass or polymers. In particular, 3D paper-based devices wick fluids and distribute microliter volumes of samples from single inlet points into arrays of detection zones (with numbers up to thousands). This capability makes it possible to carry out a range of new analytical protocols simply and inexpensively (all on a piece of paper) without external pumps. We demonstrate a prototype 3D device that tests 4 different samples for up to 4 different analytes and displays the results of the assays in a side-by-side configuration for easy comparison. Three-dimensional paper-based microfluidic devices are especially appropriate for use in distributed healthcare in the developing world and in environmental monitoring and water analysis.

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

Paper Microzone Plates

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

Fully enclosed microfluidic paper-based analytical devices.

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.

Paper Microzone Plates as Analytical Tools for Studying Enzyme Stability: A Case Study on the Stabilization of Horseradish Peroxidase Using Trehalose and SU-8 Epoxy Novolac Resin

This paper describes 96- and 384-microzone plates fabricated in paper as alternatives to conventional multiwell plates fabricated in molded polymers. Paper-based plates are functionally related to plastic well plates, but they offer new capabilities. For example, paper-microzone plates are thin (approximately 180 microm), require small volumes of sample (5 microL per zone), and can be manufactured from inexpensive materials (

Patterned Paper as a Platform for Inexpensive, Low‐Volume, Portable Bioassays

0.05 per plate). The paper-based plates are fabricated by patterning sheets of paper, using photolithography, into hydrophilic zones surrounded by hydrophobic polymeric barriers. This photolithography used an inexpensive formulation photoresist that allows rapid (approximately 15 min) prototyping of paper-based plates. These plates are compatible with conventional microplate readers for quantitative absorbance and fluorescence measurements. The limit of detection per zone loaded for fluorescence was 125 fmol for fluorescein isothiocyanate-labeled bovine serum albumin, and this level corresponds to 0.02 the quantity of analyte per well used to achieve comparable signal-to-noise in a 96-well plastic plate (using a solution of 25 nM labeled protein). The limits of detection for absorbance on paper was approximately 50 pmol per zone for both Coomassie Brilliant Blue and Amaranth dyes; these values were 0.4 that required for the plastic plate. Demonstration of quantitative colorimetric correlations using a scanner or camera to image the zones and to measure the intensity of color, makes it possible to conduct assays without a microplate reader.