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 paper describes three-dimensional microfluidic paper-based analytical devices (3-D microPADs) that can be programmed (postfabrication) by the user to generate multiple patterns of flow through them. These devices are programmed by pressing single-use 'on' buttons, using a stylus or a ballpoint pen. Pressing a button closes a small space (gap) between two vertically aligned microfluidic channels, and allows fluids to wick from one channel to the other. These devices are simple to fabricate, and are made entirely out of paper and double-sided adhesive tape. Programmable devices expand the capabilities of microPADs and provide a simple method for controlling the movement of fluids in paper-based channels. They are the conceptual equivalent of field-programmable gate arrays (FPGAs) widely used in electronics.

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# 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...

The ability to identify the precise time of ovulation is important for women who want to plan conception or practice contraception. Here, we review the current literature on various methods for detecting ovulation including a review of point‐of‐care device technology. We incorporate an examination of methods to detect ovulation that have been developed and practiced for decades and analyze the indications and limitations of each—transvaginal ultrasonography, urinary luteinizing hormone detection, serum progesterone and urinary pregnanediol 3‐glucuronide detection, urinary follicular stimulating hormone detection, basal body temperature monitoring, and cervical mucus and salivary ferning analysis. Some point‐of‐care ovulation detection devices have been developed and commercialized based on these methods, however previous research was limited by small sample size and an inconsistent standard reference to true ovulation.

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We have developed a colorimetric sensing strategy employing gold nanoparticles and a paper-based analytical platform for the diagnosis of tuberculosis (TB). By utilizing the surface plasmon resonance effect, we were able to monitor changes in the color of a gold nanoparticle colloid based on the effects of single-stranded DNA probe molecules hybridizing with targeted double-stranded TB DNA. The hybridization event changes the surface charge density of the nanoparticles, causing them to aggregate to various degrees, which modifies the color of the solution in a manner that can be readily measured to determine the concentration of the targeted DNA analyte. In order to adapt this TB diagnosis method to resource-limited settings, we extended this label-free oligonucleotide and unmodified gold nanoparticle solution-based technique to a paper-based system that can be measured using a smartphone to obtain rapid parallel colorimetric results with low reagent consumption and without the need for sophisticated analytical equipment. In this study, we investigated various assay conditions, including the denaturing temperature and time, different oligonucleotide probe sequences, as well as the ratio of single stranded probe and double stranded target DNA. After optimizing these variables, we were able to achieve a detection limit of 1.95 × 10 ng/mL for TB DNA. Furthermore, multiple tests could be performed simultaneously with a 60 min turnaround time.