Paper-based microfluidic devices for analysis of clinically relevant analytes present in urine and saliva

We report the use of paper-based microfluidic devices fabricated from a novel polymer blend for the monitoring of urinary ketones, glucose, and salivary nitrite. Paper-based devices were fabricated via photolithography in less than 3 min and were immediately ready for use for these diagnostically relevant assays. Patterned channels on filter paper as small as 90 microm wide with barriers as narrow as 250 microm could be reliably patterned to permit and block fluid wicking, respectively. Colorimetric assays for ketones and nitrite were adapted from the dipstick format to this paper microfluidic chip for the quantification of acetoacetate in artificial urine, as well as nitrite in artificial saliva. Glucose assays were based on those previously demonstrated (Martinez et al., Angew Chem Int Ed 8:1318-1320, 1; Martinez et al., Anal Chem 10:3699-3707, 2; Martinez et al., Proc Nat Acad Sci USA 50:19606-19611, 3; Lu et al., Electrophoresis 9:1497-1500, 4; Abe et al., Anal Chem 18:6928-6934, 5). Reagents were spotted on the detection pad of the paper device and allowed to dry prior to spotting of samples. The ketone test was a two-step reaction requiring a derivitization step between the sample spotting pad and the detection pad, thus for the first time, confirming the ability of these paper devices to perform online multi-step chemical reactions. Following the spotting of the reagents and sample solution onto the paper device and subsequent drying, color images of the paper chips were recorded using a flatbed scanner, and images were converted to CMYK format in Adobe Photoshop CS4 where the intensity of the color change was quantified using the same software. The limit of detection (LOD) for acetoacetate in artificial urine was 0.5 mM, while the LOD for salivary nitrite was 5 microM, placing both of these analytes within the clinically relevant range for these assays. Calibration curves for urinary ketone (5 to 16 mM) and salivary nitrite (5 to 2,000 microM) were generated. The time of device fabrication to the time of test results was about 25 min.