Point-of-Care Assay Platform for Quantifying Active Enzymes to Femtomolar Levels Using Measurements of Time as the Readout

Abstract

This Article describes a strategy for quantifying active enzyme analytes in a paper-based device by measuring the time for a reference region in the paper to turn green relative to an assay region. The assay requires a single step by the user, yet accounts for variations in sample volume, assay temperature, humidity, and contaminants in a sample that would otherwise prevent a quantitative measurement. The assay is capable of measuring enzymes in the low to mid femtomolar range with measurement times that range from ~30 s to ~15 min (lower measurement times correspond to lower quantities of the analyte). Different targets can be selected in the assay by changing a small molecule reagent within the paper-based device, and the sensitivity and dynamic range of the assays can be tuned easily by changing the composition and quantity of a signal amplification reagent or by modifying the configuration of the paper-based microfluidic device. By tuning these parameters, limits-of-detection for assays can be adjusted over an analyte concentration range of low femtomolar to low nanomolar, with dynamic ranges for the assays of at least 1 order of magnitude. Furthermore, the assay strategy is compatible with complex fluids such as serum.

Determination of Iron Ion in the Water of a Natural Hot Spring Using Microfluidic Paper-based Analytical Devices

“…4,5 μPADs have shown promise for their use in point-of-care testing during bioanalysis, as described in many publications. [6][7][8][9][10][11][12][13][14][15][16] When used in environmental analysis, μPADs have been successfully applied to the measurement of metal ions in particulate matters (PMs), [17][18][19] as well as being useful for measuring the oxidative activity of PMs. 20 We also reported the rapid use of μPADs in acid-base titrations during the on-site analysis of acidic water in a natural hot spring.…”

Paper Microfluidics in Bioanalysis

“…Lewis et al utilized time as readout to quantify enzyme analytes where time is measured for a region in the paper device that turns green relative to the assay region [16]. The technique is internally calibrated and compensates for effects of humidity, temperature, and sample viscosity on sample distribution.…”

Highly Sensitive Paper-based Analytical Devices with the Introduction of a Large-Volume Sample via Continuous Flow

“…These techniques include time-based, [9][10][11][12] distancebased, [13][14][15][16] and titration-based detection. [17][18][19] Philips and coworkers demonstrated time-based detection using an enzymatic reaction wherein a color change took place at different times depending on the concentration of the analyte.…”

“…[17][18][19] Philips and coworkers demonstrated time-based detection using an enzymatic reaction wherein a color change took place at different times depending on the concentration of the analyte. 9,10 Conversely, some research groups have developed distance-based PADs that permit the quantitation of analytes without the use of either a scanner or a digital camera, since the amount of the analyte could be determined by reading the distance of the color bar evolving on the PAD. [13][14][15][16] Zhang and coworkers employed both the time and the number of the detection zones that changed color.…”