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Point-of-Care Assay Platform for Quantifying Active Enzymes to Femtomolar Levels Using Measurements of Time as the Readout
Gregory G. Lewis, Jessica S. Robbins, and Scott T. Phillips*
Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, United States
S Supporting Information *

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). Dierent 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 amplication reagent or by modifying the conguration of the paper-based microuidic 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 uids such as serum. hile qualitative point-of-care (POC) assays are available in the form of dipsticks and lateral- ow tests, quantitative assays pose practical challenges that have been dicult to overcome in an inexpensive and convenient way.1,2 The ideal quantitative POC assay, particularly for use in extremely resource-limited environments such as remote villages in the developing world,36 not only should be inexpensive, straightforward to operate, and provide rapid and reproducible quantitative results but also should do so without the use of an external reader.714 This goal of reader-less quantitative POC assays represents a formidable scientic and technical challenge. A key question to address toward this end relates to the type of readout that should be produced by an assay so that the readout is easy to quantify without using electronic devices. Standard spectroscopic and electrochemical analyses typically require an external device to obtain a quantitative result,9,11,1316 but more recent studies have focused on measurements based on distance, time, or the number of regions that turn color on a device. All three of these outputs can be quantied, in theory, by counting, which represents a rst step toward achieving quantitative point-of-care assays that do not require electronics. Representative examples of quantitative and semiquantitative assays (not all are POC assays) based on counting include time-based assays,1719 semiquantitative2023 and quantitative24 assays that require counting colored regions, as well as distance-based measurements in the context of nanomotor,25 microuidic,2628 spot tests,29 and immunochromatographic and lateral-ow assays.3034
2013 American Chemical Society

Measurements based on time are the least developed of these unconventional readouts, yet time is a readout that can be measured in a number of ways, even without the use of electronic devices. Consequently, herein we describe the development of a new approach for quantifying enzyme analytes by measuring the time that is required for one region on a paper-based device to turn color after a rst region turns color (see the Abstract image). This strategy is demonstrated in assays for active enzymes.35 The intensity of the color in either region for this assay is not indicative of the quantity of the analyte; rather, the quantity of the analyte is directly related to the relative time required for the color to appear. This relative measurement enables assays that are internally calibrated for eects of temperature, humidity, and sample viscosity on sample distribution,3639 and the overall approach requires only that a user begin a measurement once the assay region turns color, thus leading to short measurement times (seconds to minutes), with overall assay times (from application of the sample to the completed test) requiring only 1530 min. This new assay strategy also oers a remarkable level of sensitivity (femtomolar),3 is selective, is inexpensive (the device is made from paper and microgram quantities of reagents), and is easy to use (the user need only add the sample to the device and then time how long it takes for one region to turn color after the rst turns color). Thus,
Received: August 1, 2013 Accepted: September 27, 2013 Published: September 27, 2013
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Figure 1. Detailed depiction of the assay device as well as the reagents used in the assays. (a) Photograph of a three-dimensional paper-based microuidic device19,24,36,37,4446 for quantifying active enzyme analytes by measuring the relative time required for a sample to turn a control region green (right-hand region) relative to when an assay region (left-hand region) turns green. The device is made from stacked layers of wax-patterned paper44 that are held together using spray adhesive47 and then laminated. The dimensions of the paper portion of the device are 20 mm 10 mm 1.8 mm; the black regions are hydrophobic wax, and the white regions are hydrophilic paper. The white dotted line shows the location of the crosssection depicted in (b). The left channel in (b) is the assay region and the right channel is the control region. (c) Specic substrate reagents are incorporated into the device to provide selective detection of the target enzyme analyte. (d) A hydrophobic oligomer is used to amplify signal for the detection event. Amplication arises from head-to-tail depolymerization19,4852 in response to hydrogen peroxide that is generated during the detection event (c).19,24,53,54

the strategy oers a step forward toward the goal of conducting quantitative point-of-care assays without using auxiliary instruments or electronics.

EXPERIMENTAL DESIGN An example conguration of a paper-based microuidic device4044 that we designed for this assay is depicted in Figure 1. This device has an entry point for addition of the sample, and hydrophilic channels of paper that split the sample into two equal directions (i.e., layer 2, Figure 1b). Layer 2 also includes (i) buer salts that are redissolved by the sample to control the pH of the uid as it distributes through the device,
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as well as (ii) the cofactor MgCl2, which is needed by certain enzyme analytes (other cofactors could be added for dierent enzyme targets). In the left-hand channel (the assay region) in Figure 1b, the sample redissolves a substrate for a target enzyme analyte (compound 1, Figure 1c),55 beginning in layer 3. If the target enzyme is in the sample, it reacts with this substrate and causes release of one molecule of glucose per enzymatic reaction. Once the sample continues through layers 3 and 4 and into layer 5, it encounters bead-bound glucose oxidase (dark blue in Figure 1b,c), which remains immobilized in the bers of the paper.13 The glucose oxidase (GOX) oxidizes the released
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Analytical Chemistry glucose and generates hydrogen peroxide as the sample travels laterally in layer 5 of the device. Once the sample reaches the vertical conduit on the far left-hand side of the device in Figure 1b, it encounters an oligomer (compound 2, Figure 1d) that is hydrophobic and thus alters the wetting properties of the paper from hydrophilic to hydrophobic.19,24 In the absence of hydrogen peroxide, the sample travels slowly through this hydrophobic region, but in the presence of hydrogen peroxide, reagent 2 converts to hydrophilic products through a cascade depolymerization reaction,19,48,50,56,57 thus switching the wetting properties of the paper from hydrophobic back to hydrophilic. This switching reaction amplies the eects of hydrogen peroxide on the ow rate through layers 4 and 3 by converting a large hydrophobic oligomer into hydrophilic products.19,48 This switching reaction also allows the sample to pass through the layers containing 2 with a rate that depends on the concentration of hydrogen peroxide in the sample, which ultimately reects the concentration of the target enzyme analyte. Once the sample passes through the layers containing 2, it continues to travel in the vertical direction until it redissolves dried green food coloring and carries the highly colored solution to the top layer where the bright green color becomes visible. The control region (right-hand channel in the cross-section in Figure 1b) contains the same reagents in the same order as the assay region, with the exception of bead-bound glucose oxidase. In this control region, the enzyme analyte (if present) will react with substrate 1 deposited into the channel and generate glucose,55 but hydrogen peroxide will not be generated; therefore, hydrogen peroxide will not be present to react with oligomer 2. Hence, the time required for the sample to pass through this control region (and carry the green color to the top of the device) depends on the temperature and humidity under which the assay is conducted, as well as on the viscosity of the sample.3739,58 These factors will aect sample distribution rates in the assay region as well (the left-hand channel); therefore, this control region normalizes the output of the assay for the eects of these variables on sample distribution. This normalization is implemented by measuring the time required for the control region to turn green relative to when the assay region (the left-hand region) turns green (i.e., Tmeasurement). This measurement time is dierent than the total time (Ttotal) for the sample to pass from the entrance of the device to the end of the control region (the right-hand region in Figure 1). The measurement time (Tmeasurement) also is dierent than the assay time (Tassay), which is the time required for the sample to pass from the entrance of the device to the end of the assay region (the left-hand region in Figure 1). The relationship between Tmeasurement, Ttotal, and Tassay is depicted in eq 1.
Tmeasurement = Ttotal Tassay
(1)

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RESULTS AND DISCUSSION Demonstrating Selectivity in the Assays. The performance of the assay is revealed by the calibration curves shown in Figure 2 for the model enzyme analytes alkaline phosphatase (a

Experimental details, device fabrication procedures, and tabulated data are available in the Supporting Information.

Figure 2. Calibration curves for (a) alkaline phosphatase and (b) -Dgalactosidase, both of which are model enzymes to demonstrate the quantitative assay. The calibration curves were obtained at 19 C and 20% relative humidity using 2d as the phase-switching reagent and compound 1a for alkaline phosphatase and 1b for -D-galactosidase. The data points are the average of three measurements, and the error bars reect the standard deviations of these averages. The insets for (a) and (b) provide an expanded view of the regions that are bracketed using a dotted line. The equation for the line in (a) is y = 0.591x + 0.349, and the equation for the line in (b) is y = 0.122x + 0.436. The data for alkaline phosphatase is black, catalase is green, and -Dgalactosidase is blue. Values for all of the enzyme assays, in U/L, are available in the Supporting Information.

Ttotal and Tassay are not measured during an assay and vary depending on the conditions for the assay. Typically Ttotal and Tassay range from 15 to 30 min for Ttotal and from 2 to 15 min for Tassay. The only reason to discuss either of these two times is because Tassay is the period of time in which a user must watch for the appearance of green color in the assay region (the lefthand region in Figure 1) so that Tmeasurement can be made once the assay region turns green. The actual measurement time (Tmeasurement) typically is seconds for low concentrations of the target analyte up to 15 min for high concentrations.
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marker in blood that is indicative of liver function)5961 and 62 D-galactosidase (a general marker of fecal coliforms in water). In Figure 2a, the analyte is alkaline phosphatase, which uses 1a as the small molecule substrate in the device and 2d as the phase-switching reagent. The calibration curve was generated by depositing samples of alkaline phosphatase in 40 mM HEPES buer (pH 8.0) to the top of the device and measuring Tmeasurement for the control region to turn green after the assay region turns green. The limit-of-detection for this assay is 320 pM (0.355 U/L) alkaline phosphatase, with a dynamic range of 320 pM (0.355 U/L) to 14.8 nM (16.3 U/L).63 This level of sensitivity far exceeds the sensitivity of colorimetric assays that use camera-equipped cellular phones for quantifying assays in
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Analytical Chemistry paper microuidic devices59 and is even more sensitive than comparable activity-based assays that use a glucose meter to obtain the quantitative readout.55 Perhaps more importantly, the measurement time (Tmeasurement) is proportional to the concentration of the analyte, which is a feature that enables rapid trace-level detection, even if only a qualitative result is desired. Moreover, the assay is selective for alkaline phosphatase, as revealed by the negligible response when catalase or -Dgalactosidase are added to the device instead of alkaline phosphatase (green and blue data in Figure 2a, respectively). Catalase was used for comparison to alkaline phosphatase since it decomposes hydrogen peroxide rapidly to water and oxygen64 and thus should not provide a measurable response if the mechanism of the quantitative assay relies on analyteinduced production of hydrogen peroxide. -D-Galactosidase was chosen because it belongs to a dierent enzyme family than alkaline phosphatase and therefore demonstrates that selectivity between classes can be achieved. If the substrate in the device (i.e., 1) is switched to detect an enzyme other than alkaline phosphatase, then the selectivity switches as well (Figure 2b). The calibration curve in Figure 2b is for the enzyme -D-galactosidase, which uses 1b (Figure 1c) as the substrate in the device. The limit-of-detection for this assay is comparable to the alkaline phosphatase assay (i.e., 1.94 nM; 693 U/L), with a similar dynamic range (1.94 nM (693 U/ L) to 43 nM (15 360 U/L)).63 More importantly, this second calibration curve demonstrates that the assay can be recongured easily by changing the activity-based detection reagent (i.e., 1) to target a variety of enzymes.55 Demonstrating Tolerance to Sample Volume. An important feature of the assay is its ability to provide quantitative results without requiring precise measurements of sample volume (Figure 3). The patterned hydrophilic paper in

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has a negligible eect on the results of the quantitative assay (blue data in Figure 4a), as anticipated based on inclusion of

Figure 4. Eect of humidity and temperature on the accuracy of measuring Tmeasurement when using 11 nM (12 U/L) alkaline phosphatase as the model enzyme analyte for the humidity assay and 4.0 nM (4.0 U/L) for the temperature study. The assays were conducted using devices that contained 1a and 2d. The data points are the average of three measurements, and the error bars reect the standard deviations from these averages. The inset in (b) depicts the linear region of the temperature vs. Tmeasurement graph. The equation for the line is y = 0.3079x 3.6831.

Figure 3. Eect of sample volume on Tmeasurement for a sample that contains 11 nM alkaline phosphatase (12 U/L). The assays were conducted at 19 C and 20% relative humidity using reagents 1a and 2d. The data are the average of three measurements, and the error bars are smaller than the data points. The vertical dotted line marks the lowest volume of sample that is required for the assay. The assay was stopped at 45 min regardless of whether it was complete; therefore, the data points below 25 L (x-axis) reect assays that lacked enough sample to reach completion.

the device absorbs a xed volume of sample,40,65 which provides sucient control over sample volume to enable quantitative assays, so long as a minimum quantity of sample is added to the device. The minimum volume for the device shown in Figure 1b is 25 L. Normalizing for the Eects of Humidity and Calibrating for the Eects of Temperature. While humidity has a noticeable eect on sample distribution rates in paper,36,38,39 it
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the control region. Variations in temperature, however, do aect the time required for a sample to ow through the device. Temperature-induced changes in sample viscosity aect sample distribution rates3739,58 but are likely accounted for by the control region, whereas temperature eects on enzymatic activity are not. Instead, as depicted in the black data in Figure 4a, Tmeasurement tracks with the anticipated activity of the target enzyme (alkaline phosphatase in this case).66 The eects of assay temperature on enzymatic activity (and, ultimately assay time) can be accounted for easily, however, since there are three temperature regimes (within the ranges that we tested) that aect the assay: (i) <15 C, (ii) 15 to 33 C, and (iii) >33 C. The enzymatic activity is equal within the rst temperature range, so no adjustment is needed to the calibration curve in Figure 2a with the exception of adding 1 min to Tmeasurement so that the measurement times match the calibration curve (Figure 2a), which was generated at a warmer temperature (i.e., 19 C) than this cold temperature range (<15 C). Likewise, another plateau appears above 33 C, which requires subtraction of 4 min from Tmeasurement to recalibrate the curve in Figure 2a for this warmer temperature range. Between the range of 15 and 33 C, however, the measurement times increase linearly with temperature. This linear increase is easily accommodated in a quantitative assay as well. For example, when the assay is conducted at temperatures above 19 C (the temperature for establishing the calibration curve) but below 33 C, Tmeasurement should be decreased by a factor of 0.3079 T (T is the dierence in temperature between when the assay was conducted and the calibration curve was generated).67 Likewise, when the assay is conducted at temperatures below 19 C (but above 15 C), Tmeasurement is increased by the same factor. By using these adjustments, the calibration curve in Figure 2a remains functional over a wide range of temperatures.
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Analytical Chemistry While measurements of assay temperature may sound complicated, it is worth noting that these measurements can be accomplished easily and cheaply using exible and reversible leucodye-based temperature strips,68 which can be axed to packaging that may contain hundreds of the assay devices and, therefore, can be used to measure the temperature for many assays. With limited access to temperature-controlled environments in point-of-care settings, the ability to correct for variations in temperature is crucial for reliable quantitative results. Tuning the Sensitivity of the Assays by Altering the Design of the Assay Platform. There are several straightforward ways to tune the limit-of-detection for a particular assay in these devices, one of which involves changing the length of the lateral ow channels in layer 5 of the device (Figure 5a). The lengths of these channels are

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enzyme that is required to achieve a measurable response in the assay. We recently studied this concept for hydrogen peroxide detection and found that a single layer of phase-switching reagent provides the best sensitivity for detecting hydrogen peroxide.19 In the context of the enzyme assays, one layer of the phase-switching reagent also provides the best sensitivity. For example, a device with a 5.4 mm-long lateral ow region that contains only 1 layer of 2d has a limit-of-detection for alkaline phosphatase that is 1.9 better than an equal device that contains 2 layers of 2d (LOD = 45 vs 84 pM, respectively) and 9.8 better than a device that contains 3 layers of the phaseswitching reagent (LOD = 440 pM). Tuning the Sensitivity and Dynamic Range of the Assays by Altering the Phase-Switching Reagent. A third method for altering the sensitivity of an assay involves changing the phase-switching reagent (2) used in the device. Strategies such as (i) increasing the rate of conversion of hydrophobic 2 into hydrophilic byproducts in response to hydrogen peroxide (e.g., 2a vs 2b, Figure 1d)69 and (ii) increasing the magnitude of the change by employing depolymerizable oligomers (e.g., 2b2f)19,48 both provide improvements in sensitivity for an assay for alkaline phosphatase (the model enzyme) (Table 1). In fact, use of 2f in the device instead of 2a improves the sensitivity for the assay by 4 orders of magnitude. Table 1. Eect of the Phase-Switching Reagent (i.e., Compound 2) on the Sensitivity and Dynamic Range of an Assay for Alkaline Phosphatasea
compound no. LOD (pM) 5600 342 93 45 13 0.128 relative improvement in sensitivity 1 16 60 124 431 43 750 dynamic range (pM) 560074 000 34237 000 931480 451480 13740 0.1287.4

Figure 5. Eect of the length of the lateral ow region in the paperbased device on the sensitivity of an assay for alkaline phosphatase. (a) Cross-section of the device in Figure 1a showing the lateral ow channel that was varied in length. (b) Relationship between the length of the lateral ow region and the measurement times (Tmeasurement) for assays for 11 nM (12 U/L) alkaline phosphatase in 40 mM HEPES buer (pH 8) using 1a and 2d. (c) Relationship between channel length and the limit-of-detection (LOD) for an assay for alkaline phosphatase (ALP).

2a 2b 2c 2d 2e 2f
a

inversely proportional to the measurement time (Tmeasurement) for an assay (Figure 5b), which means that shorter channels in layer 5 provide greater distinction than longer channels between the time for the sample to pass through the control region (right-hand region in Figure 1b) relative to the assay region (left-hand region in Figure 1b). As the time gap (i.e., Tmeasurement) between these two conduits increases, the limit-ofdetection for an assay decreases (Figure 5c), with improvements in sensitivity reaching as high as 28-fold for the channel lengths that we tested. A second method for tuning the sensitivity involves changing the number of layers of paper that contain compound 2 (the phase-switching reagent).19 In this case, two scenarios are envisaged: (1) multiple layers of the phase-switching reagent may provide greater interactions between the hydrogen peroxide that is generated in the assay and the phase-switching reagent and thus may improve the ability of the device to dierentiate between dierent concentrations of the target enzyme or (2) single layers of the phase-switching reagent may require little hydrogen peroxide to impart a change in wetting properties from hydrophobic to hydrophilic upon reaction with the phase-switching reagent, thus decreasing the quantity of
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The assays were conducted using a device that contained a 5.4 mmlong lateral ow region and 1 layer of compound 2. The quantity of each derivative of compound 2 was optimized to maximize the sensitivity of the assay.

Combining these three strategies for improving the sensitivity for an assay enables tuning of the assay for a relevant concentration of a target analyte over the concentration range of low femtomolar to low nanomolar, with dynamic ranges for the assays of at least 1 order of magnitude. The most sensitive assay uses a 5.4 mm-long lateral ow channel and 1 layer of 2f and has a limit-of-detection of 128 fM. In contrast, the least sensitive assay that we tested has a limitof-detection of 26 nM by using a 12.4 mm-long lateral ow channel and 3 layers of 2a . 70 Advances in polymer chemistry51,57,58,71 should provide access to new phaseswitching reagents (2) that further expand the operating range of the assays, particularly by decreasing the limit-ofdetection. Modifying the Design of the Device for Complex Fluids. All of these optimization studies were performed using buered samples that lack the complexity of various uids that may contain the analyte of interest. Depending on the uid, a variety of factors must be considered, most notably whether the
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Figure 6. Cross-section of a revised device that includes preprocessing reagents in the central channel in layer 2. These reagents remove glucose and hydrogen peroxide that may be in a sample. The location of the cross-section is depicted in the photograph in Figure 1a.

uid contains glucose or hydrogen peroxide that would interfere with the outcome of the assay. Conguration of a Modied Device. To overcome this type of interference, we created a revised device that includes a new layer (Figure 6) in which bead-bound glucose oxidase and catalase are deposited into the central channel of the device.13 The glucose oxidase oxidizes glucose in a sample and generates hydrogen peroxide, while catalase converts the hydrogen peroxide into oxygen and water. These preprocessing steps occur before the sample redissolves the buer salts in the device and distributes into the assay and reference conduits. Preprocessing of Glucose and Hydrogen Peroxide in a Sample. These scavenging reagents are placed in the modied device in layer 2 in Figure 6 and are capable of scavenging 25 mM glucose and 15 mM hydrogen peroxide when the device is challenged with a sample of alkaline phosphatase containing either of the contaminants (Figure 7). The typical concen-

Figure 8. Calibration curve for quantifying alkaline phosphatase spiked into horse serum. The calibration curve was generated using the device in Figure 6 that contained 1a and 2f. The data were acquired at 20 C and 53% relative humidity. The data points are the averages of three measurements, and the error bars reect the standard deviations of these averages. The inset provides an expanded view of the region that is bracketed with a dotted line. The equation for the line is y = 2.5498x + 5.3199.

Figure 7. Eect of the concentration of hydrogen peroxide or glucose in a sample on Tmeasurement. The black data reects Tmeasurement values for samples of 1.5 pM (1.6 mU/L) alkaline phosphatase that were spiked with hydrogen peroxide, and the blue data corresponds to the same experiment using glucose instead of hydrogen peroxide. The colorcoded vertical dotted lines reect the highest concentration of either hydrogen peroxide or glucose that is tolerated in the assay without aecting Tmeasurement.

used along with 1 layer of phase-switching reagent 2f. While this level of sensitivity is 19 higher than in pure buer,76 it still represents a highly sensitive quantitative assay, especially given that the user only adds a sample to the device and then measures the time to appearance of color in the control region relative to the assay region on time scales of 5 s to 4.5 min. This level of sensitivity for alkaline phosphatase is well below the levels of the enzyme typically found in human serum (42369 U/L).78,79 Should we wish to create an assay for relevant concentrations of alkaline phosphatase in serum,73 we could use a dierent phase-switching reagent (2), change the number of layers of 2, or lengthen the lateral ow channel to easily and predictably tune the sensitivity of the assay and bring it within the clinical range for this analyte.

tration of glucose in blood is 3.55.3 mM,72 while the highest levels of hydrogen peroxide in urine, beverages such as tea, or rainwater (in a polluted environment) are 5100,73 100,74 and 5.4 M,75 respectively. Clearly, the device will be capable of removing these types of interfering contaminants. Sensitivity of the Assay in Horse Serum. With these userindependent preprocessing reagents in place, the assay platform is compatible with complex uids such as serum, as depicted by the calibration curve for measuring the levels of alkaline phosphatase in enzyme-spiked horse serum (Figure 8). The limit-of-detection for alkaline phosphatase in this uid is 2.4 pM (2.6 mU/L) when a 5.4 mm-long lateral ow channel is
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CONCLUSION In conclusion, we describe a new type of quantitative POC assay platform that we believe oers analytical capabilities that typically are only available using specic instruments.3 Our approach requires a single operation by the user; the assay is completed in seconds to minutes, and the readout involves simple measurements of time to quantify the amount of an active enzyme analyte in a sample down to femtomolar levels in simple uids and low picomolar levels in complex uids. The assay also is inexpensive: only paper, food coloring, buer salts, and microgram quantities of reagents are needed per test. The approach is among a rare class of assays that, in principle, are capable of providing a quantitative result without use of an external electronic reader.1834 The opportunity for signal amplication by using polymers that depolymerize19 sets
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Analytical Chemistry this approach apart from others and may oer a level of sensitivity that is particularly relevant for point-of-care and point-of-use applications in resource-limited settings such as the developing world, home healthcare, emergency situations, and others. The next steps include extending this proof-of-concept approach to other classes of analytes as well as fully developing assays for specic analytes. Further improvements in sensitivity also are being sought by creating new types of oligomers/ polymers as phase-switching reagents.71

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ASSOCIATED CONTENT

S Supporting Information *

Experimental procedures, tables of data, and supporting gures. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author Author Contributions

*E-mail: sphillips@psu.edu. Fax: 814-865-5235. The manuscript was written through contributions of all authors. All authors have given approval to the nal version of the manuscript.
Notes

The authors declare no competing nancial interest.

ACKNOWLEDGMENTS This work was supported in part by NSF (CHE-1150969), Eli Lilly, and Louis Martarano. S.T.P. acknowledges support from the Alfred P. Sloan Research Fellows Program. REFERENCES
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