Point-of-Care Assay Platform for Quantifying Active Enzymes toFemtomolar Levels Using Measurements of Time as the ReadoutGregory 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 quantifyingactive enzyme analytes in a paper-based device by measuringthe time for a reference region in the paper to turn greenrelative to an assay region. The assay requires a single step bythe user, yet accounts for variations in sample volume, assaytemperature, humidity, and contaminants in a sample thatwould otherwise prevent a quantitative measurement. Theassay is capable of measuring enzymes in the low to midfemtomolar range with measurement times that range from!30 s to !15 min (lower measurement times correspond tolower quantities of the analyte). Different targets can be selected in the assay by changing a small molecule reagent within thepaper-based device, and the sensitivity and dynamic range of the assays can be tuned easily by changing the composition andquantity of a signal amplification reagent or by modifying the configuration of the paper-based microfluidic device. By tuningthese parameters, limits-of-detection for assays can be adjusted over an analyte concentration range of low femtomolar to lownanomolar, with dynamic ranges for the assays of at least 1 order of magnitude. Furthermore, the assay strategy is compatiblewith complex fluids such as serum.

While qualitative point-of-care (POC) assays are availablein the form of dipsticks and lateral-flow tests,

quantitative assays pose practical challenges that have beendifficult to overcome in an inexpensive and convenient way.1,2

The ideal quantitative POC assay, particularly for use inextremely resource-limited environments such as remotevillages in the developing world,3"6 not only should beinexpensive, straightforward to operate, and provide rapid andreproducible quantitative results but also should do so withoutthe use of an external “reader”.7"14 This goal of “reader-less”quantitative POC assays represents a formidable scientific andtechnical challenge.A key question to address toward this end relates to the type

of readout that should be produced by an assay so that thereadout is easy to quantify without using electronic devices.Standard spectroscopic and electrochemical analyses typicallyrequire an external device to obtain a quantitative re-sult,9,11,13"16 but more recent studies have focused onmeasurements based on “distance”, “time”, or “the number ofregions that turn color” on a device. All three of these outputscan be quantified, in theory, by counting, which represents afirst step toward achieving quantitative point-of-care assays thatdo not require electronics. Representative examples ofquantitative and semiquantitative assays (not all are POCassays) based on counting include time-based assays,17"19

semiquantitative20"23 and quantitative24 assays that requirecounting colored regions, as well as distance-based measure-ments in the context of nanomotor,25 microfluidic,26"28 spottests,29 and immunochromatographic and lateral-flow as-says.30"34

Measurements based on “time” are the least developed ofthese unconventional readouts, yet “time” is a readout that canbe measured in a number of ways, even without the use ofelectronic devices. Consequently, herein we describe thedevelopment of a new approach for quantifying enzymeanalytes by measuring the time that is required for one regionon a paper-based device to turn color after a first region turnscolor (see the Abstract image). This strategy is demonstrated inassays for active enzymes.35 The intensity of the color in eitherregion for this assay is not indicative of the quantity of theanalyte; rather, the quantity of the analyte is directly related tothe relative time required for the color to appear.This relative measurement enables assays that are internally

calibrated for effects of temperature, humidity, and sampleviscosity on sample distribution,36"39 and the overall approachrequires only that a user begin a measurement once the assayregion turns color, thus leading to short measurement times(seconds to minutes), with overall assay times (fromapplication of the sample to the completed test) requiringonly 15"30 min. This new assay strategy also offers aremarkable level of sensitivity (femtomolar),3 is selective, isinexpensive (the device is made from paper and microgramquantities of reagents), and is easy to use (the user need onlyadd the sample to the device and then time how long it takesfor one region to turn color after the first turns color). Thus,

Received: August 1, 2013Accepted: September 27, 2013Published: September 27, 2013

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the strategy offers a step forward toward the goal of conductingquantitative point-of-care assays without using auxiliary instru-ments or electronics.

! EXPERIMENTAL DESIGNAn example configuration of a paper-based microfluidicdevice40"44 that we designed for this assay is depicted inFigure 1. This device has an entry point for addition of thesample, and hydrophilic channels of paper that split the sampleinto two equal directions (i.e., layer 2, Figure 1b). Layer 2 alsoincludes (i) buffer salts that are redissolved by the sample tocontrol the pH of the fluid as it distributes through the device,

as well as (ii) the cofactor MgCl2, which is needed by certainenzyme analytes (other cofactors could be added for differentenzyme 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 targetenzyme is in the sample, it reacts with this substrate and causesrelease of one molecule of glucose per enzymatic reaction.Once the sample continues through layers 3 and 4 and intolayer 5, it encounters bead-bound glucose oxidase (dark blue inFigure 1b,c), which remains immobilized in the fibers of thepaper.13 The glucose oxidase (GOX) oxidizes the released

Figure 1. Detailed depiction of the assay device as well as the reagents used in the assays. (a) Photograph of a three-dimensional paper-basedmicrofluidic device19,24,36,37,44"46 for quantifying active enzyme analytes by measuring the relative time required for a sample to turn a control regiongreen (right-hand region) relative to when an assay region (left-hand region) turns green. The device is made from stacked layers of wax-patternedpaper44 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 cross-section depicted in (b). The left channel in (b) is the assay region and the right channel is the control region. (c) Specific substrate reagents areincorporated into the device to provide selective detection of the target enzyme analyte. (d) A hydrophobic oligomer is used to amplify signal for thedetection event. Amplification arises from head-to-tail depolymerization19,48"52 in response to hydrogen peroxide that is generated during thedetection event (c).19,24,53,54

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glucose and generates hydrogen peroxide as the sample travelslaterally in layer 5 of the device. Once the sample reaches thevertical conduit on the far left-hand side of the device in Figure1b, it encounters an oligomer (compound 2, Figure 1d) that ishydrophobic and thus alters the wetting properties of the paperfrom hydrophilic to hydrophobic.19,24 In the absence ofhydrogen peroxide, the sample travels slowly through thishydrophobic region, but in the presence of hydrogen peroxide,reagent 2 converts to hydrophilic products through a cascadedepolymerization reaction,19,48,50,56,57 thus switching thewetting properties of the paper from hydrophobic back tohydrophilic. This switching reaction amplifies the effects ofhydrogen peroxide on the flow rate through layers 4 and 3 byconverting a large hydrophobic oligomer into hydrophilicproducts.19,48 This switching reaction also allows the sample topass through the layers containing 2 with a rate that dependson the concentration of hydrogen peroxide in the sample,which ultimately reflects the concentration of the target enzymeanalyte. Once the sample passes through the layers containing2, it continues to travel in the vertical direction until itredissolves dried green food coloring and carries the highlycolored solution to the top layer where the bright green colorbecomes visible.The control region (right-hand channel in the cross-section

in Figure 1b) contains the same reagents in the same order asthe assay region, with the exception of bead-bound glucoseoxidase. In this control region, the enzyme analyte (if present)will react with substrate 1 deposited into the channel andgenerate glucose,55 but hydrogen peroxide will not begenerated; therefore, hydrogen peroxide will not be presentto react with oligomer 2. Hence, the time required for thesample to pass through this control region (and carry the greencolor to the top of the device) depends on the temperature andhumidity under which the assay is conducted, as well as on theviscosity of the sample.37"39,58 These factors will affect sampledistribution rates in the assay region as well (the left-handchannel); therefore, this control region normalizes the outputof the assay for the effects of these variables on sampledistribution. This normalization is implemented by measuringthe time required for the control region to turn green relative towhen the assay region (the left-hand region) turns green (i.e.,Tmeasurement).This measurement time is different than the total time

(Ttotal) for the sample to pass from the entrance of the device tothe end of the control region (the right-hand region in Figure1). The measurement time (Tmeasurement) also is different thanthe assay time (Tassay), which is the time required for the sampleto pass from the entrance of the device to the end of the assayregion (the left-hand region in Figure 1). The relationshipbetween Tmeasurement, Ttotal, and Tassay is depicted in eq 1.

= !T T Tmeasurement total assay (1)

Ttotal and Tassay are not measured during an assay and varydepending on the conditions for the assay. Typically Ttotal andTassay range from 15 to 30 min for Ttotal and from 2 to 15 minfor Tassay. The only reason to discuss either of these two times isbecause Tassay is the period of time in which a user must watchfor the appearance of green color in the assay region (the left-hand region in Figure 1) so that Tmeasurement can be made oncethe assay region turns green. The actual measurement time(Tmeasurement) typically is seconds for low concentrations of thetarget analyte up to 15 min for high concentrations.

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

! RESULTS AND DISCUSSIONDemonstrating Selectivity in the Assays. The perform-

ance of the assay is revealed by the calibration curves shown inFigure 2 for the model enzyme analytes alkaline phosphatase (a

marker in blood that is indicative of liver function)59"61 and β-D-galactosidase (a general marker of fecal coliforms in water).62

In Figure 2a, the analyte is alkaline phosphatase, which uses 1aas the small molecule substrate in the device and 2d as thephase-switching reagent. The calibration curve was generatedby depositing samples of alkaline phosphatase in 40 mMHEPES buffer (pH 8.0) to the top of the device and measuringTmeasurement for the control region to turn green after the assayregion turns green. The limit-of-detection for this assay is 320pM (0.355 U/L) alkaline phosphatase, with a dynamic range of320 pM (0.355 U/L) to 14.8 nM (16.3 U/L).63 This level ofsensitivity far exceeds the sensitivity of colorimetric assays thatuse camera-equipped cellular phones for quantifying assays in

Figure 2. Calibration curves for (a) alkaline phosphatase and (b) β-D-galactosidase, both of which are model enzymes to demonstrate thequantitative assay. The calibration curves were obtained at 19 °C and20% relative humidity using 2d as the phase-switching reagent andcompound 1a for alkaline phosphatase and 1b for β-D-galactosidase.The data points are the average of three measurements, and the errorbars reflect the standard deviations of these averages. The insets for (a)and (b) provide an expanded view of the regions that are bracketedusing 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. Thedata for alkaline phosphatase is black, catalase is green, and β-D-galactosidase is blue. Values for all of the enzyme assays, in U/L, areavailable in the Supporting Information.

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paper microfluidic devices59 and is even more sensitive thancomparable activity-based assays that use a glucose meter toobtain the quantitative readout.55 Perhaps more importantly,the measurement time (Tmeasurement) is proportional to theconcentration of the analyte, which is a feature that enablesrapid trace-level detection, even if only a qualitative result isdesired.Moreover, the assay is selective for alkaline phosphatase, as

revealed by the negligible response when catalase or β-D-galactosidase are added to the device instead of alkalinephosphatase (green and blue data in Figure 2a, respectively).Catalase was used for comparison to alkaline phosphatase sinceit decomposes hydrogen peroxide rapidly to water andoxygen64 and thus should not provide a measurable responseif the mechanism of the quantitative assay relies on analyte-induced production of hydrogen peroxide. β-D-Galactosidasewas chosen because it belongs to a different enzyme family thanalkaline phosphatase and therefore demonstrates that selectivitybetween 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 selectivityswitches as well (Figure 2b). The calibration curve in Figure 2bis for the enzyme β-D-galactosidase, which uses 1b (Figure 1c)as the substrate in the device. The limit-of-detection for thisassay is comparable to the alkaline phosphatase assay (i.e., 1.94nM; 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 secondcalibration curve demonstrates that the assay can bereconfigured easily by changing the activity-based detectionreagent (i.e., 1) to target a variety of enzymes.55

Demonstrating Tolerance to Sample Volume. Animportant feature of the assay is its ability to providequantitative results without requiring precise measurements ofsample volume (Figure 3). The patterned hydrophilic paper in

the device absorbs a fixed volume of sample,40,65 whichprovides sufficient control over sample volume to enablequantitative assays, so long as a minimum quantity of sample isadded to the device. The minimum volume for the deviceshown in Figure 1b is !25 μL.Normalizing for the Effects of Humidity and Calibrat-

ing for the Effects of Temperature. While humidity has anoticeable effect on sample distribution rates in paper,36,38,39 it

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

the control region. Variations in temperature, however, doaffect the time required for a sample to flow through the device.Temperature-induced changes in sample viscosity affect sampledistribution rates37"39,58 but are likely accounted for by thecontrol region, whereas temperature effects on enzymaticactivity 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 effects of assaytemperature on enzymatic activity (and, ultimately assay time)can be accounted for easily, however, since there are threetemperature regimes (within the ranges that we tested) thataffect the assay: (i) <15 °C, (ii) 15 to 33 °C, and (iii) >33 °C.The enzymatic activity is equal within the first temperature

range, so no adjustment is needed to the calibration curve inFigure 2a with the exception of adding 1 min to Tmeasurement sothat the measurement times match the calibration curve (Figure2a), which was generated at a warmer temperature (i.e., 19 °C)than this cold temperature range (<15 °C). Likewise, anotherplateau appears above 33 °C, which requires subtraction of 4min from Tmeasurement to recalibrate the curve in Figure 2a forthis warmer temperature range.Between the range of 15 and 33 °C, however, the

measurement times increase linearly with temperature. Thislinear increase is easily accommodated in a quantitative assay aswell. For example, when the assay is conducted at temperaturesabove 19 °C (the temperature for establishing the calibrationcurve) but below 33 °C, Tmeasurement should be decreased by afactor of 0.3079 ! ΔT (ΔT is the difference in temperaturebetween when the assay was conducted and the calibrationcurve was generated).67 Likewise, when the assay is conductedat temperatures below 19 °C (but above 15 °C), Tmeasurement isincreased by the same factor. By using these adjustments, thecalibration curve in Figure 2a remains functional over a widerange of temperatures.

Figure 3. Effect of sample volume on Tmeasurement for a sample thatcontains 11 nM alkaline phosphatase (12 U/L). The assays wereconducted at 19 °C and 20% relative humidity using reagents 1a and2d. The data are the average of three measurements, and the error barsare smaller than the data points. The vertical dotted line marks thelowest volume of sample that is required for the assay. The assay wasstopped at 45 min regardless of whether it was complete; therefore,the data points below 25 μL (x-axis) reflect assays that lacked enoughsample to reach completion.

Figure 4. Effect of humidity and temperature on the accuracy ofmeasuring Tmeasurement when using 11 nM (12 U/L) alkalinephosphatase as the model enzyme analyte for the humidity assayand 4.0 nM (4.0 U/L) for the temperature study. The assays wereconducted using devices that contained 1a and 2d. The data points arethe average of three measurements, and the error bars reflect thestandard deviations from these averages. The inset in (b) depicts thelinear region of the “temperature vs. Tmeasurement” graph. The equationfor the line is y = 0.3079x " 3.6831.

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While measurements of assay temperature may soundcomplicated, it is worth noting that these measurements canbe accomplished easily and cheaply using flexible and reversibleleucodye-based temperature strips,68 which can be affixed topackaging that may contain hundreds of the assay devices and,therefore, can be used to measure the temperature for manyassays. With limited access to temperature-controlled environ-ments in point-of-care settings, the ability to correct forvariations in temperature is crucial for reliable quantitativeresults.Tuning the Sensitivity of the Assays by Altering the

Design of the Assay Platform. There are severalstraightforward ways to tune the limit-of-detection for aparticular assay in these devices, one of which involveschanging the length of the lateral flow channels in layer 5 ofthe device (Figure 5a). The lengths of these channels are

inversely proportional to the measurement time (Tmeasurement)for an assay (Figure 5b), which means that shorter channels inlayer 5 provide greater distinction than longer channelsbetween the time for the sample to pass through the controlregion (right-hand region in Figure 1b) relative to the assayregion (left-hand region in Figure 1b). As the time gap (i.e.,Tmeasurement) between these two conduits increases, the limit-of-detection for an assay decreases (Figure 5c), with improve-ments in sensitivity reaching as high as !28-fold for the channellengths that we tested.A second method for tuning the sensitivity involves changing

the number of layers of paper that contain compound 2 (thephase-switching reagent).19 In this case, two scenarios areenvisaged: (1) multiple layers of the phase-switching reagentmay provide greater interactions between the hydrogenperoxide that is generated in the assay and the phase-switchingreagent and thus may improve the ability of the device todifferentiate between different concentrations of the targetenzyme or (2) single layers of the phase-switching reagent mayrequire little hydrogen peroxide to impart a change in wettingproperties from hydrophobic to hydrophilic upon reaction withthe phase-switching reagent, thus decreasing the quantity of

enzyme that is required to achieve a measurable response in theassay.We recently studied this concept for hydrogen peroxide

detection and found that a single layer of phase-switchingreagent provides the best sensitivity for detecting hydrogenperoxide.19 In the context of the enzyme assays, one layer of thephase-switching reagent also provides the best sensitivity. Forexample, a device with a 5.4 mm-long lateral flow region thatcontains only 1 layer of 2d has a limit-of-detection for alkalinephosphatase that is 1.9! better than an equal device thatcontains 2 layers of 2d (LOD = 45 vs 84 pM, respectively) and9.8! better than a device that contains 3 layers of the phase-switching reagent (LOD = 440 pM).

Tuning the Sensitivity and Dynamic Range of theAssays by Altering the Phase-Switching Reagent. A thirdmethod for altering the sensitivity of an assay involves changingthe phase-switching reagent (2) used in the device. Strategiessuch as (i) increasing the rate of conversion of hydrophobic 2into hydrophilic byproducts in response to hydrogen peroxide(e.g., 2a vs 2b, Figure 1d)69 and (ii) increasing the magnitudeof the change by employing depolymerizable oligomers (e.g.,2b"2f)19,48 both provide improvements in sensitivity for anassay for alkaline phosphatase (the model enzyme) (Table 1).In fact, use of 2f in the device instead of 2a improves thesensitivity for the assay by !4 orders of magnitude.

Combining these three strategies for improving thesensitivity for an assay enables tuning of the assay for arelevant concentration of a target analyte over the concen-tration range of low femtomolar to low nanomolar, withdynamic ranges for the assays of at least 1 order of magnitude.The most sensitive assay uses a 5.4 mm-long lateral flowchannel 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 limit-of-detection of 26 nM by using a 12.4 mm-long lateral flowchannel and 3 layers of 2a.70 Advances in polymerchemistry51,57,58,71 should provide access to new phase-switching reagents (2) that further expand the operatingrange of the assays, particularly by decreasing the limit-of-detection.

Modifying the Design of the Device for ComplexFluids. All of these optimization studies were performed usingbuffered samples that lack the complexity of various fluids thatmay contain the analyte of interest. Depending on the fluid, avariety of factors must be considered, most notably whether the

Figure 5. Effect of the length of the lateral flow region in the paper-based device on the sensitivity of an assay for alkaline phosphatase. (a)Cross-section of the device in Figure 1a showing the lateral flowchannel that was varied in length. (b) Relationship between the lengthof the lateral flow region and the measurement times (Tmeasurement) forassays for 11 nM (12 U/L) alkaline phosphatase in 40 mM HEPESbuffer (pH 8) using 1a and 2d. (c) Relationship between channellength and the limit-of-detection (LOD) for an assay for alkalinephosphatase (ALP).

Table 1. Effect of the Phase-Switching Reagent (i.e.,Compound 2) on the Sensitivity and Dynamic Range of anAssay for Alkaline Phosphatasea

compoundno.

LOD(pM)

relative improvement insensitivity

dynamic range(pM)

2a 5600 1 5600"74 0002b 342 16 342"37 0002c 93 60 93"14802d 45 124 45"14802e 13 431 13"7402f 0.128 43 750 0.128"7.4

aThe assays were conducted using a device that contained a 5.4 mm-long lateral flow region and 1 layer of compound 2. The quantity ofeach derivative of compound 2 was optimized to maximize thesensitivity of the assay.

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fluid contains glucose or hydrogen peroxide that wouldinterfere with the outcome of the assay.Configuration of a Modified Device. To overcome this type

of interference, we created a revised device that includes a newlayer (Figure 6) in which bead-bound glucose oxidase andcatalase are deposited into the central channel of the device.13

The glucose oxidase oxidizes glucose in a sample and generateshydrogen peroxide, while catalase converts the hydrogenperoxide into oxygen and water. These preprocessing stepsoccur before the sample redissolves the buffer salts in the deviceand distributes into the assay and reference conduits.Preprocessing of Glucose and Hydrogen Peroxide in a

Sample. These scavenging reagents are placed in the modifieddevice in layer 2 in Figure 6 and are capable of scavenging 25mM glucose and 15 mM hydrogen peroxide when the device ischallenged with a sample of alkaline phosphatase containingeither of the contaminants (Figure 7). The typical concen-

tration of glucose in blood is 3.5"5.3 mM,72 while the highestlevels of hydrogen peroxide in urine, beverages such as tea, orrainwater (in a polluted environment) are 5"100,73 100,74 and5.4 μM,75 respectively. Clearly, the device will be capable ofremoving these types of interfering contaminants.Sensitivity of the Assay in Horse Serum. With these user-

independent preprocessing reagents in place, the assay platformis compatible with complex fluids such as serum, as depicted bythe calibration curve for measuring the levels of alkalinephosphatase in enzyme-spiked horse serum (Figure 8). Thelimit-of-detection for alkaline phosphatase in this fluid is 2.4pM (2.6 mU/L) when a 5.4 mm-long lateral flow channel is

used along with 1 layer of phase-switching reagent 2f. Whilethis level of sensitivity is !19! higher than in pure buffer,76 itstill represents a highly sensitive quantitative assay, especiallygiven that the user only adds a sample to the device and thenmeasures the time to appearance of color in the control regionrelative to the assay region on time scales of 5 s to 4.5 min. Thislevel of sensitivity for alkaline phosphatase is well below thelevels of the enzyme typically found in human serum (42"369U/L).78,79 Should we wish to create an assay for relevantconcentrations of alkaline phosphatase in serum,73 we could usea different phase-switching reagent (2), change the number oflayers of 2, or lengthen the lateral flow channel to easily andpredictably tune the sensitivity of the assay and bring it withinthe clinical range for this analyte.

! CONCLUSIONIn conclusion, we describe a new type of quantitative POCassay platform that we believe offers analytical capabilities thattypically are only available using specific instruments.3 Ourapproach requires a single operation by the user; the assay iscompleted in seconds to minutes, and the readout involvessimple measurements of time to quantify the amount of anactive enzyme analyte in a sample down to femtomolar levels insimple fluids and low picomolar levels in complex fluids. Theassay also is inexpensive: only paper, food coloring, buffer 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 withoutuse of an external electronic reader.18"34 The opportunity forsignal amplification by using polymers that depolymerize19 sets

Figure 6. Cross-section of a revised device that includes preprocessing reagents in the central channel in layer 2. These reagents remove glucose andhydrogen peroxide that may be in a sample. The location of the cross-section is depicted in the photograph in Figure 1a.

Figure 7. Effect of the concentration of hydrogen peroxide or glucosein a sample on Tmeasurement. The black data reflects Tmeasurement values forsamples of 1.5 pM (1.6 mU/L) alkaline phosphatase that were spikedwith hydrogen peroxide, and the blue data corresponds to the sameexperiment using glucose instead of hydrogen peroxide. The color-coded vertical dotted lines reflect the highest concentration of eitherhydrogen peroxide or glucose that is tolerated in the assay withoutaffecting Tmeasurement.

Figure 8. Calibration curve for quantifying alkaline phosphatase spikedinto horse serum. The calibration curve was generated using the devicein Figure 6 that contained 1a and 2f. The data were acquired at 20 °Cand 53% relative humidity. The data points are the averages of threemeasurements, and the error bars reflect the standard deviations ofthese averages. The inset provides an expanded view of the region thatis bracketed with a dotted line. The equation for the line is y = 2.5498x+ 5.3199.

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this approach apart from others and may offer a level ofsensitivity that is particularly relevant for point-of-care andpoint-of-use applications in resource-limited settings such as thedeveloping world, home healthcare, emergency situations, andothers. The next steps include extending this proof-of-conceptapproach to other classes of analytes as well as fully developingassays for specific analytes. Further improvements in sensitivityalso are being sought by creating new types of oligomers/polymers as phase-switching reagents.71

! ASSOCIATED CONTENT*S Supporting InformationExperimental procedures, tables of data, and supporting figures.This material is available free of charge via the Internet athttp://pubs.acs.org.

! AUTHOR INFORMATIONCorresponding Author*E-mail: sphillips@psu.edu. Fax: 814-865-5235.Author ContributionsThe manuscript was written through contributions of allauthors. All authors have given approval to the final version ofthe manuscript.NotesThe authors declare no competing financial interest.

! ACKNOWLEDGMENTSThis work was supported in part by NSF (CHE-1150969), EliLilly, and Louis Martarano. S.T.P. acknowledges support fromthe Alfred P. Sloan Research Fellows Program.

! REFERENCES(1) Yager, P.; Domingo, G. J.; Gerdes, J. Annu. Rev. Biomed. Eng.2008, 10, 107"144.(2) Yager, P.; Edwards, T.; Fu, E.; Helton, K.; Nelson, K.; Tam, M.R.; Weigl, B. H. Nature 2006, 442, 412"418.(3) Giljohann, D. A.; Mirkin, C. A. Nature 2009, 462, 461"464.(4) Fu, E.; Yager, P.; Floriano, P. N.; Christodoulides, N.; McDevitt,J. T. IEEE Pulse 2011, 2, 40"50.(5) Urdea, M.; Penny, L. A.; Olmsted, S. S.; Giovanni, M. Y.; Kaspar,P.; Shepherd, A.; Wilson, P.; Dahl, C. A.; Buchsbaum, S.; Moeller, G.;Hay Burgess, D. C. Nature 2006, 444, 73"79.(6) Peeling, R. W.; Holmes, K. K.; Mabey, D.; Ronald, A. Sex Transm.Infect. 2006, 82, v1"6.(7) These readers include devices such as cell phone cameras,8,9

glucose meters,10,11 conductivity meters,12"14 or any number ofelectronic devices that could be paired with an assay.(8) Martinez, A. W.; Phillips, S. T.; Carrilho, E.; Thomas, S. W., III;Sindi, H.; Whitesides, G. M. Anal. Chem. 2008, 80, 3699"3707.(9) Shen, L.; Hagen, J.; Papautsky, I. Lab Chip 2012, 12, 4240"4243.(10) Xiang, Y.; Lu, Y. Anal. Chem. 2012, 84, 1975"1980.(11) Nie, Z.; Deiss, F.; Liu, X.; Akbulut, O.; Whitesides, G. M. LabChip 2010, 10, 3163"3169.(12) Nie, Z.; Nijhuis, C. A.; Gong, J.; Chen, X.; Kumachev, A.;Martinez, A. W.; Narovlyansky, M.; Whitesides, G. M. Lab Chip 2010,10, 477"483.(13) Liu, H.; Xiang, Y.; Lu, Y.; Crooks, R. M. Angew. Chem., Int. Ed.2012, 51, 6925"6928.(14) Zang, D.; Ge, L.; Yan, M.; Song, X.; Yu, J. Chem. Commun. 2012,48, 4683"4685.(15) Ellerbee, A. K.; Phillips, S. T.; Siegel, A. C.; Mirica, K. A.;Martinez, A. W.; Striehl, P.; Jain, N.; Prentiss, M.; Whitesides, G. M.Anal. Chem. 2009, 81, 8447"8452.(16) Dungchai, W.; Chailapakul, O.; Henry, C. S. Anal. Chem. 2009,81, 5821"5826.

(17) Baker, M. S.; Phillips, S. T. J. Am. Chem. Soc. 2011, 133, 5170"5173.(18) Aili, D.; Mager, M.; Roche, D.; Stevens, M. M. Nano Lett. 2011,11, 1401"1405.(19) Lewis, G. G.; Robbins, J. S.; Phillips, S. T. Macromolecules 2013,46, 5177"5183.(20) Leung, W.; Chan, C. P.; Rainer, T. H.; Ip, M.; Cautherley, G. W.H.; Renneberg, R. J. Immunol. Methods 2008, 336, 30"36.(21) Fung, K.-K.; Chan, C. P.-Y.; Renneberg, R. Anal. Chim. Acta2009, 634, 89"95.(22) Lou, S. C.; Patel, C.; Ching, S.; Gordon, J. Clin. Chem. 1993, 39,619"624.(23) Cho, J. H.; Paek, S. H. Biotechnol. Bioeng. 2001, 75, 725"732.(24) Lewis, G. G.; DiTucci, M. J.; Phillips, S. T. Angew. Chem., Int. Ed.2012, 51, 12707"12710.(25) Wu, J.; Balasubramanian, S.; Kagan, D.; Manesh, K. M.;Campuzano, S.; Wang, J. Nat. Commun. 2010, 1, 26.(26) Zhong, M.; Lee, C. Y.; Croushore, C. A.; Sweedler, J. V. LabChip 2012, 12, 2037"2045.(27) Chatterjee, D.; Mansfield, D. S.; Anderson, N. G.; Subedi, S.;Woolley, A. T. Anal. Chem. 2012, 84, 7057"7063.(28) Song, Y.; Zhang, Y.; Bernard, P. E.; Reuben, J. M.; Ueno, N. T.;Arlinghaus, R. B.; Zu, Y.; Qin, L. Nat. Commun. 2012, 3, 1283.(29) Yang, X.; Kanter, J.; Piety, N. Z.; Benton, M. S.; Vignes, S. M.;Shevkoplyas, S. S. Lab Chip 2013, 13, 1464"1467.(30) Allen, M. P.; DeLizza, A.; Ramel, U.; Jeong, H.; Singh, P. Clin.Chem. 1990, 36, 1591"1597.(31) Liu, V. Y. S.; Lin, T.-Y.; Schrier, W.; Allen, M.; Singh, P. Clin.Chem. 1993, 39, 1948"1952.(32) Zuk, R. F.; Ginsberg, V. K.; Houts, T.; Rabbie, J.; Merrick, H.;Ullman, E. F.; Fischer, M. M.; Sitzto, C. C.; Stiso, S. N.; Litman, D. J.Clin. Chem. 1985, 31, 1144"1150.(33) Chen, R.; Li, T. M.; Merrick, H.; Parrish, R. F.; Bruno, V.;Kwong, A.; Stiso, C.; Litman, D. J. Clin. Chem. 1987, 33, 1521"1525.(34) Cate, D. M.; Dungchai, W.; Cunningham, J. C.; Volckens, J.;Henry, C. S. Lab Chip 2013, 13, 2397"2404.(35) Goddard, J.-P.; Reymond, J.-L. Trends Biotechnol. 2004, 22,363"370.(36) Noh, H.; Phillips, S. T. Anal. Chem. 2010, 82, 8071"8078.(37) Noh, H.; Phillips, S. T. Anal. Chem. 2010, 82, 4181"4187.(38) Washburn, E. W. Phys. Rev. 1921, 17, 273"283.(39) Schilling, K. M.; Lepore, A. L.; Kurian, J. A.; Martinez, A. W.Anal. Chem. 2012, 84, 1579"1585.(40) Martinez, A. W.; Phillips, S. T.; Whitesides, G. M.; Carrilho, E.Anal. Chem. 2010, 82, 3"10.(41) Li, X.; Ballerini, D. R.; Shen, W. Biomicrofluidics 2012, 6, 11301.(42) Yetisen, A. K.; Akram, M. S.; Lowe, C. R. Lab Chip 2013, 13,2210"2251.(43) Martinez, A. W.; Phillips, S. T.; Whitesides, G. M. Proc. Natl.Acad. Sci. U.S.A. 2008, 105, 19606"19611.(44) (a) Lu, Y.; Shi, W.; Jiang, L.; Qin, J.; Lin, B. Electrophoresis 2009,30, 1497"1500. (b) Carrilho, E.; Martinez, A. W.; Whitesides, G. M.Anal. Chem. 2009, 81, 7091"7095.(45) Liu, H.; Crooks, R. M. J. Am. Chem. Soc. 2011, 133, 17564"17566.(46) Schilling, K. M.; Jauregui, D.; Martinez, A. W. Lab Chip 2013,13, 628"631.(47) Lewis, G. G.; DiTucci, M. J.; Baker, M. S.; Phillips, S. T. LabChip 2012, 12, 2630"2633.(48) Robbins, J. S.; Schmid, K. M; Phillips, S. T. J. Org. Chem. 2013,78, 3159"3169.(49) Sagi, A.; Weinstain, R.; Karton, N.; Shabat, D. J. Am. Chem. Soc.2008, 130, 5434"5435.(50) Esser-Kahn, A. P.; Sottos, N. R.; White, S. R.; Moore, J. S. J. Am.Chem. Soc. 2010, 132, 10266"10268.(51) DeWit, M. A.; Gillies, E. R. J. Am. Chem. Soc. 2009, 131, 18327"18334.(52) Esser-Kahn, A. P.; Odom, S. A.; Sottos, N. R.; White, S. R.;Moore, J. S. Macromolecules 2011, 44, 5539"5553.

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dx.doi.org/10.1021/ac402415v | Anal. Chem. 2013, 85, 10432"1043910438

(53) Lippert, A. R.; Van de Bittner, G. C.; Chang, C. J. Acc. Chem.Res. 2011, 44, 793"804.(54) Cho, D.-G.; Sessler, J. L. Chem. Soc. Rev. 2009, 38, 1647"1662.(55) Mohapatra, H.; Phillips, S. T. Chem. Commun. 2013, 49, 6134"6136.(56) Peterson, G. I.; Larsen, M. B.; Boydston, A. J. Macromolecules2012, 45, 7317"7328.(57) Wong, A. D.; DeWit, M. A.; Gillies, E. R. Adv. Drug Delivery Rev.2012, 64, 1031"1045.(58) Masoodi, R.; Pillai, K. M. AIChE J. 2010, 56, 2257"2267.(59) Vella, S. J.; Beattie, P.; Cademartiri, R.; Laromaine, A.; Martinez,A. W.; Phillips, S. T.; Mirica, K. A.; Whitesides, G. M. Anal. Chem.2012, 84, 2883"2981.(60) Pekarthy, J. M.; Short, J.; Lansing, A. I.; Lieberman, I. J. Biol.Chem. 1972, 247, 1767"1774.(61) Reichling, J. J.; Kaplan, M. M. Dig. Dis. Sci. 1988, 33, 1601"1614.(62) Tryland, I.; Fiksdal, L. Appl. Environ. Microbiol. 1998, 64, 1018"1023.(63) The limit of detection was defined as 3! the standard deviationof the lowest measured sample (the blank) divided by the slope of thelinear dynamic region.(64) Keilin, D.; Hartree, E. F. Biochem. J. 1945, 39, 293"301.(65) Phillips, S. T.; Lewis, G. G. MRS Bull. 2013, 38, 315"319.(66) Boulanger, R. R., Jr.; Kantrowitz, E. R. J. Biol. Chem. 2003, 278,23497"23501.(67) 0.3079 is the slope of the linear region in Figure 4b.(68) www.thermometersite.com; $10 for a pack of 10 devices;accessed 7/31/13.(69) Schmid, K. M.; Jensen, L.; Phillips, S. T. J. Org. Chem. 2012, 77,4363"4374.(70) The assay could be made even less sensitive by changing thequantity of 2a used per layer in the device.(71) Olah, M. G.; Robbins, J. S.; Baker, M. S.; Phillips, S. T.Macromolecules 2013, 46, 5924"5928.(72) Tietz, N. W. Clinical Guide to Laboratory Tests; W. B. SaundersCompany: Philadelphia, PA, 1995.(73) Halliwell, B.; Clement, M. V.; Long, L. H. FEBS Lett. 2000, 486,10"13.(74) Chai, P. C.; Long, L. H.; Halliwell, B. Biochem. Biophys. Res.Commun. 2003, 304, 650"654.(75) Ortiz, V.; Rubio, M. A.; Lissi, E. A. Atmos. Environ. 2000, 34,1139"1146.(76) We have two working hypotheses for this observed decrease insensitivity relative to assays in buffer. First, perhaps the decreasedsensitivity for detecting alkaline phosphatase in serum vs buffer is theresult of other components in serum that increase the solubility ofphase-switching reagent 2f in relation to buffer. Second, perhapscatalase in serum (!50 kU/L)77 is decomposing some of the hydrogenperoxide that is generated in response to the analyte, thus decreasingthe observed Tmeasurement. Experiments are in progress to test thesehypotheses and further improve the sensitivity of assays in complexfluids.(77) Got́h, L. Clin. Chim. Acta 1991, 196, 143"151.(78) Wolf, P. L. J. Clin. Pathol. 1975, 28, 587"591.(79) Bowers, G. N., Jr.; McComb, R. B. Clin. Chem. 1966, 12, 70"89.

Analytical Chemistry Article

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