Real-Time Droplet DNA Amplification with a New Tablet PlatformStephanie L. Angione,† Anuj Chauhan,‡ and Anubhav Tripathi*,†

†Center for Biomedical Engineering, School of Engineering and Division of Biology and Medicine, Brown University, Providence,Rhode Island, United States‡Department of Chemical Engineering, University of Florida, Gainesville, Florida, United States

*S Supporting Information

ABSTRACT: We present a novel droplet-based tablet platform fortemporal polymerase chain reaction (PCR) in microliter droplets. Thesimple design of the device does not require extensive processing orexternal equipment, which allows for greater ease of use and integration asa point-of-care diagnostic. We demonstrate its functionality to performboth PCR and reverse-transcription PCR for λ phage DNA and H3influenza RNA with ramp rates and cycle times consistent with traditionalPCR thermal cyclers. We additionally investigate the effect of performingPCR in small volumes on the reaction performance by specificallyexamining adsorption of reagents at the oil/water interface. Wedetermined that adsorption of Taq polymerase at the biphasic interfacereduces yield and impairs reaction performance at standard concen-trations. Thus, microdroplet PCR reactions require additional polymeraseto achieve sufficient amplification and we project for applications utilizing nanodroplets or picodroplets like digital applications,even greater concentrations of polymerase are required to achieve desired results. Following the adsorption investigation, weevaluated the sensitivity of λ phage PCR on our platform to be less than 2.0 copies/μL with an efficiency of 104.4% and similarsensitivity for reverse-transcription PCR for influenza H3 RNA.

The ability to bring rapid diagnosis to developing countrieshas been an objective for many microfluidic technologies

as well as a crucial challenge. There is a growing public healthneed for portable, low-cost diagnostic devices in resource-limited settings, which presents many different designchallenges.1 Rapid and sensitive diagnosis of diseases likehuman immunodeficiency virus (HIV), influenza, and bacterialinfections by use of compact point-of-care devices has thepotential to transform health care, in both hospital and fieldsettings. Due to the typically low concentrations of pathogennucleic acids in patient samples, many diagnostic assays dependupon DNA amplification though polymerase chain reaction(PCR), which exponentially amplifies the number of DNAmolecules. Real-time monitoring of amplified DNA is criticalfor gene identification and mutation detection.Since PCR requires temperature cycling, it is typically carried

out in benchtop units, which are slow and bulky and requirerelatively large volumes to perform the thermal cyclingrequired. Recently, with the emergence of microfluidic PCRformats, smaller sample volumes are used and faster heatingand cooling times have been achieved.2−4 Utilizing the conceptsof both spatial and temporal microfluidic PCR, droplet-basedmicrofluidic technologies have been emerging as alternatives tosingle-phase reactions. The advantage of droplet formats lies inlow reagent usage and faster heat transfer and thermalequilibrium, as well as limiting interactions of channel wallswith polymerase and DNA. Channel-based single-phasemethods often require specific surface treatments or

passivation5,6 to lessen adsorption of reagents. Additionally,by carrying out the reaction in isolated droplets, PCR inhibitionand carryover contamination can be mitigated.7 Dropletformats also allow for the possibility of increased throughputand sensitivity8,9 since droplets are discrete small volumes thatcan be easily adapted for parallel amplification at lowconcentrations. There are many examples of flow-throughsystems that utilize aqueous droplets surrounded in anonaqueous oil or solvent. Several droplet PCR microdeviceshave been adapted from the traditional spatial thermal cyclingformat to include droplet-generating technologies for boththree zones and two zones.9−12 There have been severalpressure-driven flow systems that employ microdroplets,nanodroplets, or picodroplets8,13−15 to perform both PCRand reverse transcription (RT) PCR. These flow-throughtechnologies have proved successful in generating high-throughput results as well as single-molecule sensitivity. Similardigital microfluidic platforms have used electrowettingtechnology to move microdroplets in place of pressure-drivensystems,16 while other investigators have used actuatingmagnets to manipulate droplets containing magnetic beads toperform PCR temperature cycling or surface acoustic wavepump (SAW),17 to alleviate the need for pressure pumps.18,19

However, all of the above approaches are complex to create and

Received: October 1, 2011Accepted: February 6, 2012Published: February 6, 2012

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nontrivial to operate. Many require continuous flow of liquidsin microchannels, often driven by pressure pumps4,20 orutilizing valving,3,4,21 both of which add complexity to thedevice and operation. Additionally, fabrication of these devicesis often time-consuming and expensive. Many require photo-lithography and wet etching methods for glass, quartz, or plasticmaterials.2,3,22 As an alternative to continuous flow technolo-gies, several investigators have employed stationary dropletplatforms23−28 or other forms of stationary droplets like theSlipChip to perform PCR.29,30

Although droplet technologies help mitigate the adverseaffects due to the large surface to volume ratios associated withmicrofluidics, use of an immiscible fluid introduces a newinterface for possible adsorption of reagents. Thus, despite theobvious use of droplet PCR methods, little has been reportedregarding the transport and behavior in droplets surrounded byoil. The majority of publications have focused on hardware andmethodology for droplet-based PCR, and less insight has beengiven regarding differences in performance or amplificationefficiency. Some investigators have provided analyses of heattransfer properties and temperature distributions,10 but littlehas been reported regarding fundamental mass transport. Inexamining the results of representative droplet-based tech-nologies, like the radial PCR device designed by Schaerli et al.8

that utilizes picoliter-sized droplets, there is an apparentdifference between chip-based amplification and thermal cyclercontrols. The amplification factor of the device-based PCRcompared to thermal cycler was up to 52-fold lower. Thisindicates that there is an inherent difference between dropletPCR and conventional single-phase PCR, but contributingfactors such as adsorption of reagents have not been rigorouslyinvestigated. Some investigators have looked at alteringparameters, such as Wang and Burns,27 who investigatednanoliter-sized droplet performance parameters includingconcentrations of Mg2+, Taq polymerase, and DNA templateas well as temperature hold times and cycling conditions.Insight into the fact that there are factors at work in droplet-based microreactions was established but not fundamentallyinvestigated. Since droplet in oil technologies are growing andhave found a niche in point-of-care diagnostic development, itis useful to understand the principles that govern the systemand how various parameters differ for droplet PCR comparedto bulk reactions.Here we present a novel droplet-based tablet platform that

utilizes low power and a straightforward setup, thus offering aneasy-to-use PCR chip that offers greater simplicity over othermethods. The simplicity of the device will make it easy tointegrate into a point-of-care device for PCR diagnosis ofnumerous diseases. We demonstrate the platform’s usefulnessin both standard DNA amplification and reverse transcriptasePCR in real time for identification of H3 influenza RNA.Additionally, we explore the inherent differences in the dropletPCR format compared to traditional PCR.

■ EXPERIMENTAL PROCEDURESThe New “Tablet” Platform. The tablet platform, as

shown in Figure 1a, consists of a droplet of PCR mixsurrounded by mineral oil, producing a disk-shaped compounddroplet. A hydrophobic adhesive imaging spacer provides thecylindrical wall to create a tablet-like chamber for the PCRreaction, which has a diameter of 9 mm and is 1 mm thick. Thebottom surface of the tablet is an indium tin oxide (ITO)coated soda-lime glass microscope slide. ITO is an optically

transparent angstrom-thick thin film that provides rapid andtime-dependent resistive heating. The top surface of the tabletis an optically clear glass. The glass surface is covered with

Figure 1. (a) Schematic of droplet platform: The ITO chip with thecompound droplet is surrounded by an imaging spacer and coveredwith a coverslip. The droplet is placed at the center of the chip andcovered in mineral oil. The spacer seals the compound droplet in achamber with the coverslip to prevent evaporation. Each surface incontact with the droplet is covered with Teflon tape to preventadsorption of DNA polymerase. (b) Overall control system: Theentire droplet platform system, including the microscope andfluorescence acquisition, is shown. The droplet is mounted on acustom-made stage to accommodate connection to the voltage supplyand cooling by a fan. The entire system is controlled by a LabViewprogram that provides temperature feedback and fluorescenceacquisition. (c) Temperature cycling profile for five representativecycles: Black line, PID-controlled surface temperature; red line,calibrated droplet temperature, measured by thermocouple.

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optically transparent Teflon fluorinated ethylene propylene(FEP) tape to prevent adsorption of PCR reagents to thesurface of the glass. A Teflon surface was chosen on the basis ofevidence that Taq polymerase does not adsorb to fluorinatedsurfaces like Teflon.31 On the ITO side of the chip, athermocouple is placed at the center of the tablet. A droplet(1−4 μL) of PCR mix is placed at the center of the tablet onthe FEP tape-covered side directly above and opposite thethermocouple. Four times as much mineral oil is then placed ontop of the droplet. A glass coverslip also covered with FEP tapeis then placed on the imaging spacer, effectively sealing thedroplet of PCR mix inside the larger droplet of oil. Presently,the tablet platform is flipped and placed on a custom-mademicroscope stage with a small cooling fan. The PCR reactioncan be monitored in real time from the top and bottomsurfaces.The platform we present is unique and specifically

advantageous in its inherent simplicity. It does not requireany extensive fabrication methods, photolithography, or wetetching techniques, which are time-consuming and expensiveand must be done in clean-room conditions with harshchemicals. The fact that the PCR surface and heater areencompassed by the ITO-coated glass means that there are noexternal heating devices and no heater fabrication is necessary.Imaging is also extremely simple and straightforward since ITOis optically transparent. The platform is also very easy to use,both to assemble and to run PCR reactions. The computerinterface is very user-friendly and easily adapted to manydifferent cycling conditions or PCR reactions. Additionally, byimplementing a fluorinated tape surface, which is removablebetween reactions, there are no specialized cleaning stepsrequired between samples, and the removable surfaceeliminates sample carryover. By utilizing this platform withsmall volumes (1−4 μL), real-time PCR can be done utilizing astandard inverted microscope and photomultiplier tube (PMT)at a fraction of the cost of a specialized real-time PCRinstrument without compromising sensitivity and improvingthe time required for temperature cycling.Instrumentation and Experimental Setup. Figure 1b

shows the instrumentation required to operate the tabletplatform. The ITO tablet platform is controlled by a custom-written proportional integral derivative (PID) temperaturecontroller in LabVIEW (National Instruments). The thermo-couple controls the temperature of the ITO surface byadjusting the voltage according to the feedback information.Temperature control was achieved via a computer with a dataacquisition card (National Instruments) and USB voltagecontrollers from Matsusada Inc. Both temperature andfluorescence signals are collected during program operation.Since the ITO is deposited on only one side, the temperature ofthe reaction mixture is lower than the thermocouple reading onthe ITO side. The temperatures were calibrated with athermocouple placed in an oil droplet on the reaction surface.Materials and Preparation. Tablet Platform Prepara-

tion. Indium tin oxide (ITO) coated soda-lime glass micro-scope slides were obtained from SPI Supplies, and transparentTeflon fluorinated ethylene propylene (FEP) tape was obtainedfrom McMaster. Hydrophobic adhesive imaging spacers werepurchased from Grace Biolabs, and adhesive type Ethermocouples from Omega. The cooling fan was obtainedfrom Imbpapst and the coverslips from Fischer.λ Phage Polymerase Chain Reaction. A 106 bp amplicon of

λ phage DNA (New England Biolabs) was amplified by PCR.

The primer set 5′-GATTGCCAGGCTTAAATGAGTC-3′ and5′-GTTTCCGGATAAAAACGTCGAT-3′ was used. Primerswere obtained from IDT. SYBR Green I dye (AppliedBiosystems) was used for real-time fluorescence detection.Each 50 μL PCR mix consisted of 1× Taq buffer, 200 μMdNTPs, 0.4 μM both forward and reverse primers, 0.05 unit ofTaq polymerase (unless otherwise noted for the polymeraseinvestigation), a dilution of λ phage DNA template (500 bp),and 0.1× SYBR Green I. PCR was performed for 40 cycles,both on the PCR tablet and a control in a conventionalthermocycler (Bio-Rad). Each cycle consisted of three stages:95 °C for 10 s, 49 °C for 20 s, and 72 °C for 40 s. An initialdenature was done at the beginning of cycling for 30 s, and afinal extension was performed for 60 s. Sizing of the ampliconwas done with the Agilent Bioanalyzer 2100.

H3 Viral RNA Reverse Transcription Polymerase ChainReaction. For influenza H3 RNA amplification, synthetic viralRNA (vRNA) of full-length H3 RNA was used as template.The sequence of the vRNA was identical to the strain withNCBI accession number AF348176. Primers specific for theindividual strain were designed and include 5′-CTTTTAA-GATCTGCTGCTTGTCCT-3′ and 5′-AGAAACAAACTA-GAGGCCTATTCG-3′. Primers were obtained from IDT.Each RT PCR reaction consisted of 50 μL of RT-PCR mixfrom the Superscript III RT PCR kit, which includesSuperscript III reverse transcriptase and Platinum Taq DNApolymerase (Invitrogen). Both reverse transcription and PCRwere carried out in the same tube or on the droplet platform.This included the 1× reaction mix, containing buffer, MgCl2and dNTPs, and 0.2 μM of both the forward and reverseprimers, and 0.1× SYBR Green I dye was utilized forfluorescence quantification. Cycling consisted of a 30 min RTstep at 50 °C, followed by 40 cycles of 94 °C for 10 s, 54 °C for20 s, and 68 °C for 40 s. An initial denature was done followingthe RT step, 10 min for off-chip controls and 2 min forplatform droplet amplification.

■ RESULTS AND DISCUSSIONTemperature Cycling. Figure 1c shows the measured

temperature inside the PCR droplet. The ITO tablet platformwas used to perform temperature cycling for the PCR mix viathe PID temperature controller. The temperature cyclingprogram displayed is 95 °C for 10 s, 49 °C for 20 s, and 72°C for 40 s. The maximum error of the system was found to be1.3 ± 0.7 °C at the extension isothermal step. This is likely dueto the fact that the droplet is heated through the glass surface,so error is generated in the feedback system. For the mosttemperature-dependent step, the annealing phase had anaverage error of 0.1 ± 0.4 °C, which is exceptionally low andbetter than most traditional thermal cyclers. The average cycletime is 117 ± 1.5 s, and the ramp rate for heating is 1.6 ± 0.2°C/s and for cooling is 1.8 ± 0.1 °C/s. The ramp rate oftraditional benchtop thermal cyclers is ∼2.0 °C/s. Although arepresentative five cycles is displayed, cycling was done out to40 cycles for each PCR droplet. For RT-PCR for H3 RNA, theaverage error for the reverse transcription step was 0.8 ± 0.5°C. The annealing step at 54 °C for the RT-PCR had a averageerror of 0.3 ± 0.5 °C. The measured temperature cycling isclear proof that the tablet platform can offer PCR cycling withramp rates and cycle times consistent with or better than thoseobserved for traditional bulky PCR thermal cyclers.

DNA Amplification Controls. DNA amplification wascarried out on the tablet platform with the cycling conditions

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previously described in a 2 μL droplet. These results werecompared to a control with the same PCR mix in aconventional benchtop thermal cycler. Both a full-volume (50μL) control and a droplet control were performed in thethermal cycler as described in Figure 2a. The droplet controlconsists of a droplet of the same experimental size covered in 4times as much mineral oil amplified in a standard PCR tube.The electropherograms for the tablet, droplet control, and full-volume samples are also shown in Figure 2a, which shows thatamplification occurred for both the ITO tablet platform and theoff-chip control for a DNA concentration of 0.01 ng/μL, allgenerating a similar yield with concentrations of 292.9, 221.4,and 251.5 nM for the full-volume, droplet control, and tabletsamples, respectively. The peak displays the 106 bp ampliconthat is also visible on the corresponding gel plot in Figure 2bfrom the Agilent 2100 bioanalyzer. Lanes 2, 3, and 4 displayamplification for the full-volume, droplet control, and tabletPCR reactions, respectively. This also confirms that the in-tubedroplet controls appropriately model amplification via thetablet platform. Negative controls were also performed for boththe tablet platform and in-tube, which show no amplification ofthe 106 bp segment. In Figure 2b, lanes 5−7 display thecorresponding negative controls for each platform. Thenegative control results are primers which have not beenused in amplification. Overall, the tablet platform performs aswell as off-chip thermal cycling with a fraction of the volume, ata faster speed.

Real-Time Tablet PCR. Real-time PCR was conducted byfluorescence acquisition during the extension step of the PCRreaction with SYBR Green I. The characteristic qPCRamplification curve is shown in Figure 2b along with a negativecontrol that displays no change in fluorescence, with minimalfluctuation in fluorescence of 0.006 (AU). The linear phase ofamplification occurs very early for the DNA startingconcentration of 0.01 ng/μL, with a calculated cycle threshold(Ct) value of 6.3 ± 1.0. The Ct value was found by determiningthe intersection where the fluorescence intensity crosses thecritical background signal. The critical background signal wasdetermined as 10 times the standard deviation of thebackground signal. Additionally, the slight decrease influorescence displayed at the plateau phase is attributed tophotobleaching of the SYBR Green dye. However, the generaltrend of the PCR is as expected, demonstrating that the ITOplatform is ideal for small-volume real-time PCR.

Sensitivity and Efficiency. Although polymerase adsorp-tion resulting in anemic cycles is an issue that must beaddressed for droplet PCR, sensitivity and efficiency ofamplification is not compromised on the droplet platform atappropriate polymerase concentrations. To determine thesensitivity of the tablet platform, serial dilutions of templateconcentrations were amplified via the tablet system. Figure 3adisplays the real-time data for serial dilutions of 2.0 × 105, 2.0 ×

Figure 2. (a) Electropherograms of tablet, droplet control, and full-volume PCR: time vs fluorescence plots. The peak near 38s representsthe 106bp amplicon of the lambda phage PCR. The smaller earlierpeak is the marker for the Agilent bioanalyzer DNA 1000 assay. It isevident that each sample was amplified sufficiently and within thesame amplification factor based on DNA production. Baselineflucutation in the droplet electropherogram is an artifact of theinstrument and represents a 2.6% baseline error. (Inset) Schematic ofthe droplet in oil control setup compared to a regular in-tube assay.The droplet control consists of a 2 μL droplet of PCR mix covered in4 times as much mineral oil. Both the full-volume regular assay and thedroplet control are then amplified by use of a traditional thermalcycler. (b) Real-time PCR of positive and negative controls: PCRamplification plot for 0.01 ng/μL with a Ct value of 6.7 cycles isdisplayed for on-chip amplification and the corresponding negativecontrol, with no significant change in fluorescence. (c) Gel plot of full

Figure 2. continued

volume, droplet control and tablet amplified samples: The gel plotinset displays the full-volume thermal cycler control with 0.01 ng/μLin lane 2; droplet and tablet samples in lanes 3 and 4, respectively; andfull-volume, droplet, and tablet negative controls in lanes 5−7. The106 bp amplicon is clearly visible in lanes 2−4, and the light band inlanes 5−7 represents unused primers. Concentrations of the ampliconin lanes 2, 3, and 4 are 292.9, 221.4, and 251.5 nM, respectively. Theshading in lane 4 is background fluctuation.

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104, 2.0 × 103, 2.0 × 102, 2.0 × 101, and finally 2.0 copies/μL.The critical background signal was calculated as describedpreviously, and corresponding Ct values were found to be 12.4,17.1, 20.0, 23.1, 26.2, and 28.8 for the series. Thus, it isapparent that single-molecule amplification is entirely possiblewith the droplet PCR system. Although our results are for shortmodel DNA, high-sensitivity results are also possible for longerfragments of clinically relevant samples as displayed with theH3 RT-PCR. By plotting the Ct values for each of the serialdilutions, the efficiency of the PCR can be found. Figure 3bdisplays the efficiency of the reaction, with a linear regression fitof 99%. The slope of the line is −3.22, indicating the tabletPCR is 104.4% efficient. The higher than 100% efficiencyindicates some error associated with creating dilutions,especially at low starting concentrations. We determined thelowest concentration to be subject to statistical fluctuationsdisplayed by a Ct of 28.8 ± 0.62. Utilizing a slope of −3.3 for100% efficiency, the efficiency was calculated as E = 10(−1/slope)

− 1. Typical and acceptable efficiencies are between 90% and

110%, demonstrating that the droplet system operates as well asconventional systems with very high sensitivity. Our systemperforms consistently if not better than other platforms, withthe obvious advantage of remarkably high efficiency at thelowest functional polymerase concentration. For a comparisonwith other droplet systems, please refer to Table S-1 in theSupporting Information.

Influenza H3 Reverse Transcription−PolymeraseChain Reaction. Clinically relevant concentrations ofinfluenza RNA are 104−106 copies/ml for patient samplesand therefore require highly sensitive assays for subtypingdetection. We designed a reverse-transcriptase PCR to amplifya segment of H3 RNA for detection of RNA via the dropletPCR platform. From a starting concentration of 107 copies/μL,RT-PCR was carried out in a 2 μL droplet, and the real-timeamplification plot is displayed in Figure 4. This template

concentration was chosen to ensure amplification, but anadditional experiment displays a sensitivity of 100 copies/μLRNA template in Figure 4. The results display an appropriateamplification curve with a Ct value of 15.1 for 107 copies/μLand 29 for 102 copies/μL. Our Ct values are similar to theamplification performed for H5 RNA with an amplicon of 114bp in a 500 nL droplet utilizing a similar droplet system.26 Thereported Ct value at a concentration of 2.4 × 107 copies/μLwas approximately 15, and Ct was approximately 33 for 2.4 ×102 copies/μL.

Role of Oil−Water Interface on PCR Reaction. Theeffect of droplet size was investigated to determine the effect ofsurface to volume ratio of the PCR reaction. There is a trade-offbetween the number of molecules of polymerase in the dropletand the amount of molecules that become adsorbed at thesurface of the oil/water interface. Protein adsorption at the oil/water interface is a well-known phenomenon as investigated byBeverung et al.32 It is therefore of interest to investigate theeffect of interfacial adsorption of Taq polymerase in dropletsfor PCR, which has only been briefly examined.27 Weinvestigated the effect of polymerase adsorption at the interfaceby examining various polymerase concentrations for threedifferent droplet sizes, 1, 2, and 4 μL. These experiments were

Figure 3. (a) Real-time PCR plots for 1/10 dilution series of template:Threshold is 10× standard deviation of background. The lowestconcentration amplified was 2.0 copies/μL, whose Ct was determinedto be 28.8 ± 0.62 for triplicate experiments. (b) Efficiency plot of real-time data: log concentration value vs Ct values calculated from Figure5. The data have a correlation of 99% with an intercept of 30.3 and aslope of −3.22, with a calculated efficiency of 104.4% from E =10(−1/slope)−1.

Figure 4. Real-time reverse transcription PCR for H3 influenza:amplification plot for 107 and 102 copies/μL full-length influenza H3RNA with corresponding Ct values of 15.1 and 29.

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done with the in-tube droplet controls, whose end-point DNAconcentrations match the tablet-amplified droplets well asdemonstrated in Figure 2a. The results displayed in Figure 5

display the final DNA concentration for each droplet at varyingpolymerase concentrations as measured on the Agilent 2100bioanalyzer after 40 cycles of PCR. The data display the well-known phenomenon of protein adsorption at the water/oilinterface. The only volume that displayed amplification for allthe polymerase concentrations examined, 0.01−0.05 unit/μL,was the full-volume 50 μL PCR controls. The effect ofinterfacial adsorption was pronounced for all the dropletsinvestigated, although significant differences between thedroplet volumes were not observed. The reason not muchdifference is observed between 1, 2, and 4 μL droplets is thatthe relevant controlling parameter is the surface to volumeratio, which is A/V ∼ 3/R for a spherical interface. Thus it isthe radius, not the volume, that contributes and this does notvary appreciably in the small volume changes considered. Therespective radii for 1, 2, and 4 μL droplets are 0.62, 0.78, and0.98 mm. We examined the data points by developing thefollowing mass balance equation to calculate Γ, the interfacialenzyme concentration:

= − ΓC V C V SB 0 (1)

where CB is the effective or bulk enzyme concentration, C0 isthe initial enzyme concentration, V is the volume of the droplet,and S is the surface area of the droplet. If a spherical dropletshape is assumed, the mass balance reduces to

Γ = −d C C( )/30 B (2)

where d is the diameter of the droplet.The loss of enzyme is assumed to occur early in the PCR

reaction, most likely during the initial denaturation phase. Thisis evidenced by utilizing the following expression32 to calculatethe time t for Taq polymerase to adsorb at the interface:

Γ = πt C Dt( ) 2 /B (3)

where D is the diffusion coefficient. When a typical proteindiffusion coefficient of 5 × 10−7 cm2/s is utilized, adsorption ofa monolayer occurs between 2.2 and 13.6 min for bulk enzymebetween 0.05 and 0.02 unit/μL. Protein adsorption at an oilinterface is attributed to a short induction time, when diffusionto the interface is important and the molecules irreversiblyadsorb to the interface due to exposure of hydrophobic residuescausing conformational changes.32 Equation 3 does not accountfor interfacial composition or hydrophobic interactionsassociated with an oil/water interface, so it is likely that thetime required for a monolayer to form is more than thepredicted values.To evaluate monolayer formation at the interface, the

thermodynamic variable Γ for polymerase was calculated tobe 0.0046 unit/mm2 or 3.65 × 108 molecules/mm2 from eq 2.Here, we have used the experimental data as shown in Figure 5to evaluate CB ≈ 0.0074 unit/μL with C0 ≈ 0.027, 0.026, and0.0215 unit/μL for the 1, 2, and 4 μL droplets, respectively.This was done by utilizing the bulk polymerase concentrationassociated with the same DNA production in the droplets aswas achieved for the full-volume controls. A fixed DNAproduction concentration of 150 nM was utilized forevaluation. To verify this calculation, we assumed that theenzyme creates a monolayer at the biphasic interface anddetermined that the area occupied per molecule for a 2 μLdroplet would be 2.74 × 10−9 mm2. In comparing this area permolecule to the area per molecule determined from thereported radius of gyration of native polymerase (38.3 Å), thearea occupied is significantly smaller, only 1.88 × 10−10 mm2.33

However, it is known from the literature that the enzyme wouldnot be in its native state but denatured at the interface withexposure of hydrophobic residues of the 832 amino acid chain,as it is accepted that proteins unfold at liquid/liquidinterfaces.32,34 Studies of proteins at the oil/water interfaceaccount for the fact that the protein likely adopts differentconformations. Specifically, at low concentrations, the protein/oil interaction can lead to further unfolding of the proteinstructure.34 We can calculate the denatured Rg for Taqpolymerase using the scaling law provided by Flory:35

= νR R Ng 0 (4)

where N is the number of residues, R0 is a constant related topersistence length, and ν is the scaling factor, which isdependent on solvent quality. If we utilize accepted values forR0 = 2.08 ± 0.19 Å and ν = 0.598 ± 0.029,36 we determine thatthe random coil radius of gyration for Taq polymerase is 116.0± 10.7 Å. The error for the radius of gyration and subsequentcalculations was done by standard propagation of errorformulas. For verification we utilized these values of R0 and νfor a smaller protein, phosphoglycerate kinase (PGK), whosenative Rg is reported as 23 Å and unfolded Rg as 78 Å.37 Usingthe length of PGK as 417 residues, we determined the unfoldedRg for PGK to be 76.7 ± 7.1 Å, which correlates with thereported value. Thus, using the calculated unfolded Rg = 116.0± 10.7 Å, the area/molecule for Taq polymerase in our systemis 1.69 × 10−9 ± 3.12 × 10−10 mm2, which provides a morereasonable approximation of the interfacial coverage of Taqpolymerase. It would then seem that the enzyme is at leastpartially unfolded at the interface in a denatured conformation,as would be expected. It should be noted, however, that thevalues for R0 and ν are from correlations of small-anglescattering studies of proteins, which provide approximate values

Figure 5. Polymerase investigation: final DNA concentration after 40cycles of PCR for droplet volumes of 1, 2, and 4 μL with enzymeconcentrations ranging from 0.01 to 0.05 unit/μL. Each experimentwas carried out in triplicate. The trends display a marked differencebetween full-volume (FV) reactions and droplet experiments.

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for evaluation. Additional studies provide insight into the factthat different denatured states of proteins will demonstratedifferent Rg values, up to 10−25% different for certainproteins.38 Additionally, it is of interest that, at the oil/waterinterface, a decrease in interfacial area occupied by the proteinwas observed and thought to occur due to extension of theprotein molecules and better solvency of the hydrophobicregions, resulting in greater extension into the oil phase.34 Thisobservation for β-casein may account for our result ofapproximately 61.7% ± 11.4% monolayer formation at thepolymerase concentrations utilized.These results indicate that, for droplet applications involving

enzymatic reactions like PCR, higher concentrations of enzymeare required. The typical full-volume PCR reaction utilizes0.02−0.025 unit/μL polymerase, whereas our results indicatethat, for a 1 μL droplet to get maximum amplification, 0.04−0.045 unit/μL is required, as displayed in Figure 5 and ingreater detail in Figure S-1 (Supporting Information). It isapparent that DNA production plateaus near 0.02 unit/μL forthe full-volume reactions, but this is not the case for thesignificantly smaller 1 μL volume. It is well-known that, at lowenzyme concentrations, PCR results in anemic cycles.39 Ananemic cycle refers to a cycle where Taq polymerase moleculesare exceeded by primed templates, resulting in templates thatdo not get amplified. It is thought that anemia begins aroundcycle 15 for most protocols, which we project can besignificantly lower for low enzyme droplets, resulting in eitherlittle or no amplification. For other droplet applications thatutilize nanoliter or even picoliter droplets, even greaterconcentrations of polymerase are required.Our results indicate that the majority of adsorption of Taq

polymerase likely occurs at the oil/water interface and not tothe Teflon surface. The experiments displayed in Figure 5 weredone without an adsorptive surface present, and our in-tubedroplet controls match our on-chip amplification results veryconsistently (Figure 2). While there is evidence that Taqpolymerase adsorbs to Teflon, the indicated results are forconcentrations far above the operating parameters for PCR (0.3mg/mL, or 24.0 units/μL) and the authors subsequently reportTeflon as an optimal surface for droplet-based PCR.31

Additionally, with the hydrophobic nature of Teflon, it is likelythat a thin layer of oil surrounds the disk-shaped droplet onboth surfaces, due to the higher affinity the surface holds for oilover water and the working volume of oil as 4 times that of thePCR droplet. This affinity is affirmed by the contact angles forwater and oil on Teflon surfaces, with water having a contactangle near 120° and oil near 60°.40 It has also been reportedthat, at the Teflon/oil/water/air interface, typically oil willdisplace water on the Teflon surface, resulting in waterdisplacement from the solid so that the resulting interfacialinteractions are limited to the oil/water interface.41 This wouldthen indicate that adsorption for our tablet-based PCR occursalmost exclusively at the oil/water interface. However, it shouldbe noted that adsorption to surfaces is a larger concern forchannel-based microfluidic PCR, due to the fact that eitherdroplets or a single phase is in greater contact with theadsorptive surface due to the high surface to volume ratio ofthese devices.Table S-1 (Supporting Information) displays a summary of

several droplet PCR platforms with their representativesensitivities, efficiencies, and polymerase concentrations asprovided. It is of note that most investigators are utilizingsignificantly higher enzyme concentrations than our optimized

results require. It is also of interest that for a 150 nL droplet, aconcentration of 0.175 unit/μL Taq polymerase was employedas optimal, which by utilizing our calculated Γ value and massbalance equation provides a bulk enzyme concentration greater(0.13 unit/μL) than required to accommodate loss at theinterface.27 This indicates that for several of these technologiesthere may be additional adsorption to microchip surfaces. Inorder to reduce the relatively large concentration of polymeraserequired to achieve an appropriate yield for microliter topicoliter droplets, a blocking protein such as bovine serumalbumin (BSA) can be added to the PCR master mix to occupyadsorption sites at the interface and reduce the loss ofpolymerase from the bulk.

■ CONCLUSIONSWe present a simple high-efficiency PCR tablet platformcapable of amplifying DNA with a starting concentration of 2.0copies/μL and full-length influenza RNA with 100 copies/μL.Additionally, we determined that loss of polymerase at thebiphasic interface in droplet-based techniques reducesamplification yield and sensitivity. These results highlight thepotential of this device to be useful for point-of-careapplications with the obvious requirement that the need for amicroscope be eliminated. We have additionally highlighted afundamental difference between droplet-based amplificationand batch reactions that can help future designs for PCRmicrodroplet devices account for polymerase loss and optimizethe amount required when utilizing microliter- to picoliter-sizeddroplets for PCR.

■ ASSOCIATED CONTENT

*S Supporting InformationOne table, with a summary of droplet PCR methods andrelevant parameters for comparison with our results, and onefigure, with graphs of full-volume and 1 μL polymeraseexperiments that correspond with Figure 5 for furtherclarification. This material is available free of charge via theInternet at http://pubs.acs.org.

■ AUTHOR INFORMATION

Corresponding Author*Email: anubhav_tripathi@brown.edu.

■ ACKNOWLEDGMENTSA.T. acknowledges the support of the National ScienceFoundation (CBET 0653835), and S.L.A. acknowledges thefinancial support of the Brown University Graduate Fellowshipand RISG NASA Graduate Fellowship for this research.

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