Time-Resolved Analysis of Biological Reactions Based onHeterogeneous Assays in Liquid Plugs of Nanoliter VolumeMartin Rendl, Thomas Brandstetter, and Jurgen Ruhe*

Laboratory for Chemistry and Physics of Interfaces, Department of Microsystems Engineering (IMTEK), University of Freiburg,Georges-Kohler-Allee 103, D-79110 Freiburg, Germany

*S Supporting Information

ABSTRACT: In this article, we present a concept which uses liquid plugs asreaction volumes for heterogeneous assay reactions to facilitate time-resolvedanalysis of biomolecular reactions. For this purpose, the reaction is firstcompartmentalized to a train of many identical plugs. Therefore, weestablished a simple fluidic setup build from off-the-shelf available tubing andconnectors. It permits reliable formation of plugs and successive dosing offurther assay reagents to these compartments (plug volume <5% CV). Thetime course of the reaction is obtained by routing the plugs successivelythrough a detector. Thereby, the arrival time of a given plug at the detector represents the reaction time of the overall reaction atthat moment. Thus, each analyzed plug represents a discrete state of the overall reaction. With this approach, we can achieve atemporal resolution as small as one second, which hardly can be met by conventional analytical methods for analysis ofendogenous biological compounds. For analysis of the content of the plugs, we developed a method which allows forheterogeneous assays in two-phase flow. For this purpose, functionalized superparamagnetic beads are enclosed in the plugs forspecific binding of the assay product. Purification from supernatant species is achieved by transferring the beads with boundanalyte across the phase boundary between aqueous plugs and water-immiscible carrier fluid. We demonstrate this assay principleexemplarily for a sandwich immunoassay (cytokine IL-8). Time-resolved analysis is validated by monitoring a cell-free in vitroexpression reaction (turboGFP) in plugs and conventionally in bulk solution. We show that our approach allows for analyzing theentire course of a reaction in a single run. It permits kinetic studies of biological processes with significantly reduced experimentaleffort and consumption of costly reagents.

The rise of novel disciplines in biology, such as systemsbiology where molecular interactions and regulatory

mechanisms of biological systems (e.g., of cell signalingpathways)1,2 are elucidated, has caused an increasing demandfor novel high-throughput analytical methods. These inves-tigations frequently target cellular regulatory mechanisms bydetermining protein expression levels and modifications, as wellas temporal changes thereof. However, conventionallyemployed techniques to monitor changes in concentration ofcertain proteins, such as Western Blotting or mass spectrosco-py, frequently do not meet these requirements. They involverather labor-intensive, time-consuming, and costly proceduresand equipment.1

At the same time, a broad spectrum of miniaturized systemsfor biomedical and bioanalytical applications have beendeveloped.1,3 Microfluidic systems are ideal candidates fordevices which permit time and cost efficient analysis due totypical dimensions at the micrometer scale and large surface-to-volume ratios.3−5 Advances in microfabrication allow forcomplex fluidic structures which foster high-throughput analysisof proteins. Measurements of enzyme kinetics have beenperformed in continuous flow microfluidic systems.6,7 Arrays offemtoliter chambers were used to measure activities of singleenzyme molecules.8 Systems with integrated fluid controlelements such as valves and pumps allow for assays whichrequire more complex fluid handling, such as immuno-

assays.9−13 Also “digital microfluidic” devices based on fluidicactuation via EWOD (electrowetting on dielectrics) have beenused for this type of assay.14

However, a drawback of many techniques is the rathersophisticated systems design which is required for typical assayworkflows (i.e., the successive handling of different liquids, forexample, for washing steps). Therefore, frequently valves10,15 orelectrodes14,16 need to be integrated into the microfluidic chip.Additionally, the throughput of most of these continuous flowapproaches is still rather limited.An alternative microfluidic approach with great potential for

high-throughput analysis is based on two-phase flow. Here,aqueous reagents are confined to small droplets or plugssurrounded by a water-immiscible continuous phase. Theprocess of formation of such compartments in microfluidicstructures (e.g., T-junctions) is well-established.4,17,18 Two-phase flow is not subject to Taylor dispersion18 and even at lowReynolds numbers, efficient means of mixing can beachieved.19,20 Different reagents can be transported and storedwithout cross contamination, which is important for analyticalapplications.21−24 A variety of different chemical and

Received: January 25, 2013Accepted: September 8, 2013

Article

pubs.acs.org/ac

© XXXX American Chemical Society A dx.doi.org/10.1021/ac401752j | Anal. Chem. XXXX, XXX, XXX−XXX

biochemical reactions based on two-phase systems have alreadybeen reported. These include protein expression,25,26 in vitrodirected evolution,27 and cell-based assays.28 A first approach totime-resolved analysis of biological reactions in plug-basedmicrofluidic systems has been described by Song et.al.29 Theyused the principle of compartmentalization of reactions tomeasure enzyme kinetic with millisecond resolution. Courtoiset al. monitored time-dependent in vitro expression of GFP(green fluorescent protein) in picoliter droplets.26 Furthermore,Mazutis et al. analyzed kinetics of an in vitro translated enzymeusing multistep microfluidic droplet processing.30

However, to our knowledge, the potential of two-phase flowfor bioanalytical tests like immunoassays has sparsely been usedso far. An interesting concept of a droplet-based immunoassayfor intracellular protein determination has recently beenpresented by Martino et al.31 Detection and quantification ofintracellular proteins obtained by on-chip lysis was achieved bylocal enrichment of the analyte via binding to beads withoutfurther purification. However, this concept is limited to single-stage immunoassays for the detection of fusion proteins. It is

not capable of detecting endogenous proteins.31 The assays thathave been reported to date for two-phase flow systems areessentially all homogeneous reactions (i.e., reactions where allreactants including the reaction product remain in the liquidphase). This type of assay requires that the assay product canbe visualized in situ (e.g., through a color change or afluorescence event). However, especially in the field ofbioanalysis, heterogeneous assays play a dominant role. Here,the assay product is bound to a solid phase. This allows forsimple purification, isolation, and labeling of the reactionproduct for downstream detection and analysis.In this contribution, we present a concept which allows one

to perform heterogeneous assays in two-phase flow. It aims attime-resolved analysis of biological processes. The concept isbased on parallel performing of many redundant experiments.This can be achieved easily in two-phase flow by compartmen-talization of the assay reactions to trains of plugs. The plugs aresuccessively routed through a detector, which effectively assignseach compartment a distinct reaction time. Thus, each plugrepresents a discrete temporal state of the overall reaction,

Figure 1. Schematic illustrations of (A) a typical conventional workflow of a heterogeneous assay, exemplified by a sandwich immunoassay. (B)Workflow of the corresponding plug-based assay. Reagents are added successively to the plugs. The reactions are performed on magneticmicroparticles. After completion of the assay, the reaction product is separated from supernatant species by a transfer of the bead-bound analyteacross the interface between the two phases. Simultaneously, detection of the thus purified analyte molecules is performed. (C) Process ofentrapping of beads in the magnetic separation setup. A larger amount of nonmodified beads is placed in the area where separation occurs forming abed of beads. Then, beads which were contained in the plugs are deposited in adjacent layers as plugs flow through the magnetic separation deviceone after another. (D) Image sequence showing the process of bead extraction at different time points.

Analytical Chemistry Article

dx.doi.org/10.1021/ac401752j | Anal. Chem. XXXX, XXX, XXX−XXXB

which allows for facile monitoring of temporal changes of atarget compound.For analysis of the content of the plugs, we developed a

method which allows one to perform heterogeneous assays fordetection of endogenous proteins in segmented flow. Super-paramagnetic beads are enclosed in the plugs for specificbinding of the analyte complexes. A strong magnetic fieldgradient is used to transfer the beads from the aqueous phaseinto the water-immiscible continuous, thus separating it fromthe supernatant species.As a first example, we follow this approach to carry out a

simple sandwich immunoassay (cytokine IL-8). The applic-ability for time-resolved analysis of biological processes isshown for protein synthesis of turboGFP, using a cell-free invitro expression reaction.

■ CONCEPTHeterogeneous Assays in Liquid Plugs. To allow

performing of heterogeneous immunoassays in a two-phaseflow, we developed a simplified assay workflow. Therefore, weadopt the fluid handling procedures of conventional assays tosegmented flow of plugs. In a first step, superparamagneticbeads functionalized with capture antibodies are confined toaqueous plugs, which are separated from each other by afluorocarbon liquid. These plugs represent discrete reactionvolumes analogous to wells of well plates used in conventionalimmunoassay systems (compare Figure 1, panels B and C).Subsequently, analyte is added to these plugs in a T-junction. Inthe same way, secondary antibodies, as well as labelingsubstances, are introduced into the reaction volumes. Thefluidic conditions necessary for reliable plug generation andplug merging processes have been described by Shestopalov etal.32 Key parameters are the viscosities of the respective liquids,the interfacial tension, and the flow rates.In conventional assays, several washing procedures are

applied after each reaction step to remove nonbound species(Figure 1A). To simplify the assay workflow, we reduced thewashing procedures to one final purification step. This step iscrucial for downstream detection, as it significantly influencesthe background signal. Bound-labeled immunocomplexes mustbe purified from nonbound marker compounds. To allow forsimple purification, the analyte complex is bound to a solidsubstrate. In many microfluidic systems, functionalized micro-particles are used for specific binding of the assay product.Isolation from free compounds is achieved by keeping thebeads stationary, while the supernatant solution is removed.This is frequently achieved by means of magnetophoresis [i.e.,the manipulation of magnetic beads (and bound analytes) viastrong permanent magnets].33,34 With the use of the samemethod, separation can be performed in two-phase systems.Therefore, the beads are present in the dispersed aqueousphase containing the reaction. Separation from nonboundspecies is achieved by transferring the beads across the interfacebetween the two phases into the continuous, water-immisciblephase. Similar concepts have already been presented for theextraction and purification of nucleic acids.35,36 In our setup, weuse a simple magnetic configuration for immobilization ofsuperparamagnetic particles. Two permanent magnets wereplaced at either sides of the tubing attracting each other.(Figure 1, panels B and C). They generate a strong magneticfield gradient in the direction of flow, which “traps” themagnetic particles in the channel section between the twomagnets. To guarantee effective extraction of the particles, first,

a larger amount of nonfunctionalized beads is flown in anddeposited in the separator (Figure 1C). These beads intensifythe gradient of the magnetic field and form an obstacle for thefunctionalized beads contained in the plugs.37,38 Thus, whenplugs flow through the area between the two magnets, thecontained beads accumulate successively behind that barrier.During this process, beads are deposited in the order of thearriving plugs (Figure 1C). The separation of beads fromaqueous plugs is shown in a series of pictures taken at differenttime points (Figure 1D).Intermediate washing as applied in conventional assays is

difficult to carry out in two-phase flow. It would requireunfavorably large amounts of beads in the plugs to facilitateextraction of the beads. This is due to the large surface energyof the interface between the two phases, which impedes anextraction at the microscale. Following the extraction,resuspension of beads into plugs containing the next reagentwould be required.Thus, to avoid intermediate washing steps, we chose reaction

conditions which drive the reaction closer to completion byusing slightly higher concentrations of detection antibodies andmarker compounds to compensate for competitive binding tononbound species. Practically all homogeneous assays followthe same strategy. A similar concept for heterogeneous assayshas been suggested in form of a no-wash assay protocol for aconventional bead-based assay.39

The reaction scheme of immunoassays requires successiveaddition of several different reactants. Thus, a dedicated fluidhandling is required (compare Figure 1). Therefore, a series ofT-junctions placed one after another could be used. In such aconfiguration, the first T-junction is used for generation ofplugs and each subsequent T-junction would be used foraddition of a further reactant solution. Thus, for addition ofn reagents, n T-junctions, and n + 1 syringe pumps would berequired. However, to reduce the complexity of the fluidicsetup, we used a setup, which is based on two independentlycontrolled liquid streams and which uses only one single T-junction. In this configuration, the same T-junction is used forthe initial generation of the plugs and for addition of furtherreagents. The corresponding procedure is illustrated in Figure2: After initial formation of the plugs, the train of plugs is drawnback into the horizontal inlet (compare Figure 2) until behindthe T-junction. In the meantime, solution containing the nextreagent is placed in the vertical (orthogonal) inlet of the T-junction. Then, this solution and the train of plugs are flowninto the T-junction, where the reagent solution adds to theplugs. This procedure is repeated for each reaction step with afurther reactant. With dependence on the number of plugs(typically ∼200), this process takes about 1 min per additionstep.

Time-Resolved Analysis. Two-phase flow is ideally suitedfor monitoring the time course of reactions. It allows forconfinement of reactions in space as well as in time. Therefore,the reaction of interest is allocated to many identicalcompartments (i.e., plugs or droplets). These compartmentsare ordered back-to-back in a microfluidic channel or tubingsimilarly to a pearl necklace (Figure 3). In this arrangement,each compartment has a unique distance to a particular point ofthe channel. Thus, when the plugs are flown through thechannel at a constant flow rate, this distance defines the timerequired for a given plug to reach this location.Hence, the reaction time available for the reagents in such a

compartment is determined by the distance between the

Analytical Chemistry Article

dx.doi.org/10.1021/ac401752j | Anal. Chem. XXXX, XXX, XXX−XXXC

location where the plugs are formed and the reagents arecombined and the point of detection along the channel. The

use of this time-distance relationship for time-resolved analysisof reactions has previously been demonstrated by Song etal.29,40

In our approach, the time ti available for a reaction in a plug iis essentially determined by the distance di of the plug relativeto the detector at the beginning of the experiment (t = 0). It isthe time, ti=di/v, the plug needs to reach the detector whenflown at a constant velocity (v). Therefore, the assay reaction isin a first step confined to a train of hundreds of identical plugs.This process takes about 1−2 min, which is short compared tothe total reaction time. Thus, reactions in all plugs are initiatedessentially in parallel. The time-course of the reaction isobtained by routing this train of plugs slowly through thedetector. During this process, plugs arrive at the detector at adifferent time ti (Figure 3B). There, the content of individualcompartments is analyzed. Thus, each plug represents a discretetemporal state S(ti) of the overall reaction [i.e., the amount ofreaction product generated during ti (Figure 3C)].Note that the time t = 0 corresponds to the time when the

first plug has passed through the detector. In the presentconfiguration, the reaction in this plug has already beenincubated for about 2 min when it arrives at this point. Thistime is required for transport of the first plug from the point itwas generated to the detector. However, this time is typicallyrather short compared to the total reaction time.The temporal resolution (Δt) of the measurement can be

varied by adjusting the flow rate and thus the frequency of plugspassing through the detector in a given time. The minimumtemporal resolution (Δtmin) under constant flow conditions isdetermined by the number of plugs (N) and the total reactiontime (ttotal) as Δtmin = (ttotal)/(N − 1).

■ EXPERIMENTAL SECTIONMaterials. Beads coated with streptavidin and anti-rabbit

IgGs were obtained from Chemicell, Germany. Beads withepoxy-modified surfaces (Dynabeads) were obtained fromInvitrogen GmbH, Germany. Rabbit anti-turboGFP antibodies(AB514) were obtained from Evrogen (Russia). Anti-TNFIgGs (ab21576) and corresponding Cy5-conjugated secondaryantibody (ab6563), as well as biotinylated anti-rabbit IgGs(ab6720), were obtained from Abcam, U.K. Streptavidin andanti-His antibodies, both labeled with Dy647, were purchasedfrom GE Healthcare and Qiagen, Germany, respectively. Forthe sandwich immunoassays, an ELISA kit specific for cytokineIL-8 (CHC1303 Human IL-8 CytoSet, Invitrogen GmbH,Germany) was used. Cell-free protein expression wasperformed with a one-step human coupled IVT (DNA) kitobtained from Thermo Scientific. It includes the template DNApCFE-GFP for cell-free turboGFP synthesis. PBS buffer (10mM phosphate-buffered saline) was obtained by Sigma-Aldrich.PBST refers to PBS buffer containing 0.1% (v/v) Tween 20.Western Blotting reagents such as precast gels (567-1094),sample buffer (161-0737), nitrocellulose membrane (162-0115), and running buffer (161-0732) were obtained fromBiorad, Germany. 3,3′,5,5′-Tetramethylbenzidine (TMB sub-strate solution, T0565) was obtained from Sigma-Aldrich.Streptavidin conjugated with HRP (horse radish peroxidase)was obtained from Invitrogen.

Functionalization of Beads with Capture Molecules.Chemicell beads were washed twice and diluted to aconcentration of 1 mg/mL. Antibodies were added in amountsexceeding the theoretical-binding capacity of the beads.Solutions were incubated for several hours (>5 h) at room

Figure 2. Schematic illustration of the fluidic protocol employed forgeneration of the plugs and subsequent addition of further reagents.Step (1): generation of plugs, both immiscible phases are flown in theT-junction. Step (2): flow into the vertical inlet is stopped, the train ofplugs is drawn back into the horizontal inlet until behind the T-junction. Step (3): flow in horizontal inlet is stopped; vertical inlet isfilled with solution containing the next reagent. Step (4): train of plugsand reagent solution are flown in T-junction where reagents is addedto the plugs. For successive addition of multiple reagents, steps 2 to 4are iterated.

Figure 3. Schematic illustration of the principle of the time−distancerelationship, which is used for time-resolved analysis. (A) Plugscontaining an identical reaction are arranged successively in thechannel. Thereby, each plug has a unique distance to the detector.This allows us to assign each compartment a certain reaction time, ifplugs flow through the detector shown in (B). Accordingly, each plugcorresponds to a discrete state of the overall reaction.

Analytical Chemistry Article

dx.doi.org/10.1021/ac401752j | Anal. Chem. XXXX, XXX, XXX−XXXD

temperature with gentle agitation, using a shaker to allow forbinding of the biomolecules to the beads. During this process,the sample was protected from light. Nonbound biomoleculeswere removed by three washes with PBS. Beads werereconstituted to a final concentration of 2 mg/mL with PBS.Conjugation of antibodies to Dynabeads was carried out

according to the manufacturer’s instruction. Antibodies areimmobilized to the microparticles through reaction of theiramine groups with epoxy groups at the surface of the beads.The amount of bound capture antibodies (assuming 100%binding efficiency) was 7 μg per 1 mg of beads. Beads werestored at 4 °C prior to usage. A more detailed description of theconjugation protocols can be found in the SupportingInformation.Fluidic setup. HPLC tee fittings (PEEK, 0.25 mm bore)

obtained from Macherey-Nagel GmbH (Germany) were usedas T-junctions for the generation of aqueous plugs. Pressure-driven flow of carrier and reactant fluids was provided bysyringe pumps (Cetoni GmbH, Germany). FC-43 (3M,Germany) was used as a water-immiscible carrier fluid. Forstabilization of the plugs and to prevent unspecific adsorptionof biomolecules to surfaces of the fluidic system, afluorosurfactant solution based on Zonyl FSO-100 (SigmaAldrich) was added as previously described by Roach et al.41

Transparent FEP (fluorinated ethylene propylene) tubing(1/16″, 0.25 mm i.d.) was obtained from LIQUID-scanGmbH & Co KG (Germany) and served as a channel forstorage and transport of plugs.Generation of Plugs/Addition of Further Reactants to

the Plugs. Plugs were generated at a flow rate of thefluorocarbon solution of 25 μL/min and 5 μL/min of solutioncontaining beads with bound capture antibodies.The flow rate of segmented flow during addition of the

reagents was 25 μL/min in all cases. The flow rates of thecontinuous solutions added through the T-junction werechosen to be values between 1 and 3 μL/min. Variation ofthis flow rate allows for the control of the added liquid volumeand thus the final concentration in the plug. After completionof the addition of all assay components, the flow rate wasadjusted to the temporal resolution and overall measurementtime required for the respective assay. In any case, themaximum value applied was 2 μL/min to allow for sensitivefluorescence detection. A list of the fluidic conditions used forthe different biological reactions can be found in the SupportingInformation.Isolation and Purification of Beads and Bead-Bound

Reactants. Extraction of beads and simultaneous removal ofsupernatant species was performed in a magnetic separationsetup. It consists of two NdFeB permanent magnets (5 × 5 × 5mm3, N50, 1.4−1.46 T, Webcraft GmbH, Switzerland) placedon either side of the channel/tubing with a spacing of 1.6 mmattracting each other, as shown schematically in Figure 1B.Detection and Image Analysis. Detection of fluorescence

signals was performed with a setup similar to an epi-fluorescence microscope consisting of a CCD camera (Guppy038B, Allied Vision Technologies, Germany), which has beenequipped with a Cy3/Cy5-specific dual band filterset and a c-mount lens (focal length: 25 mm, f/1.4). Image analysis wasperformed with the open-source software ImageJ (NationalInstitute of Mental Health, MD). Fluorescence intensities weremeasured as the average pixel value for a rectangular area ofmeasurement. For characterization of size and conformity of

the plugs, an epi-fluorescence microscope (Eclipse TS-100F,Nikon GmbH, Germany) was used.

■ WESTERN BLOTTING/IMMUNOSTAINING

Samples were analyzed with SDS−polyacrylamide gel electro-phoresis (SDS−PAGE) gels. Proteins were transferred tonitrocellulose membranes, blocked with blocking solution [5%bovine serum albumin (BSA), 0.5% milk powder in PBST]overnight at 4 °C. Incubation with the primary antibody (anti-turboGFP, 1:10000 diluted) was performed overnight at 4 °C.Blots were washed three times with PBST and incubated withsecondary antibodies (biotinylated anti-rabbit IgGs, 1:10000diluted) for 2 h. After 3 washes with PBST, blots wereincubated with streptavidin-HRP (1:1000 diluted) and washedthree times with PBST before detection. All incubationreactions were performed in the blocking solution.HRP was detected by adding 3 mL of TMB substrate

solution onto the membrane. After 10 min of incubation, themembrane was washed 3 times with ddH2O. Imaging of thestained membrane was performed with a flatbed scanner.Obtained images were analyzed with the software ImageJ. Thelocal background was subtracted.

■ RESULTS AND DISCUSSION

Heterogeneous Assay. To test our concept for heteroge-neous assays in segmented flow, we applied it to a sandwichimmunoassay. We chose the inflammatory marker IL-8(interleukin 8) as an example for a clinically relevant parameter.Therefore, we adopted the fluid handling procedures of theconventional assay to microsegmented flow. The process isillustrated in Figure 1B.In the first step, plugs containing beads with capture

antibodies against IL-8 are generated. The volume per plugwas 25 nL, and the concentration of beads was 1 mg/mL. Afterall the plugs have passed the T-junction, the plugs are drawnback behind the junction for addition of further reagent to thetrain of plugs (compare Figure 2). This process takes about 1min. In the second step, the analyte is added to this train ofplugs. Thus, a continuous solution containing IL-8 is flown inone inlet of the T-junction. There it adds to the train of plugsflown in via the perpendicular inlet. The resulting volume perplug was approximately 50 nL (step 2). Similarly, biotinylatedsecondary antibodies (2 μg/mL) are added in step 3, resultingin a volume of about 70 nL per plug. Finally (step 4), thesolution containing Dy647-conjugated streptavidin (10 μg/mL)was added to the plugs for fluorescent labeling of theimmunocomplexes. The volume after these three mergingsteps with different reagents was 90 nL, and the coefficient ofvariation was below 5%. After addition of all reagents, the plugswere flown through the magnetic separation setup to separatethe bead-bound immunocomplexes from supernatant, fluores-cent species (step 5). Simultaneously, fluorescence read-out ofthe labeled immunocomplexes was performed. This procedurewas performed for different concentrations of the IL-8 standard(10 ng/mL to 300 pg/mL). The fluorescence intensitiesobtained during passage of the plugs through the detector showa linear increase with the numbers of plugs flown through theseparation setup (Figure 4). This linear increase and the smallvariability of the signal indicate that each plug contains verysimilar amounts of bound fluorescently labeled analytes(integration of a constant amount of analyte over time).Thus, although different plugs have had different incubation

Analytical Chemistry Article

dx.doi.org/10.1021/ac401752j | Anal. Chem. XXXX, XXX, XXX−XXXE

times, rather identical results were obtained for each plug.Therefore, it can be concluded that owing to the smalldimensions and the good mixing of the plug-based assays, theanalytical reaction (i.e., the binding of the antibodies to theanalyte) has already reached an equilibrium state when the firstplugs arrive at the detector. This is important for the time-resolved experiments, as it allows one to perform the analyticalreaction in parallel to the reaction of interest. Moreover, thelinear increase demonstrates that the process of plug generationas well as the subsequent merging steps are sufficientlyreproducible, which is an important prerequisite for performingsuch assays. Furthermore, it can be observed that the slope ofthe fluorescence signals is proportional to the amount of

analyte contained in the plugs. This allows for quantification ofthe analyte concentration.Additionally, we performed a control experiment “off-chip”

to assess potential negative effects of the omission ofintermediate washing steps. Therefore, the identical assay wascarried out in a conventional well plate format withintermediate washing steps. The obtained results (Figure 4C)showed a slightly lower limit of detection (∼100 vs 300 pg/mL) and a lower background fluorescence (i.e., signal offset)compared to the plugs-based assay (Figure 4B) but were overallin good agreement. It should be emphasized that the goal ofthese experiments was to show the principle applicability of thisplug-based concept for heterogeneous assays rather thanspecific optimization for immunoassay applications (e.g.,lowering the limit of detection). Thus, for analysis of morecomplex biological samples such as whole blood, furtheroptimization of the method might be required. Note, that noadsorption of beads to the tubing was observed throughout theexperiments.

Time-Resolved Analysis. To test whether temporalchanges in concentration of target molecules can be measuredaccurately, we first performed a simple model study. We variedthe amount of analyte added to the plugs over time. Solutionwith fluorescently labeled analyte (Cy5-IgG, 1 μg/mL) wasadded in pulses to a train of plugs containing beads with thematching capture antibodies (Figure 5A).

The resulting signal (Figure 5B) shows a linear increaseduring periods when labeled species were added to the plugs.This is caused by the accumulation of beads and attachedfluorescent species in the magnetic separator during theexperiment. Accordingly, the fluorescence signal remainedconstant during times when no analyte was added. Thus, theobtained signal represents the integral over the amount ofanalyte added to the plug at a certain point in time.

Figure 4. Comparison of an immunoassay [performed in plugs (A &B) and “off-chip” in a microtiter-plate (C)]. A) Data measured duringthe assay. IL-8. Data points represent fluorescence intensities obtainedfor trains of plugs of different IL-8 concentrations (300 pg/mL to 10ng/mL). (B) End-point intensities measured after all plugs havepassed through the detector. (C) End-point intensities obtained forthe identical assay performed “off-chip” in a microtiter-plate withintermediate washing steps. Straight black lines represent linear fits ofthe data. Dashed lines correspond to the signal when no IL-8 waspresent (negative control).

Figure 5. Schematic illustration of the process of pulse-wise additionof analyte to the plugs. (B) Fluorescence intensities obtained duringpassage of a train of plugs that was subject to pulse-wise addition of thefluorescent analyte. All plugs contained beads with bound matchingcapture antibodies.

Analytical Chemistry Article

dx.doi.org/10.1021/ac401752j | Anal. Chem. XXXX, XXX, XXX−XXXF

Accordingly, the amount of analyte contained in a particularplug is obtained as the slope of the fluorescence signal at thisinstant. The close correlation between variations in the processof analyte addition and the fluorescence signals shows thattemporal changes of analyte concentrations can be monitoredusing this concept.Next, we extended our experiments to the analysis of

dynamic biological processes (i.e., reactions where theconcentration of a target compound is changing over time).As a demonstration example, we used our approach formonitoring the concentration of a synthesized protein during acell-free protein expression reaction. Therefore, we chose an invitro protein expression system, which allows expression offunctional proteins from protein-encoding plasmid DNA bycoupled transcription/translation reactions in a single step. Astarget protein turboGFP was chosen, a variant of the greenfluorescent protein (GFP) cloned from the copepod Pontellinaplumata.42 In its native conformation, turboGFP exhibits brightgreen fluorescence when excited with light in the blue range ofthe spectrum. This is referred to as “autofluorescence” in thefollowing. This property facilitates analysis of the synthesizedprotein and its functionality (appropriate folding). We used itto first test the compatibility of the reaction with the plug-basedsystem. Accordingly, similar reactions were performed in plugsas well as in bulk solution, and the fluorescence of bothreactions was recorded over time. The obtained results areshown in Figure 6. A developing fluorescence signal can bedetected after 60 to 90 min of reaction. Comparable resultswere reported by Courtois et al. for a similar reactionperformed in picoliter droplets.26 Overall, good qualitative

agreement between the bulk experiment and the correspondingreaction in plugs can be observed. This indicates that themicrosegmented flow is suitable for such relatively complexreactions. Unlike other reports, no hindering effects wereobserved.30

In the next step, we aimed to use our plug-basedimmunoassay concept for monitoring the concentration ofsynthesized target protein. Analogous to the previous experi-ments, beads functionalized with antibodies against turboGFPwere used for binding the target protein. However, for thisreaction we had to modify our assay protocol slightly. Weobserved that the efficiency of the IVT reaction is very sensitiveto dilution with other buffers. Therefore, we resuspended thefunctionalized beads with freshly prepared IVT solution “off-chip”. Additionally, Dy647-labeled detection antibodies againstthe Penta-His-tag of the synthesized target protein were added(effective dilution: 1:1250). The latter step is required fordetection of nonfluorescent endogenous proteins. The differentspectral characteristics of the chromophore of turboGFP andthe fluorophores of the secondary antibody allow for thediscrimination between the autofluorescence of turboGFP andthe immunoassay (labeled detection antibodies).This suspension was then compartmentalized and analyzed

“on-chip” following the procedure described before. When thetwo sets of data are compared, the data obtained by the plug-based immunoassay show a distinctively different kineticscompared to the autofluorescence intensities of turboGFP(Figure 6B). The signal originating from the secondaryantibodies shows an increase after t > 0.5 h and reaches aplateau for t > 2.5 h, whereas the signal originating from theautofluorescence of turboGFP does not increase until t > 1.5 hand continues to increase for t > 2.5 h. The delay in the lattersignal can be related to the maturation of the chromophore ofturboGFP. This relatively slow process involves complex foldingprocesses to yield the native conformation of turboGFP, uponwhich autofluorescence can be observed. The rate-limiting stepof this process is the oxidation of the translated turboGFPmolecules.42,43 However, the effect of maturation is notreflected by the results of the employed immunoassay. This iscaused by the anti-turboGFP antibodies, which already bind tofreshly translated, nonmatured turboGFP.To verify the results obtained by the immunoassay, we

performed a control experiment using Western Blotting. Anidentical cell-free expression reaction was performed in bulksolution and samples were taken after different reaction times.The obtained signal shows no presence of synthesized targetproteins for t < 0.5 h (Figure 7). This corresponds to the initialphase of the reaction, when plasmid DNA is transcribed intoRNA. During this phase of the coupled transcription−translation reaction, no complete protein has been synthesizedyet. For t > 0.5 h, an increase of the fluorescence signal can beobserved. It arises from proteins synthesized during thetranslation process from RNA generated in the upstreamtranscription process. For t > 2 h, the signal plateaus due todepletion of reactants such as amino acids and nucleosidetriphosphates. The results are in excellent agreement with thedata of the plug-based assay. This proves that the plug-basedsystem gives comparable results in a much simpler and fasterway compared to the tedious conventional approach.In the given experiment, the reaction was monitored over 3.5

h with a temporal resolution of 1 min. To obtain similartemporal resolution with a conventional microwell plateimmunoassay system, hundreds of wells must be processed.

Figure 6. (A) Comparison of the autofluorescence from synthesizedturboGFP molecules performed in bulk (◊) and plugs (■). (B)Comparison of autofluorescence (■) originating from maturedturboGFP molecules during cell-free protein synthesis and thefluorescence signal from the secondary antibodies used in the plug-based immunoassay (○).

Analytical Chemistry Article

dx.doi.org/10.1021/ac401752j | Anal. Chem. XXXX, XXX, XXX−XXXG

For efficient handling and automation, this would requirecomplex and expensive laboratory equipment. Anotheradvantage of miniaturization is a significantly shorter assaytime. This short assay time allows one to run the assay reactionsimultaneously to the reaction, which is to be analyzed. Thus,once the reaction in a plug is stopped, the outcome can bedetected instantaneously. Conventional workflows requiretypically more than 4 h until a result is obtained. Furthermore,the total volume of an assay in a single plug is less than 100 nL.This is 3 orders of magnitude smaller compared to theconventional reaction in a single well of a microwell plate.Another advantage of confined, miniaturized reactions is thatthe reaction product is released in a minute volume and thuscan result in an intrinsically higher concentration.44 Thisfacilitates detection of rare reaction events and low-abundancecompounds.

■ CONCLUSION & OUTLOOKIn this contribution, we have presented a concept which allowsus to perform heterogeneous assays in microsegmented flow.We used this approach to measure temporal changes in proteinconcentration. This was demonstrated for a protein which wassynthesized in a cell-free protein expression reaction. Weachieved comparable results to those performed conventionallyin microtiter-plates and analysis by Western Blotting. However,by transferring the assay reactions to the two-phase flow, wecould achieve much better temporal resolution with signifi-cantly reduced experimental effort and time. This greatlyfacilitates analysis of biomolecular processes under differentreaction conditions. It helps to cover larger parameter spaces, asonly limited numbers of serially performed experimental runsare required [e.g., for monitoring the course of a reaction under

different environmental conditions (temperature and pH)].The temporal resolution can be adapted to the reaction rate ofthe biological process by simply adjusting the flow rate of theplugs during detection. Thus, biological processes of durationsof few minutes to several hours can be investigated. In thepresent configuration, the minimum achievable temporalresolution is about 1 s.In future work, we aim to expand the capacity of our system

from the monitoring of a single biological parameter tomultiplexed detection of several parameters using barcodedbeads or differently labeled detection antibodies. Furthermore,we aim to extend our experiments toward cell-based assays.Cultivation of cells in plugs/droplets has already beenreported.28,45 Accordingly, living cells could be confined toplugs, and their temporal response (e.g., endocrine signaling:secretion of hormones) upon a given stimulus could beanalyzed. Here, minute reaction volumes are advantageous,since the compound of interest is secreted in a very smallvolume.44 This allows for simple and sensitive detection.

■ ASSOCIATED CONTENT

*S Supporting InformationAdditional information as noted in text. This material isavailable free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATION

Corresponding Author*E-mail: ruehe@imtek.de. http://www.imtek.de/cpi.

NotesThe authors declare no competing financial interest.

■ REFERENCES(1) Breslauer, D. N.; Lee, P. J.; Lee, L. P. Mol. BioSyst. 2006, 2 (2),97.(2) Ideker, T.; Galitski, T.; Hood, L. Annu. Rev. Genomics Hum. Genet.2001, 2, 343−72.(3) Ng, A. H. C.; Uddayasankar, U.; Wheeler, A. R. Anal. Bioanal.Chem. 2010, 397 (3), 991−1007.(4) Teh, S. Y.; Lin, R.; Hung, L. H.; Lee, A. P. Lab Chip 2008, 8 (2),198−220.(5) Dittrich, P. S.; Tachikawa, K.; Manz, A. Anal. Chem. 2006, 78(12), 3887−3907.(6) Seong, G. H.; Heo, J.; Crooks, R. M. Anal. Chem. 2003, 75 (13),3161−7.(7) Kerby, M. B.; Legge, R. S.; Tripathi, A. Anal. Chem. 2006, 78(24), 8273−80.(8) Rondelez, Y.; Tresset, G.; Tabata, K. V.; Arata, H.; Fujita, H.;Takeuchi, S.; Noji, H. Nat. Biotechnol. 2005, 23 (3), 361−365.(9) Hermann, M.; Veres, T.; Tabrizian, M. Anal. Chem. 2008, 80(13), 5160−5167.(10) Herrmann, M.; Roy, E.; Veres, T.; Tabrizian, M. Lab Chip 2007,7 (11), 1546−1552.(11) Hong, J. W.; Quake, S. R. Nat. Biotechnol. 2003, 21 (10), 1179−1183.(12) Kartalov, E.; Zhong, J.; Scherer, A.; Quake, S.; Taylor, C.;French Anderson, W. BioTechniques 2006, 40 (1), 85−90.(13) Hayes, M. A.; Polson, N. A.; Phayre, A. N.; Garcia, A. A. Anal.Chem. 2001, 73 (24), 5896−5902.(14) Sista, R. S.; Eckhardt, A. E.; Srinivasan, V.; Pollack, M. G.;Palanki, S.; Pamula, V. K. Lab Chip 2008, 8 (12), 2188−2196.(15) Diercks, A. H.; Ozinsky, A.; Hansen, C. L.; Spotts, J. M.;Rodriguez, D. J.; Aderem, A. Anal. Biochem. 2009, 386 (1), 30−35.(16) Lehmann, U.; Hadjidj, S.; Parashar, V. K.; Vandevyver, C.; Rida,A.; Gijs, M. A. M. Sens. Actuators, B 2006, 117 (2), 457−463.

Figure 7. (A) Comparison of signals obtained using the plug-basedsystem (○) and conventionally by Western Blotting (▲). In the caseof the plug-based immunoassay, each of the data points (○) representthe relative change in fluorescence intensity over time between 10successive plugs. (B) Western Blot of solutions containing turboGFPtaken from cell-free protein expression performed in bulk for differentreaction times (0 to 5 h). NTC = no template control.

Analytical Chemistry Article

dx.doi.org/10.1021/ac401752j | Anal. Chem. XXXX, XXX, XXX−XXXH

(17) Garstecki, P.; Fuerstman, M. J.; Stone, H. A.; Whitesides, G. M.Lab Chip 2006, 6 (5), 693−693.(18) Song, H.; Tice, J. D.; Ismagilov, R. F. Angew. Chem., Int. Ed.2003, 42 (7), 768−772.(19) Song, H.; Ismagilov, R. F. J. Am. Chem. Soc. 2003, 125 (47),14613−14619.(20) Bringer, M. R.; Gerdts, C. J.; Song, H.; Tice, J. D.; Ismagilov, R.F. Philos. Trans. R. Soc., A 2004, 362 (1818), 1087−1104.(21) Adamson, D. N.; Mustafi, D.; Zhang, J. X. J.; Zheng, B.;Ismagilov, R. F. Lab Chip 2006, 6 (9), 1178−1186.(22) Song, H.; Chen, D. L.; Ismagilov, R. F. Angew. Chem., Int. Ed.2006, 45 (44), 7336−7356.(23) Zheng, B.; Tice, J. D.; Ismagilov, R. F. Anal. Chem. 2004, 76(17), 4977−4982.(24) Zheng, B.; Ismagilov, R. F. Angew. Chem., Int. Ed. 2005, 44 (17),2520−2523.(25) Dittrich, P. S.; Jahnz, M.; Schwille, P.; New, A. ChemBioChem2005, 6 (5), 811−814.(26) Courtois, F.; Olguin, L. F.; Whyte, G.; Bratton, D.; Huck, W. T.S.; Abell, C.; Hollfelder, F. ChemBioChem 2008, 9 (3), 439−446.(27) Fallah-Araghi, A.; Baret, J.-C.; Ryckelynck, M.; Griffiths, A. D.Lab Chip 2012, 12 (5), 882.(28) Funfak, A.; Cao, J.; Wolfbeis, O. S.; Martin, K.; Kohler, J. M.Microchim. Acta 2008, 164 (3−4), 279−286.(29) Song, H.; Ismagilov, R. F. J. Am. Chem. Soc. 2003, 125 (47),14613−9.(30) Mazutis, L.; Baret, J.-C.; Treacy, P.; Skhiri, Y.; Araghi, A. F.;Ryckelynck, M.; Taly, V.; Griffiths, A. D. Lab Chip 2009, 9 (20), 2902.(31) Martino, C.; Zagnoni, M.; Sandison, M. E.; Chanasakulniyom,M.; Pitt, A. R.; Cooper, J. M. Anal. Chem. 2011, 83 (13), 5361−5368.(32) Shestopalov, I.; Tice, J. D.; Ismagilov, R. F. Lab Chip 2004, 4(4), 316−321.(33) Peyman, S. A.; Iles, A.; Pamme, N. Lab Chip 2009, 9 (21),3110−3117.(34) Pamme, N. Lab Chip 2007, 7 (12), 1644−1659.(35) Beebe, D. J.; Berry, S. M.; Alarid, E. T. Lab Chip 2011, 11 (10),1747−1753.(36) Shikida, M.; Takayanagi, K.; Inouchi, K.; Honda, H.; Sato, K.Sens. Actuators, B 2006, 113 (1), 563−569.(37) Pamme, N. Lab Chip 2006, 6 (1), 24−38.(38) Descroix, S.; Teste, B.; Malloggi, F.; Gassner, A. L.; Georgelin,T.; Siaugue, J. M.; Varenne, A.; Girault, H. Lab Chip 2011, 11 (5),833−840.(39) Luminex_Corporation Sample protocol for no wash capturesandwich immunoassay. http://www.luminexcorp.com/prod/groups/public/documents/lmnxcorp/no-wash-capture-sandwich-immun.pdf(accessed April 24, 2013).(40) Bringer, M. R.; Gerdts, C. J.; Song, H.; Tice, J. D.; Ismagilov, R.F. Philos. Trans. R. Soc., A 2004, 362 (1818), 1087−1104.(41) Roach, L. S.; Song, H.; Ismagilov, R. F. Anal. Chem. 2005, 77(3), 785−796.(42) Evdokimov, A. G.; Pokross, M. E.; Egorov, N. S.; Zaraisky, A.G.; Yampolsky, I. V.; Merzlyak, E. M.; Shkoporov, A. N.; Sander, I.;Lukyanov, K. A.; Chudakov, D. M. EMBO Rep. 2006, 7 (10), 1006−1012.(43) Li, B.; Shahid, R.; Peshkepija, P.; Zimmer, M. Chem. Phys. 2012,392 (1), 143−148.(44) Boedicker, J. Q.; Li, L.; Kline, T. R.; Ismagilov, R. F. Lab Chip2008, 8 (8), 1265.(45) He, M.; Edgar, J. S.; Jeffries, G. D.; Lorenz, R. M.; Shelby, J. P.;Chiu, D. T. Anal. Chem. 2005, 77 (6), 1539−44.

Analytical Chemistry Article

dx.doi.org/10.1021/ac401752j | Anal. Chem. XXXX, XXX, XXX−XXXI