Microfluidic bead-based sensing platform for monitoringkinase activity

Seung Hwan Lee a, Hyun-Woo Rhee b, Danny van Noort c, Hong Jai Lee a, Hee Ho Park a,Ik-Soo Shin d, Jong-In Hong e,n, Tai Hyun Park a,f,nn

a School of Chemical and Biological Engineering, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-744, Republic of Koreab School of Nano-Bioscience and Chemical Engineering, Ulsan National Institute of Science and Technology, 100 Banyeon-ri, Ulju-gun, Ulsan 689-798,Republic of Koreac Department of Chemistry and Nano Sciences, Ewha Womans University, 11-1 Daehyun-dong, Seodaemun-gu, Seoul 120-750, Republic of Koread Department of Chemistry, Soongsil University, 369 Sangdo-ro, Dongjak-gu, Seoul 156-743, Republic of Koreae Department of Chemistry, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-747, Republic of Koreaf Advanced Institutes of Convergence Technology, Suwon, Gyeonggi-do 443-270, Republic of Korea

a r t i c l e i n f o

Article history:Received 14 November 2013Received in revised form18 January 2014Accepted 20 January 2014Available online 31 January 2014

Keywords:Protein kinase activityChemosensorKinase assayMicrofluidic device

a b s t r a c t

Protein kinases control cellular functions by regulating protein phosphorylation. Monitoring proteinkinase activity is essential for medical diagnosis and drug screening. Here, we present a novelmicrofluidic device for performing simple and versatile protein kinase assays, which utilizes amicrobead-based chemosensor. An automatic mix-and-measure technique was achieved using inte-grated pneumatic valves. After mixing each reagent for the kinase assay, the mixture was transferred tothe sensing chamber. Then, phosphorylated and fluorescence-labeled peptides were captured anddetected by the chemosensor. A fluorescence signal was observed depending on the presence of thekinase. Furthermore, activities of various kinases in the cell lysate and the inhibitory effect of specificchemicals on the kinases were monitored. These results indicate that chemosensor-based microfluidicchips can be developed as a versatile kinase assay system.

& 2014 Elsevier B.V. All rights reserved.

1. Introduction

Since protein kinases were first identified in the late 1930s, morethan 500 protein kinases have been discovered from the humankinome (Cohen, 2002a; Manning et al., 2002). Protein kinasescontrol protein phosphorylation, a form of post-translational mod-ification, which plays a crucial role in various cellular functions(Schweppe et al., 2003). Abnormal regulation of protein phosphor-ylation has been associated with many diseases including cancer(Blume-Jensen and Hunter, 2001). Thus, monitoring protein phos-phorylation is important for medical diagnosis and drug screening(Cohen, 2002b). For this purpose, diverse methods have beendeveloped and used for detecting protein kinase activity andscreening of inhibitors of specific kinases. Traditionally, gel-basedassays, filter-binding assays, or enzyme-linked immunosorbentassays (ELISAs) have been used for such purposes (Chen et al.,2004; von Ahsen and Bömer, 2005). However, these assays have

some disadvantages such as being time-consuming procedures andlabor-intensive washing steps.

As the need for screening inhibitors for various protein kinasesincreases, traditional assay methods have been replaced by simplehomogeneous mix-and-measure techniques (von Ahsen and Bömer,2005) including the scintillation proximity assay (SPA) (Glickman et al.,2008; Park et al., 1999; Sills et al., 2002; Wu, 2002), fluorescenceresonance energy transfer (FRET) (Park et al., 1999; Wang et al., 2006;Wu, 2002; Zhang and Allen, 2007), and fluorescence polarization (FP)(Beasley et al., 2003; Fowler et al., 2002; Seethala and Menzel, 1997;Sharlow et al., 2008; Sportsman et al., 2004; Turek-Etienne et al.,2004). From an economic point of view, sample volume is one of thecritical factors. To reduce the cost of drug screening, the number ofwells of plate has been increased to reduce the sample volumes (vonAhsen and Bömer, 2005). To keep pacewith these trends, microfluidicshave recently emerged as a promising platform for medical diagnosisand anti-cancer drug screening (Lee et al., 2012a; Wlodkowic andDarzynkiewicz, 2010; Baac et al., 2004). Because this technology onlyrequires nanoliter volumes of reagents, it has the advantages of lowsample consumption and low reagent costs (Hong and Quake, 2003;Squires and Quake, 2005). Furthermore, automation can be achievedusing computer-controlled valves and pumps in the fluidic network(Lee et al., 2012b; Melin and Quake, 2007).

Contents lists available at ScienceDirect

journal homepage: www.elsevier.com/locate/bios

Biosensors and Bioelectronics

0956-5663/$ - see front matter & 2014 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.bios.2014.01.039

n Corresponding author. Tel.: þ82 2 880 6682; fax: þ82 2 889 1568.nn Corresponding author at: School of Chemical and Biological Engineering,

Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-744, Republic ofKorea. Tel.: þ82 2 880 8020; fax: þ82 2 875 9348.

E-mail addresses: jihong@snu.ac.kr (J.-I. Hong), thpark@snu.ac.kr (T.H. Park).

Biosensors and Bioelectronics 57 (2014) 1–9

Recently, several groups have proposed the use of microfluidicdevices for kinase activity assays, and radiometric system andhydrogel-based kinase assay platforms have been developed (Fanget al., 2010; Lee et al., 2012a). However, as these systems useantibodies to detect kinase activity, their versatility and economicfeasibility is insufficient. A pre-concentration (electrokinetic trap-ping) technique has been reported to increase sensitivity (Leeet al., 2009a). Although this system shows high sensitivity, itrequires complicated fabrication steps and additional componentssuch as nanochannels, Nafion film, and electronic systems. Toapply a simple mix-and-measure method on a chip, we previouslyreported a kinase detection method based on the use of selectivefluorescence quencher probes for phosphorylated fluorescentpeptides (Rhee et al., 2010). However, that detection method wasbased on a turn-off system rather than a turn-on system. A turn-on sensor system is more preferable because external factors caninterfere with turn-off systems (Du et al., 2012; Lu et al., 2011;Neupane et al., 2013; Pourghaz et al., 2011).

In this study, we report a novel microfluidic system to monitorkinase activity using a bead-based turn-on chemosensor. Themicrofluidic chip was designed for a kinase assay using polydi-methylsiloxane (PDMS)-based pneumatic valves to control fluidflow with a chemosensor immobilized on the microbeads toachieve a versatile kinase activity assay. Using this system, thekinase reaction, transfer of the reaction mixture to the chemo-sensor-bead chamber, and detection of the fluorescence signalwere performed sequentially and automatically with nanolitervolumes of reagents.

2. Materials and methods

2.1. Preparation of reagents

All fluorescence-labeled peptide substrates (Table S1 in theSupporting information) for the kinase reaction were synthesizedby Anaspec (Fremont, CA, USA). Stock peptide solutions at aconcentration of 1 mM were prepared using distilled water andstored at -20 1C. All protein kinases, alkaline phosphatase, and10 mM ATP were purchased from New England Biolabs (Ipswich,MA, USA) and stored at �20 1C or �70 1C. Dimethyl sulfoxide(DMSO), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC),zinc perchlorate hexahydrate, H-89 (N-[2-(p-bromocinnamyla-mino)ethyl]-5-isoquinoline-sulfonamide), HEPES, sucrose, pro-tease inhibitor cocktail, and phosphatase inhibitor cocktail werepurchased from Sigma-Aldrich (St. Louis, MO, USA).

2.2. Synthesis of chemosensor beads

Bis(Zn2þ-dipicolylamine) complex (DPA) was used as thechemosensor. DPA is a synthetic receptor that can bind tophosphate groups strongly and selectively in solution (Rheeet al., 2007, 2008, 2009, 2010). DPA was prepared according to aprocedure reported previously (Kwon et al., 2008) and dissolved inDMSO to obtain a 10 mM stock solution. Then, 1 ml of carboxy-lated 20 μm beads (Polyscience, Warrington, PA, USA) was cen-trifuged for 1 min at 10,000 rpm and washed twice with distilledwater. EDC (30 mg) and DPA (10 μl) were added and stirredovernight. The resulting mixture was centrifuged for 5 min at13,000 rpm and washed twice with distilled water. Zinc perchlo-rate hexahydrate (final concentration, 1 mM) was added, and thereaction mixture was stirred at room temperature for 1 h. Thestructure of the DPA beads is shown Fig. 1.

2.3. Cell culture and preparation of cell lysate

HeLa cells were cultured in Dulbecco's Modified Eagle’s Med-ium (Hyclone, Logan, UT, USA) with 10% fetal bovine serum (Gibco,Grand Island, NY, USA). The cells were incubated at 37 1C under a5% CO2 atmosphere. The cultured cells were collected by trypsin/EDTA (Gibco) digestion and centrifuged for 5 min at 1200 rpm.After the supernatant was removed, the collected cells werewashed three times with dPBS (Welgene, Daegu, Korea) solution.The cells were lysed by sonication for 20 s in buffer (10 mMHEPES, 250 mM sucrose, protease inhibitor cocktail, and phospha-tase inhibitor cocktail). Then, the samples were centrifuged at12,000 rpm at 4 1C for 10 min. The supernatant was used forphosphorylation of the substrate peptides.

2.4. Microfluidic chip design and fabrication

Fig. 2a shows the design of the microfluidic chip that was usedto monitor kinase activity. This microfluidic device consisted oftwo layers of PDMS (Sylgard 184, Dow-Corning, MI, USA) and wasfabricated using multilayer soft lithography (Lee et al., 2009b;Unger et al., 2000). The top pneumatic layer contained 14 micro-channels (50 μm wide, 12.5 μm deep) as pneumatic valves. Thebottom fluidic layer had a fluidic channel (100–200 μm wide,10 μm deep) and a bead-loading channel (200 μm wide, 25 μmdeep). The chamber for the beads had a depth of 25 μm, whereasthe barrier region had a depth of 10 μm. The barrier was placed atthe end of the chamber to keep the 20 μm beads retained in thechamber, and the valve in the bead-loading channels preventedthe beads from flowing out of the reactor when closed.

The microfluidic chip consisted of a mixing part containing aperistaltic pump and a sensing part containing chemosensor beads(Fig. 2a, b). The mixing part was comprised of an elliptical channel(chamber 1) and two additional channels (chambers 2 and 3). Theelliptical channel was segmented by a pneumatic valve (valve S1)to allow solutions to be introduced through the inlet and outletchannel, which was controlled by valves (valves I1 and O1). Thetwo additional channels and the elliptical channel were segmen-ted by valves (valves S2 and S3). To function as a chamber, eachadditional channel had an inlet and outlet, which were controlledby pneumatic twin valves (valves T1 and T2). Therefore, theelliptical channel and two additional channels could be used aschambers (chambers 1, 2, and 3) for loading different solutions(Fig. 2b). The peristaltic valves (valves P1, P2, P3, P4, and P5) weredesigned to mix the solutions in the chambers. In the sensing part,the chamber for the chemosensor beads (chamber 4) had a loadingchannel that was controlled by a valve (valve B1) (Fig. 2b). Themixing part and sensing part were connected by a channel to

Fig. 1. Structure of the bis(Zn2þ-dipicolylamine) (DPA) beads.

S.H. Lee et al. / Biosensors and Bioelectronics 57 (2014) 1–92

transfer the mixed solution to the sensing part. The transfer wascontrolled by valves (valves I2 and O2).

2.5. Kinase activity assay using the microfluidic chip

Five steps were performed sequentially on a single chip:(1) reagent (protein kinase or cell lysate, ATP, and fluorescence-labeled peptide) loading; (2) reagent mixing; (3) DPA beadloading; (4) capture of the phosphorylated peptide by the DPAbeads; and (5) washing with distilled water. Chamber 1 (theelliptical channel) was used to load the protein kinase (or celllysate) and kinase buffer (Fig. 2b). After the segmentation valves(valves S1, S2, and S3) and transfer valves (I2 and O2) wereclosed, kinase buffer containing 1 unit/μl of protein kinase wasinjected. Then, 2 mM ATP and 1 mM fluorescence-labeled peptidewere introduced into chambers 2 and 3 by opening twin valves(valves T1 and T2), respectively (Fig. 2b). Then, all inlet and outletcontrol valves were closed to mix the solution. After the S1, S2,and S3 segmentation valves were opened, each solution was

mixed by the peristaltic pump for 1 h for the kinase reaction(Fig. 2c). DPA beads were loaded into chamber 4 by opening valveB1. The mixed solution was transferred to the bead-loadedchamber (chamber 4) to capture the phosphorylated peptides(Fig. 2d) by injecting distilled water through the inlet channel,which was controlled by valves I2 and O2. Distilled water wasinjected continuously for 30 s to remove non-specifically boundpeptide. The fluorescence signal of DPA beads was monitoredusing fluorescent stereo microscope (SV-6, Zeiss Optics, Jena,Germany) equipped with a Peltier cooled CCD camera. Thefluorescence intensity was quantified using the ImageJ software.For the repeatable use of this system, the DPA beads were washedwith 1 mM N,N,N0,N0-tetrakis(2-picolyl)ethylenediamine (TPEN)for 5 min to remove the captured peptides and zinc ions. Then,100 mM zinc perchlorate hexahydrate solution was injected intothe bead chamber for 30 min. Through this process, the zinc-removed beads were recovered to become zinc-containing beads.Using this method, the DPA beads could be used repeatedly atleast 5 times.

Fig. 2. (a) Schematic diagram of the microfluidic chip for monitoring kinase activity. The microfluidic chip has two layers. The top layer (red) has pneumatic valves (12.5 μmdeep). The bottom layer (green) is a fluidic layer (10 μm deep). The chamber for the beads (blue) is in the fluidic layer (25 μm deep). The flow control valves for the ellipticalchannel are called I1, inlet 1 and O1, outlet 1. The valves for segmentation are numbered S1, S2, and S3. The valves for peristaltic mixing were termed P1 to P5. The twinvalves for controlling the inlet and outlet of the additional chamber are called T1 and T2. The valve for bead-loading is called B1. The valves for controlling injection ofdistilled water are called I2 and O2. (b) The chip is separated into a mixing part and a sensing part. The mixing part has an elliptical channel (chamber 1) and two additionalchannels (chambers 2 and 3), which are segmented by pneumatic valves. The orange color represents the protein kinase. The cyan and purple colors represent ATP and thefluorescence-labeled peptide, respectively. The sensing part is the bead-loading chamber and is called chamber 4. (c) The kinase reaction occurs in the mixing part; when thesolutions of chambers 1, 2, and 3 are mixed. Phosphate is transferred to the fluorescence-labeled peptide substrate by the kinase. (d) Substrate capture occurs in the sensingpart: the peptide phosphorylated by the kinase in the mixing part is captured by bis(Zn2þ-dipicolylamine) (DPA) beads in the sensing part (chamber 4). Non-phosphorylatedpeptides are washed away with distilled water. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

S.H. Lee et al. / Biosensors and Bioelectronics 57 (2014) 1–9 3

3. Results and discussion

3.1. Device operation for kinase activity monitoring

Food dyes of different colors were used to check the operationof the microfluidic chip. Mixing of reagents loaded in the cham-bers and transfer of the mixed solution to the bead chamber wereexamined. First, to verify the mixing of solutions from chambers 1,2, and 3, all segmentation valves (valves S1, S2, and S3) and valvesI2 and O2 were closed. Then, food dyes (red, blue, and orange)were introduced into each chamber by opening valves T1, T2, I1,and O1 (Fig. S1a). Then, these valves were closed again. Afteropening the segmentation valves, the samples were mixed usingthe peristaltic valves (P1–P5). As shown in Fig. S1b, the food dyeswere completely mixed and their color changed in each chamber.Next, we monitored transfer of the mixed solution. After the20 μm beads were loaded into chamber 4 by controlling valveB1, the I2 and O2 valves were opened, and distilled water wasinjected. All of the mixed solution in chambers 1, 2, and 3 wastransferred to the bead-loaded chamber (chamber 4), and cham-bers 1, 2, and 3 were filled with water (Fig. S1c). Fig. S1d shows theflow of the mixed solution through the loaded beads in chamber 4.As time progressed, the mixed solution flowed out through theoutlet channel, and water was used to wash the beads. Movies S1and S2 in the Supporting information show the mixing, transfer,and washing processes in the mixing and sensing parts, respec-tively, of the designed chip. Therefore, the operation of designedchip was demonstrated to be ideally suited for the protein kinasereaction and sensing of phosphorylated peptide, which wasgenerated by the protein kinase.

Supporting information related to this article can be foundonline at http://dx.doi.org/10.1016/j.bios.2014.01.039.

3.2. Detection of phosphorylated peptides

Phosphorylated and non-phosphorylated kemptide, which aresubstrates of cAMP-dependent protein kinase (PKA), were pre-pared to evaluate specificity of the DPA beads (Table S1 in theSupporting information). Various concentrations of phosphory-lated and non-phosphorylated kemptide were loaded into cham-ber 3 in different chips, respectively. Chambers 1 and 2 were filledwith distilled water. After mixing, transfer of the solution to thebead chamber, and washing, the fluorescence signal of the beadchamber was observed. Fig. S2 shows a comparison of thefluorescence signal of DPA beads between phosphorylated andnon-phosphorylated kemptide at various concentrations (1, 10,and 100 μM). When non-phosphorylated and phosphorylatedkemptide were used at a concentration of 1 μM, the change inthe fluorescence signal was somewhat weak, but a difference wasobserved (Fig. S2a, b). A clear difference in the fluorescence signalbetween phosphorylated and non-phosphorylated kemptide wasobserved when 10 μM kemptide was passed through the DPAbeads (Fig. S2c, d). A strong fluorescence signal was detected whena concentration of 100 μM was used for phosphorylated kemptide,and the difference from the signal of non-phosphorylated kemp-tide was distinct (Fig. S2e, f). These results demonstrate that theDPA beads on the chip distinguished between phosphorylated andnon-phosphorylated peptides using a fluorescence signal.

3.3. Monitoring of peptide phosphorylation and de-phosphorylationusing purified enzymes

We prepared various combinations of samples to verify thefeasibility of a kinase activity assay using our DPA bead-basedmicrofluidic system. In each case, protein kinase buffer containing1 unit/μl of enzyme, 2 mM ATP (final concentration, 200 μM), and

1 mM fluorescence-labeled peptide (final concentration, 100 μM)were injected into each chamber. After the reaction, transfer, andwashing steps, the fluorescence signal of the DPA beads wasobserved. A fluorescence signal was not detected when cAMP-dependent protein kinase A (PKA) was reacted with abltide, whichis a target substrate of Abl protein tyrosine kinase (Abl) (Fig. 3a,Table S1 in the Supporting information). Next, PKA was mixedwith autocamtide-2, which is a target substrate of Ca2þ/calmodu-lin-dependent protein kinase II (CaMKII). Again, a fluorescencesignal was not detected (Fig. 3b, Table S1 in the Supportinginformation). When kemptide was subjected to a reaction withoutPKA, a fluorescence signal was not detected (Fig. 3c). However, asshown Fig. 3d, a strong fluorescence signal was observed whenPKA was reacted with kemptide. Moreover, the phosphorylationreaction was performed and the fluorescence signal was moni-tored for different kinase and substrate pairs, which were CaMKIIwith autocamtide and Abl kinase with abltide (Fig. S3, Table S1 inthe Supporting information). We also tested the phosphorylationreaction in our system using alkaline phosphatase (ALP), whichhas nonspecific dephosphorylation activity (Narayan et al., 2011;Rhee et al., 2010). ALP was injected into chamber 1 and phos-phorylated kemptide was injected into chamber 3. After thereagent was mixed, the reacted solution was transferred to thebead chamber following washing to remove non-specificallybound peptides. When ALP was not included in the reaction, thephosphorylated kemptide was captured by the DPA beads, and astrong fluorescence signal was detected (Fig. 3e). However, afterthe phosphorylated kemptide was treated with ALP, most of thepeptides were dephosphorylated. As a result, no fluorescencesignal was detected (Fig. 3f). Based on these results, the DPAbead-based microfluidic chip can be effectively used for detectingkinase activity. Furthermore, dose-dependent time-course assaywas conducted, in which different concentrations of PKA (1, 5 and10 units/μl) and kemptide were mixed and the fluorescence signalwas monitored (Fig. 3g). The peptide phosphorylation rateincreased with the PKA concentrations. The fluorescence signalsof the DPA beads were significantly different depending on eachkinase dose until 10 min. However, all the fluorescence signalseventually reached a plateau regardless of the PKA dose. Theseresults show that 10 min incubation is appropriate for the quanti-tative analysis in this reaction condition. In our system, both ATPand phosphorylated peptides can bind to DPA beads, and theirbinding depends on their ratio. In order to measure the phos-phorylated peptides, excess amount of ATP was used. When highconcentration of ATP is used, the binding of ATP is fundamentallythe same. On the other hand, as the amount of phosphorylatedpeptides increases, the molecular ratio between the phosphory-lated peptides and ATP increases. Therefore, the fluorescencesignal increases with the amount of phosphorylated peptides.The correlation between the fluorescence signal of DPA beadsand kinase activity is shown in Fig. 3h.

3.4. Detection of protein kinase activity in cell lysates

To deal with a real biological problem, HeLa cell line wasselected as a cellular model system and intracellular protein kinaseactivity was monitored. There lies a difficulty in obtaining realsamples from patients. Moreover, ethical constraints may beinvolved from the use of real samples. HeLa cell line is widelyused for understanding of cellular signal transduction and biolo-gical processes. The cell lysate was collected from 4�107 cells. Theconcentration of total protein was approximately 25 μg/μl, asdetermined by the bicinchoninic acid assay (BCA Protein AssayKit, Thermo Scientific, Rockford, IL, USA). The cell lysate waspumped into chamber 1 of the microfluidic platform. Variousprotein kinase substrates were injected into chamber 3 and

S.H. Lee et al. / Biosensors and Bioelectronics 57 (2014) 1–94

reacted with the cell lysates, respectively. After the reactionmixture was passed through the loaded DPA beads, kinase activitywas monitored using the fluorescence signal of DPA beads. Whenkemptide was reacted without the cell lysate, no fluorescencesignal was detected (Fig. 4a). However, when kemptide wasreacted with the cell lysate, a fluorescence signal was detected(Fig. 4b). Next, autocamtide-2 was tested with and without the celllysate. As observed for kemptide, a fluorescence signal was notdetected without the cell lysate (Fig. 4c). In contrast, whenautocamtide-2 was reacted with the cell lysate, a fluorescencesignal was detected (Fig. 4d). Furthermore, the substrate ofmitogen-activated protein kinase (MAPK) was tested using thesame method. When the cell lysate was not present in the reactionmixture, a fluorescence signal was not detected (Fig. 4e). When thecell lysate was mixed with the MAPK substrate in the microfluidicchannel, a fluorescence signal was detected (Fig. 4f). Cell lysatescontain a lot of biomolecules and impurities that can affect proteinkinase activity (Kalesh et al., 2010; Kang et al., 2010). Thus,compared with the purified enzyme experiments, the fluorescencesignal was a little weaker in the cell lysate experiments. Never-theless, phosphorylation of intracellular kinases was clearly

detected using the DPA bead-based microfluidic chip. These resultsshow that our system can be effectively used to monitor intracel-lular kinase activity.

3.5. Effect of inhibitors on kinase activity

Protein kinases regulate most aspects of cellular life, whereasabnormal regulation causes disease. Therefore, protein kinasescomprise an important group of drug targets (Cohen, 2002b;Zhang et al., 2009). To investigate the potential of our microfluidicsystem as a drug screening tool, we introduced H89, which is awell-established PKA inhibitor that competitively binds to the ATPpocket on the kinase catalytic subunit (Engh et al., 1996). There-fore, H89 occupies ATP binding site of PKA; thus, ATP cannot reactwith the PKA. As a result, PKA cannot phosphorylate the substratepeptide (Pflug et al., 2012). After H89 was loaded with ATP intochamber 2 of our chip, the reaction was performed as describedabove. The effect of the inhibitor was evaluated by comparing thefluorescence signals of DPA beads from samples reacted withdifferent concentrations of H89 against those obtained fromsamples without any inhibitor.

Fig. 3. Monitoring peptide phosphorylation and dephosphorylation. (a) cAMP-dependent protein kinase A (PKA) was reacted with the substrate of Abl protein tyrosinekinase (abltide). (b) PKA was reacted with the substrate of Ca2þ/calmodulin-dependent protein kinase II (autocamtide-2). (c) Only the PKA substrate (kemptide) wasincluded without PKA. (d) Kemptide and PKAwere reacted, and a strong signal was detected. (e) Only phosphorylated kemptide was included without any kinase. (f) Alkalinephosphatase was reacted with phosphorylated kemptide. (g) Various concentrations of PKA were reacted with the kemptide. (h) Correlation between the fluorescence signalof DPA beads and kinase activity. One unit of PKA is defined as the amount of PKA catalytic subunit required to catalyze the transfer of 1 pmol of phosphate to Kemptide,LRRASLG (100 mM) in 1 min at 30 1C in a total reaction volume of 25 μL.

S.H. Lee et al. / Biosensors and Bioelectronics 57 (2014) 1–9 5

Fig. 5 shows the fluorescence signal of DPA beads as a functionof H89 concentration. As a control, Fig. 5a-i shows that a strongfluorescence signal was observed when the inhibitor was notinvolved in the reaction. The IC50 value of H89 is known to be50–140 nM (Davies et al., 2000; Lochner and Moolman, 2006), andin the present study, 135 nM H89 caused a decrease of approxi-mately 40–45% in the fluorescence signal (Fig. 5a-ii), indicatingthat our result is comparable with other reports. As shown inFig. 5a-iii, when 10 μM H89 was added to the phosphorylation

reaction, the detected fluorescence signal of the beads decreasedto 15–20% of the level observed without the inhibitor case.Furthermore, when 100 μM H89 was used in the kinase reaction,a fluorescence signal was not detected at all (Fig. 5a-iv). Theseresults show that the fluorescence of the DPA beads decreased asthe concentration of the inhibitor increased (Fig. 5b). Based onthese results, we suggest that our platform can detect the effects ofprotein kinase inhibitors and can be applied as a kinase inhibitorscreening tool.

Fig. 4. Detection of the activity of various kinases in HeLa cell lysate using the bis(Zn2þ-dipicolylamine) (DPA) bead-based microfluidic platform. (a) Kemptide (proteinkinase A [PKA] substrate) was used to monitor PKA (cAMP-dependent protein kinase) activity in the kinase reaction, but the cell lysate was not included. (b) Kemptide andthe cell lysate were included in the kinase reaction. (c) Autocamtide-2 (CaMKII substrate) was used to monitor Ca2þ /calmodulin-dependent protein kinase II (CaMKII) activitywithout the cell lysate. (d) Autocamtide-2 and the cell lysate were reacted. (e) The substrate of mitogen-activated protein kinase (MAPK) was reacted without the cell lysate.(f) The MAPK substrate and the cell lysates were reacted.

S.H. Lee et al. / Biosensors and Bioelectronics 57 (2014) 1–96

3.6. Repeatable use of the kinase activity monitoring systemfor multiple assays

In this system, the bis(Zn2þ-dipicolylamine) complex (DPA)beads were used to capture the phosphorylated peptides. Thecaptured peptides and zinc ions can be washed using N,N,N0,N0-tetrakis(2-picolyl)ethylenediamine (TPEN), which is known as azinc chelator (Xue et al. 2012). Also, the zinc-removed beads canbe recovered to become zinc-containing beads by treating themwith zinc perchlorate hexahydrate solution. Through these zincremoval and addition processes, the DPA beads can be repeatedlyused. We performed a repeated assay on the same chip to checkthe repeatability of the kinase activity monitoring platform todiscriminate between phosphorylated and non-phosphorylatedpeptides.

First, the 100 μM phosphorylated and fluorescence-labeledkemptide was injected into the bead chamber, as describedpreviously. The phosphorylated kemptide was captured by theDPA beads and a fluorescence signal was detected (Fig. 6a). Then,1 mM TPEN was passed through the bead chamber for 5 min. Thefluorescence signal disappeared, which means the TPEN washed

out the zinc and the captured peptides (Fig. 6b). 100 mM zincperchlorate hexahydrate solution was injected through the beadchamber for 30 min to supply the zinc. When the 100 μMphosphorylated and fluorescence-labeled peptides were addedagain into the bead chamber, the fluorescence signal was detected(Fig. 6c). When the zinc was not supplied to the DPA beads, thephosphorylated kemptide was not captured by the DPA beads(Fig. 6d). This indicates that zinc plays an important role incapturing the phosphorylated peptides in this system. Even withthe zinc added to the DPA beads, the non-phosphorylated peptidesdid not show any fluorescence signal (Fig. 6e). We repeated thisprocess five times, and the results are shown in Fig. 6f. The signalintensity decreased about 10% when repeated 5 times. However, itdid not cause any problem in discrimination between the phos-phorylated and the non-phosphorylated peptides.

3.7. Advantages over the conventional assay

Our DPA bead-based microfluidic platform has advantages overthe conventional assay. First, DPA bead system is versatile syn-thetic fluorescent kinase probe. Other conventional technologies

Fig. 5. (a) Effect of H89 (a protein kinase A inhibitor) on kinase activity and the fluorescence intensity of bis(Zn2þ-dipicolylamine) (DPA) beads. As the concentration of H89increased, fluorescence intensity decreased. (a-i) Without inhibitor: H89 was not included in the kinase reaction, and a strong fluorescence signal was detected. (a-ii) 135 nMH89: The IC50 of H89 is well known to be 50–140 nM. When we added 135 nM H89 to the kinase reaction, the fluorescence intensity was decreased by 40–45%. (a-iii) 10 μMH89: H89 (10 μM) was used for the kinase reaction. As a result, the fluorescence signal decreased to 10–15% of the signal obtained without the inhibitor case. (a-iv) 100 μMH89: The fluorescence signal was not detected. (b) Correlation between the fluorescence intensity of DPA beads and H89 concentration.

S.H. Lee et al. / Biosensors and Bioelectronics 57 (2014) 1–9 7

require specific materials such as radio-labeled ATP (Zhu et al.,2000), a phospho-specific antibody (Seethala and Menzel, 1997) orphosphopeptide (or phosphoprotein)-binding nanoparticles(IMAP) (Sharlow et al., 2008). These expensive materials shouldbe prepared when we change our target kinase. Therefore, ageneral, versatile and economical strategy has been desired formonitoring of the kinase activity. We developed a system thatsatisfies these demands by chemical synthesis. Furthermore, werealized “turn-on” fluorescence detection by using DPA beadsystem to overcome the disadvantage of the “turn-off” systemthat we presented in our previous study (Rhee et al., 2010). Theturn-on system is more preferable because external factors cannotinterfere with the signal in such system, unlike in the turn-offsystem. For example, in our previous study, we directly diluted(�100) the reaction mixture with a chemosensor to control theinterference (Rhee et al., 2010). However, this turn-on system does

not require the dilution step and is more useful for detectingkinase activity.

With respect to the amount of necessary reagents, our systemhas an advantage. The small amount of the reaction sample is oneof the greatest advantages of the microfluidic system (Hong andQuake, 2003; Squires and Quake, 2005). In the mixing part inFig. 2, the volume of the reaction mixture is only 30 nl. In theconventional microtube-based kinase reaction assay, 100 μl isnormally used for 384-well microplate-based detection. Therefore,the amount of necessary reagents can be reduced by using ourmicrofluidic system. For example, we used 1 unit/μl of PKA toshow the same kinase activity in the conventional assay, whichcan be prepared by dilution to 1/2500 of the commercial PKA(NEB, Cat No. P6000S). Although the concentration of PKA is thesame, the used sample volume is different. As a result, theconventional assay requires 100 units of enzyme. However, our

Fig. 6. Repeated use of the microfluidic bis(Zn2þ-dipicolylamine) (DPA) bead-based system to monitor kinase activity. (a) First detection of phosphorylated peptides: 100 μMphosphorylated kemptide was captured by the bis(Zn2þ-dipicolylamine) (DPA) beads and a fluorescence signal was detected. (b) Washing out of the zinc ion and thephosphorylated kemptide with N,N,N0 ,N0-tetrakis(2-picolyl)ethylenediamine (TPEN): As the captured kemptide was washed out using TPEN solution from the beads, thefluorescence signal disappeared. (c) Redetection of phosphorylated peptides with a zinc ion: The zinc ion was re-coordinated in the DPA beads with the zinc perchloratehexahydrate solution. Phosphorylated kemptide was recaptured by DPA beads and a fluorescence signal was detected. (d) Detection of phosphorylated peptides without azinc ion: After the zinc and the phosphorylated kemptide were washed out by TPEN, the phosphorylated kemptide was passed through the beads without the zinccoordination step. The phosphorylated kemptide was not captured and fluorescence signal was not detected. (e) Detection of non-phosphorylated peptides with a zinc ion:After zinc ion was re-coordinated in the DPA beads, non-phosphorylated kemptide was tested and fluorescence signal was not detected. (f) Fluorescence signal ofphosphorylated and non-phosphorylated kemptide with repeated use of the system: Capturing of the peptides and detection, washout by TPEN, and zinc coordination withthe DPA were repeated 5 times.

S.H. Lee et al. / Biosensors and Bioelectronics 57 (2014) 1–98

system requires only 30�10�3 units. Therefore, less kinase is usedin the microfluidic system than in the conventional system. Inview of the time, we incubated the reaction mixture in the mixingpart for 1 h to complete the reaction of the kinase. However, inFig. 3g, the results of the dose-response relationship and the time-course of the assay showed that the incubation time can beshortened depending on the purpose of the experiment. 10 minincubation is recommended for quantitative analysis. In thisregard, even with the smaller volume of sample, the incubationtime is comparable with the conventional assay. In summary, theadvantages of this assay system are its versatility, turn-on fluor-escence detection system, low sample consumption, practicalincubation time and use for multiple assays.

4. Conclusions

We developed a novel device for monitoring kinase activity, byintegrating a chemosensor (DPA) on microbeads in a microfluidicchip. The fabricated two-layer microfluidic chip was controlled bypneumatic valves. After the protein kinase, ATP, and fluorescence-labeled peptide were injected, the kinase reaction was performedby peristaltic mixing in the mixing part. Then, transfer of thereacted mixture to the sensing part, capture of the phosphorylatedpeptides by the DPA beads, and washing were performed sequen-tially and automatically. These sequential steps, based on a simplemix-and-measure technique, were demonstrated using food dyes.The integrated DPA beads discriminated between phosphorylatedand non-phosphorylated peptides based on their fluorescencesignal and could detect kinase activity of purified protein kinasesand also of cell lysates. Finally, the effects of inhibitors on kinaseactivity were also detected. This system provides a simple,versatile kinase assay using nanoliter volumes of reagents. Basedon these results, our system is suitable for a kinase activity assay.Furthermore, it can be applied to anti-cancer drug screening andmedical diagnosis.

Acknowledgments

This study was supported by the National Research Foundationof Korea (NRF) Grant funded by the Ministry of Education, Science,and Technology (MEST) (Nos. 2013069511, 2013005843, and2013003890). D. van Noort was supported by Brain Pool 2012through KOFST in Korea.

Appendix. Supporting information

Supporting information associated with this article can befound in the online version at http://dx.doi.org/10.1016/j.bios.2014.01.039.

References

Baac, H., Lee, J.-H., Seo, J.-M., Park, T.H., Chung, H., Lee, S.-D., Kim, S.J., 2004. Mater.Sci. Eng. C 24 (1-2), 209–212.

Beasley, J.R., Dunn, D.A., Walker, T.L., Parlato, S.M., Lehrach, J.M., Auld, D.S., 2003.Assay Drug Dev. Technol. 1 (3), 455–459.

Blume-Jensen, P., Hunter, T., 2001. Nature 411 (6835), 355–365.Chen, C.-A., Yeh, R.-H., Yan, X., Lawrence, D.S., 2004. Biochim. Biophys. Acta 1697

(1–2), 39–51.

Cohen, P., 2002a. Nat. Cell Biol. 4 (5), 127–130.Cohen, P., 2002b. Nat. Rev. Drug Discov. 1 (4), 309–315.Davies, S.P., Reddy, H., Caivano, M., Cohen, P., 2000. Biochem. J. 351 (1), 95–105.Du, J., Liu, M., Lou, X., Zhao, T., Wang, Z., Xue, Y., Zhao, J., Xu, Y., 2012. Anal. Chem. 84

(18), 8060–8066.Engh, R.A., Girod, A., Kinzel, V., Huber, R., Bossemeyer, D., 1996. J. Biol. Chem. 271

(42), 26157–26164.Fang, C., Wang, Y., Vu, N.T., Lin, W.-Y., Hsieh, Y.-T., Rubbi, L., Phelps, M.E., Müschen,

M., Kim, Y.-M., Chatziioannou, A.F., Tseng, H.-R., Graeber, T.G., 2010. Cancer Res.70 (21), 8299–8308.

Fowler, A., Swift, D., Longman, E., Acornley, A., Hemsley, P., Murray, D., Unitt, J.,Dale, I., Sullivan, E., Coldwell, M., 2002. Anal. Biochem. 308 (2), 223–231.

Glickman, J.F., Schmid, A., Ferrand, S., 2008. Assay Drug Dev. Technol. 6 (3),433–455.

Hong, J.W., Quake, S.R., 2003. Nat. Biotechnol. 21 (10), 1179–1183.Kalesh, K.A., Sim, D.S.B., Wang, J., Liu, K., Lin, Q., Yao, S.Q., 2010. Chem. Commun. 46

(7), 1118–1120.Kang, J.-H., Asami, Y., Murata, M., Kitazaki, H., Sadanaga, N., Tokunaga, E., Shiotani,

S., Okada, S., Maehara, Y., Niidome, T., Hashizume, M., Mori, T., Katayama, Y.,2010. Biosens. Bioelectron. 25 (8), 1869–1874.

Kwon, T.-H., Kim, H.J., Hong, J.-I., 2008. Chem.—Eur. J. 14 (31), 9613–9619.Lee, A.G., Beebe, D.J., Palecek, S.P., 2012a. Biomed. Microdevices 14 (2), 247–257.Lee, J.H., Cosgrove, B.D., Lauffenburger, D.A., Han, J., 2009a. J. Am. Chem. Soci. 131

(30), 10340–10341.Lee, S.H., van Noort, D., Lee, J.Y., Zhang, B.-T., Park, T.H., 2009b. Lab Chip 9 (3),

479–482.Lee, S.H., van Noort, D., Yang, K.-A., Lee, I.-H., Zhang, B.-T., Park, T.H., 2012b. Lab Chip

12 (10), 1841–1848.Lochner, A., Moolman, J.A., 2006. Cardiovasc. Drug Rev.24 (3–4), 261–274.Lu, M., Ma, X., Fan, Y.-J., Fang, C.-J., Fu, X.-F., Zhao, M., Peng, S.-Q., Yan, C.-H., 2011.

Inorg. Chem. Commun. 14 (12), 1864–1867.Manning, G., Whyte, D.B., Martinez, R., Hunter, T., Sudarsanam, S., 2002. Science

298 (5600), 1912–1934.Melin, J., Quake, S.R., 2007. Annu. Rev. Biophys. Biomol. Struct. 36, 213–231.Narayan, M., Mirza, S.P., Twining, S.S., 2011. Proteomics 11 (8), 1382–1390.Neupane, L.N., Park, J.-Y., Park, J.H., Lee, K.-H., 2013. Org. Lett. 15 (2), 254–257.Park, Y.-W., Cummings, R.T., Wu, L., Zheng, S., Cameron, P.M., Woods, A., Zaller, D.M.,

Marcy, A.I., Hermes, J.D., 1999. Anal. Biochem. 269 (1), 94–104.Pflug, A., Johnson, K.A., Engh, R.A., 2012. Acta crystallographica section F. Struct.

Biol. Cryst. Commun. 68 (8), 873–877.Pourghaz, Y., Dongare, P., Thompson, D.W., Zhao, Y., 2011. Chem. Commun. 47 (39),

11014–11016.Rhee, H.-W., Choi, H.-Y., Han, K., Hong, J.-I., 2007. J. Am. Chem. Soc. 129 (15),

4524–4525.Rhee, H.-W., Choi, S.J., Yoo, S.H., Jang, Y.O., Park, H.H., Pinto, R.M., Cameselle, J.C.,

Sandoval, F.J., Roje, S., Han, K., Chung, D.S., Suh, J., Hong, J.-I., 2009. J. Am. Chem.Soc. 131 (29), 10107–10112.

Rhee, H.-W., Lee, C.-R., Cho, S.-H., Song, M.-R., Cashel, M., Choy, H.E., Seok, Y.-J.,Hong, J.-I., 2008. J. Am. Chem. Soc. 130 (3), 784–785.

Rhee, H.W., Lee, S.H., Shin, I.S., Choi, S.J., Park, H.H., Han, K., Park, T.H., Hong, J.I.,2010. Angew. Chem. Int. Ed. 49 (29), 4919–4923.

Schweppe, R.E., Haydon, C.E., Lewis, T.S., Resing, K.A., Ahn, N.G., 2003. Acc. Chem.Res. 36 (6), 453–461.

Seethala, R., Menzel, R., 1997. Anal. Biochem. 253 (2), 210–218.Sharlow, E.R., Leimgruber, S., Yellow-Duke, A., Barrett, R., Wang, Q.J., Lazo, J.S., 2008.

Nat. Protocols 3 (8), 1350–1363.Sills, M.A., Weiss, D., Pham, Q., Schweitzer, R., Wu, X., Wu, J.J., 2002. J. Biomol.

Screen. 7 (3), 191–214.Sportsman, J.R., Gaudet, E.A., Boge, A., 2004. Assay Drug Dev. Technol. 2 (2),

205–214.Squires, T.M., Quake, S.R., 2005. Rev. Mod. Phys. 77 (3), 977–1026.Turek-Etienne, T.C., Lei, M., Terracciano, J.S., Langsdorf, E.F., Bryant, R.W., Hart, R.F.,

Horan, A.C., 2004. J. Biomol. Screen. 9 (1), 52–61.Unger, M.A., Chou, H.-P., Thorsen, T., Scherer, A., Quake, S.R., 2000. Science 288

(5463), 113–116.von Ahsen, O., Bömer, U., 2005. ChemBioChem 6 (3), 481–490.Wang, Q., Dai, Z., Cahill, S.M., Blumenstein, M., Lawrence, D.S., 2006. J. Am. Chem.

Soc. 128 (43), 14016–14017.Wlodkowic, D., Darzynkiewicz, Z., 2010. World J. Clin. Oncol. 1 (1), 18–23.Wu, J.J., 2002. Comparison of SPA, FRET, and FP for kinase assay. In: Janzen, W.P.

(Ed.), High Throughput Screening Methods and protocols. Humana Press, NewYork, pp. 65–86

Xue, L., Li, G., Zhu, D., Liu, Q., Jiang, H., 2012. Inorg. Chem. 51 (20), 10842–10849.Zhang, J., Allen, M.D., 2007. Mol. Biosyst. 3 (11), 759–765.Zhang, J., Yang, P.L., Gray, N.S., 2009. Nat. Rev. Cancer 9 (1), 28–39.Zhu, H., Klemic, J.F., Chang, S., Bertone, P., Casamayor, A., Klemic, K.G., Smith, D.,

Gerstein, M., Reed, M.A., Snyder, M., 2000. Nat. Genet. 26 (3), 283–289.

S.H. Lee et al. / Biosensors and Bioelectronics 57 (2014) 1–9 9