High-Throughput Analysis of Alzheimer’s �-AmyloidAggregation Using a Microfluidic Self-Assembly ofMonomersf

Joon Seok Lee, Jungki Ryu, and Chan Beum Park*

Institute for the BioCentury and Department of Materials Science and Engineering, Korea Advanced Institute ofScience and Technology, 335 Gwahangno, Yuseong-gu, Daejeon 305-701, Republic of Korea

The principal histopathological feature of Alzheimer’sdisease is the presence of �-amyloid (A�) aggregates inthe gray matter of the brain, and researchers believe thatvarious environmental factors play significant roles in theconformational change and self-assembly of A� peptides.Therefore, discovering a rapid and convenient analyticalmethod of evaluating the environmental factors on A�aggregation would have a considerable impact. Herein wereport our development of a novel microfluidic screeningsystem enabling high-throughput analysis, low-consump-tion of reagents, and short analytical time. Microchannelswith a cross-sectional dimension of 100 µm × 100 µmwere immobilized with A� monomers via N-hydroxysuc-cinimide ester activation of the internal surfaces, and thena fresh A� monomer solution mixed with different smallmolecules or metal ions was continuously introduced intothe microchannels to induce A� aggregation. In this work,we investigated (1) the temporal evolution of A� aggrega-tion within microchannels, (2) the high-throughput screen-ing of the inhibitory effect of 12 small molecules againstA� aggregation, and (3) the effect of different metal ions(Fe3+, Cu2+, Zn2+, and Al3+) on A� aggregation by usingthioflavin T (ThT)-induced fluorescence microscopyand ex situ atomic force microscopy. The microfluidicsystem should contribute to a simultaneous analysisof multiple environmental factors affecting amyloidaggregates in a parallel manner and to screen thera-peutic small molecules prior to their in vivo evaluation.

Alzheimer’s disease (AD) is the most common neurodegen-erative disorder, which causes a loss of memory and cognitionand affects 27 million people worldwide according to a recentestimate.1 A major pathological hallmark of AD is the formationof senile plaques consisting of �-amyloid (A�) aggregates in thebrain tissues.2,3 A� is a peptide with ∼42 amino acid residues,produced from a membrane-bound amyloid precursor protein(APP) through proteolytic actions of �- and γ-secretases (Figure

1A).4 A�42, the most predominant component in A� plaques, isknown to self-assemble into amyloid aggregates more readily thanothers.5 Despite numerous evidence suggesting close correlationsbetween A� aggregation and AD,3,4,6 we are still behind indeveloping a therapeutic method and screening drug candidatesfor AD due to the limited number of efficient analytical tools aswell as the lack of clear understanding of the underlying mech-anism of A� aggregation.2 The analytical situation becomes evenmore complex with various environmental factors affecting theaggregation of A�, such as temperature, pH, shear flow, ionicstrength, surface features, and the presence of metal ions or smallmolecules.7-10 For example, it was reported that the rate andmorphology of A� aggregation is significantly influenced by thetype of metal ions and their concentrations.8,11-15

Thus, it is critically important to develop an efficient analyticalsystem that enables high-throughput screening of drug candidatesfor Alzheimer’s disease. A successful system should satisfy thefollowing criteria: consume few reagents, require little work, allowready access, and provide a quick and easy evaluation of results.Many researchers have worked on the development of newscreening assay methods that include capillary electrophoresis,16

mass spectrometry,17 enzyme-linked immunosorbent assay(ELISA),18 surface plasmon resonance spectrometry,19 and cell-

* To whom correspondence should be addressed. Phone: +82-42-350-3340.Fax: +82-42-350-3310. E-mail: parkcb@kaist.ac.kr.

(1) Brookmeyer, R.; Johnson, E.; Ziegler-Graham, K.; Arrighi, M. Alzheimer’sDementia 2007, 3, 186–191.

(2) Murphy, R. M. Annu. Rev. Biomed. Eng. 2002, 4, 155–174.(3) Lahiri, D. K.; Farlow, M. R.; Greig, N. H.; Sambamurti, K. Drug Dev. Res.

2002, 56, 267–281.

(4) Hardy, J.; Selkoe, D. J. Science 2002, 297, 353–356.(5) Bitan, G.; Kirkitadze, M. D.; Lomakin, A.; Vollers, S. S.; Benedek, G. B.;

Teplow, D. B. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 330–335.(6) Wolfe, M. S. Nat. Rev. Drug Discovery 2002, 1, 859–866.(7) Kelly, J. W. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 930–932.(8) Bush, A. I. Trends Neurosci. 2003, 26, 207–214.(9) Petkova, A. T.; Leapman, R. D.; Guo, Z.; Yau, W.-M.; Mattson, M. P.; Tycko,

R. Science 2005, 307, 262–265.(10) Hill, E. K.; Krebs, B.; Goodall, D. G.; Howlett, G. J.; DunstanRozga, D. E.

Biomacromolecules 2006, 7, 10–13.(11) Lovell, M. A.; Robertson, J. D.; Teesdale, W. J.; Campbell, J. L.; Markesbery,

W. R. J. Neurol. Sci. 1998, 158, 47–52.(12) Suzuki, K.; Miura, T.; Takeuchi, H. Biochem. Biophys. Res. Commun. 2001,

285, 991–996.(13) House, E.; Collingwood, J.; Khan, A.; Korchazkina, O.; Berthon, G.; Exley,

C. J. Alzheimer’s Dis. 2004, 6, 291–301.(14) Ha, C.; Ryu, J.; Park, C. B. Biochemistry 2007, 46, 6118–6125.(15) Ryu, J.; Girigoswami, K.; Ha, C.; Ku, S. H.; Park, C. B. Biochemistry 2008,

47, 5328–5335.(16) Sabella, S.; Quaglia, M.; Lanni, C.; Racchi, M.; Govoni, S.; Caccialanza, G.;

Calligaro, A.; Bellotti, V.; De Lorenzi, E. Electrophoresis 2004, 25, 3186–3194.

(17) Cheng, X.; van Breemen, R. B. Anal. Chem. 2005, 77, 7012–7015.(18) Inbar, P.; Bautista, M. R.; Takayama, S. A.; Yang, J. Anal. Chem. 2008,

80, 3502–3506.(19) Ryu, J.; Joung, H.-A.; Kim, M.-G.; Park, C. B. Anal. Chem. 2008, 80, 2400–

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10.1021/ac802701z CCC: $40.75 2009 American Chemical Society 2751Analytical Chemistry, Vol. 81, No. 7, April 1, 2009Published on Web 03/10/2009

based assay,20 but the assay systems could not fully satisfy suchcriteria. They are often labor-intensive and require special instru-ments. We suggest that a microfluidic analytical system would

be a good platform to solve those problems since a microfluidicsystemcanbereadilypreparedusingsoft-lithographictechniques,21-23

(20) Apostol, B. L.; Kazantsev, A.; Raffioni, S.; Illes, K.; Pallos, J.; Bodai, L.; Slepko,N.; Bear, J. E.; Gertler, F. B.; Hersch, S.; Housma, D. E.; Marsh, J. L.;Thompson, L. M. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 5950–5955.

(21) Whitesides, G. M. Nature 2006, 442, 368–373.(22) Zhang, Z. L.; Crozaiter, C.; Le Berre, M.; Chen, Y. Microelectron. Eng. 2005,

78-79, 556–562.

Figure 1. (A) Amyloid precursor protein and the amino acid sequence of �-amyloid peptide produced through proteolytic actions of �- andγ-secretases. (B) Chemical structures of small molecules tested for the inhibition of �-amyloid aggregation. (C) Experimental scheme for theimmobilization of A�42 seeds within microchannels.

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and it would satisfy most criteria mentioned above.24

In the present work, we report the development of a microf-luidic analytical system for aggregation in vitro from fresh A�monomers to fully grown fibrils. Recently, we successfullydemonstrated that a microfluidic platform can be effectively usedto monitor the self-assembly of bovine insulin into amyloid fibrilswithin microchannels.25 Here we have applied the microfluidicsystem to the high-throughput screening of small-molecule inhibi-tors, listed in Figure 1B, and the effect of metal ions onAlzheimer’s A� aggregation. Our results show that ibuprofen,curcumin, and simvastatin have higher inhibition efficiency thanothers and that the incubation of A� monomers with Al3+/Fe3+

and Cu2+/Zn2+ leads to the formation of fibrillar and nonfibrillaraggregates, respectively. The results were highly consistentwith previous works conducted in bulk systems, demonstratingthe effectiveness and efficiency of the microfluidic analyticalsystem. We also found that the shear stress generated fromthe continuous flow of A� solution in the microfluidic systemfacilitates and accelerates the aggregation of A�, which isbeneficial because it leads to low consumption of the expensivereagents. We believe that our system not only provides anefficient analytical tool to study amyloid formation but alsoenables high-throughput screening of inhibitors against A�aggregation and thus drug candidates against Alzheimer’sdisease.

EXPERIMENTAL SECTIONMaterials. Human A�42 peptide was obtained from rPeptide

Co. (Athens, GA). Hemin and trehalose were purchased fromFluka (Buchs, Switzerland). Ectoine and hydroxyectoine wereprovided by Bitop AG (Witten, Germany). The rest of chemicalsthat were all ACS grade were purchased from Sigma-Aldrich (St.Louis, MO).

Preparation of A�42 Solution in the Presence or Absenceof Additives. Monomeric A�42 peptides were prepared asreported previously.26 Briefly, as-received A�42 peptides were firstdissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and thensonicated in a water bath to disintegrate preformed peptideaggregates. A�42 peptides were then aliquoted into microtubesand dried in a vacuum desiccator before being stored at -20 °C.Immediately prior to use, the A�42 peptides were dissolved indimethyl sulfoxide (Me2SO) and further diluted with a buffersolution to the desired concentration. Because small moleculeshave a low solubility in aqueous media, they were first dissolvedin Me2SO and then diluted with a buffer solution. In most cases,we used a 10 mM phosphate buffer (pH 7.4), but in theexperiment with metal ions, we used a 20 mM Tris-HCl buffer(pH 7.4) to prevent the precipitation of metal ions. Chloridesalts such as FeCl3, AlCl3, CuCl2, and ZnCl2 were used to supplyrespective metal ions. Throughout the experiment, the finalconcentration of Me2SO in the A� solution was kept constantat 5% (v/v).

Fabrication of PDMS Microchannels. Microchannels werefabricatedusingthereplicamoldingprocessdescribedelsewhere.25,27

Briefly, SU-8 (MicroChem, Newton, MA) was spun on a siliconwafer and patterned using photolithography. The microchannelswere then obtained by poly(dimethylsiloxane) (PDMS) (DowCorning, Midland, MI) molding on the patterned silicon wafer.The dimension of each microchannel was 100 µm (width) × 100µm (depth) × 15 000 µm (length). Glass slides were cleaned priorto use with a piranha solution of 70% H2SO4/30% H2O2 (7:3, v/v)for 15 min at 60 °C, rigorously rinsed with deionized water,and then dried with N2 gas. Microchannels were formed bybonding the PDMS film to a glass slide using either reversibleor irreversible method as follows. For the fluorescence analysisof amyloid aggregates, both the glass slide and PDMS micro-channels were treated with oxygen plasma and then they wereirreversibly bound. For the visualization of amyloid aggregatesformed on the microchannel surface using ex situ atomic forcemicroscopy (AFM), PDMS microchannels were physicallypressed onto the glass slide with aluminum slabs and screwsfor easy detachment, rather than using irreversible plasmabonding.

Immobilization of Monomeric A�42 Seeds within Micro-channels. For the covalent attachment of A�42 monomer seeds,microchannels were activated with an NHS functional group asillustratively described in Figure 1C. 7-Octenyltrichlorosilane (7-OTS, 1 mM) in toluene was introduced into microchannels at aflow rate of 5 µL/h for silanization using an 11 Plus syringe pump(Harvard Apparatus, Holliston, MA) for 10 min. Microchannelswere then washed with toluene and cured at 80 °C. Newlydeveloped vinyl groups within the microchannels were im-mediately oxidized to a carboxylic group by the injection of asolution mixture (pH 7.5) of KMnO4 (0.5 mM), K2CO3 (1.8 mM),and NaIO4 (19.5 mM). After 1 h of the carboxylation treatment,they were rinsed with 0.1 M NaHSO3, water, 0.1 N HCl, andwater. Finally, they were activated with the injection of anaqueous mixture of 0.1 M 1-ethyl-3-(3-dimethylaminopropyl-)carbodiimide hydrochloride (EDC) and 0.025 M N-hydrox-ysuccinimide (NHS) (1:1) for 20 min at room temperature andthen washed with deionized water. A fresh A�42 solution (10µM) was injected into the NHS-activated microchannels for 10min to covalently immobilize monomeric A�42 peptides, andthe microchannels were rinsed and ready for A� immobiliza-tion. Remaining NHS-active sites were blocked with 0.1% bovineserum albumin (BSA) solution in a 50 mM phosphate bufferfor 40 min. Physically adsorbed peptides were removed bywashing the microchannels with a phosphate buffer anddeionized water for 30 min.

Formation and Characterization of A� Aggregates withinMicrochannels. Microchannels immobilized with fresh A�42seeds were incubated with an A�42 feeding solution under acontinuous-flow condition (5 µL/h) to induce the A� aggregation.According to our previous work,25 amyloid aggregation wasaccelerated with an increased flow rate, but amyloid aggregateswere not formed uniformly in the microchannel when the flowrate exceeded over 5 µL/h. After 24 h of incubation at 37 °C withor without additives such as small molecules or metal ions, themicrochannels were washed with phosphate buffer and deionized

(23) Wu, H.; Zhai, J.; Tian, Y.; Lu, H.; Wang, X.; Jia, W.; Liu, B.; Yang, P.; Xu,Y.; Wang, H. Lab Chip 2004, 4, 588–597.

(24) Weibel, D. B.; Whitesides, G. M. Curr. Opin. Chem. Biol. 2006, 10, 584–591.

(25) Lee, J. S.; Um, E.; Park, J.-K.; Park, C. B. Langmuir 2008, 24, 7068–7071.(26) Ha, C.; Park, C. B. Langmuir 2006, 22, 6977–6985. (27) Xia, Y.; Whitesides, G. M. Angew. Chem., Int. Ed. 1998, 37, 550–575.

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water and then dried with N2 for further analysis. In order tocompare the amyloid aggregation in the microfluidic systemwith that in a bulk solution, slide glasses (7 mm × 7 mm) withor without immobilized A� seeds were incubated in the bottomof a 24-well plate containing fresh A�42 solution (500 µL) asreported previously.26 Characterization of A� aggregates formedwithin microchannels was carried out with a fluorescencemicroscope and AFM. For the fluorescence microscopicanalysis, each microchannel was treated with thioflavin T (ThT)solution (50 µM) in Tris-HCl buffer (pH 8.0, 20 mM) afterthe amyloid aggregation, subsequently rinsed, and then dried.The fluorescence microscope (Eclipse 80i; Nikon, Japan) wasfocused on the bottom surface of the microchannels to obtainuniform images of each microchannel. The filter had anexcitation wavelength of 430 nm and an emission wavelengthof 490 nm. Note that the binding of ThT to the �-sheet structureleads to a shift of the excitation/emission peak of ThT from350/450 nm to 450/482 nm, respectively, and ThT fluorescenceintensity at an emission of 482 nm is proportional to the densityof A� fibrils.28 The fluorescence micrograph was furtherprocessed with ImageJ software (freely available at the follow-ing address: http://rsb.info.nih.gov/ij/) to quantify the relativeprofile of florescence from ThT binding. The morphology ofamyloid aggregates formed within microchannels was analyzedusing ex situ AFM. AFM images were acquired in a tapping-mode in air with an NCHR silicon cantilever (Nanosensors Inc.,Switzerland) by using a Multimode AFM instrument equippedwith a Nanoscope III controller and “E”-type scanner (DigitalInstruments Inc., Santa Barbara, CA) under the followingconditions: scan rate of 1-1.5 Hz, cantilever resonant frequencyrange of 250-350 kHz, pixel numbers of 512 × 512.

RESULTS AND DISCUSSIONFormation of A� Aggregates within Microchannels. We

studied the temporal evolution of the A� aggregation withinmicrochannels by using A�42 as a model Alzheimer’s A� peptidefor the formation of amyloid aggregates because A�42 aggregatesmore readily and is more amyloidogenic than A�40.5 Prior to theinjection of A�42 feeding solution into the microchannels to induceA� aggregation, fresh A�42 monomers were covalently im-mobilized within microchannels as nucleation sites for amyloidformation.25 After further incubation of seeded microchannels withA�42 feeding solution under a continuous-flow condition (5 µL/h) for 0, 3, 12, and 24 h at 37 °C, we observed the temporalevolution of A� aggregation within microchannels using fluores-cence microscopy (Figure 2, parts A and B). For the fluorescencemicroscopic analysis, each microchannel was treated after the A�aggregation with a solution containing ThT, a fluorescent dye thatspecifically binds only to a cross-�-sheet structure of amyloidfibrils.28 As shown in Figure 2B, the microchannel incubated for3 h exhibited slightly stronger fluorescence than the one withoutincubation (i.e., 0 h), indicating the start of aggregate formationby the conformational change through an interaction betweenA�42 peptides dissolved in the flow solution and immobilizedmonomeric seeds. With further incubation of more than 12 h, wecould observe a significant increase in fluorescence intensity. Exsitu AFM analysis of the bottom surface of the microchannels

supported the results obtained with fluorescence microscopy. Atthe start of incubation, surface features smaller than a couple ofnanometers were observed, which should correspond to theseeded A�42 monomers. When we further investigated themicrochannels incubated for 3 h, there was still no detectable A�fibril on the surface, indicating that the kinetics of amyloidfibrillation exhibits an initial lag phase; but small, nonfibrillaraggregates were observed, which may have been formed by theoligomerization of A�42. After 12 h of incubation, much largeramorphous aggregates and amyloid fibrils started to appearoccasionally on the surface, and numerous A� fibrils covered theentire surface of the microchannels like a dense forest after 24 hof incubation under the continuous-flow condition. We did notobserve any spherulitic aggregates that were clearly visible in ourprevious study with bovine insulin,25 which might be due to themuch lower concentration of A�42 than insulin.

Comparison between A� Aggregation in Microfluidic andBulk Systems. We investigated the A� aggregation withinmicrochannels in comparison to that observed in a bulk solutionsystem. The bulk counterpart was prepared, as described in ourprevious report,26 by immobilizing monomeric A�42 seeds ontothe entire solid surface without the confinement supplied bymicrochannels and then incubating the solid substrate in a freshA�42 solution. After incubation for 24 h under no-flow conditions,only a few long fibrils were observed on the surface of the bulksystem, together with amorphous clusters having quite irregularmorphologies with bends and kinks (upper panel in Figure 3). Incontrast, we could observe numerous, uniform fibrils on the samesurface when we employed the microfluidic system (lower panelin Figure 3). Note that, according to our results, it took morethan 3 days for the bulk system to deposit amyloid fibrils in anamount comparable to that in the microfluidic system. Wespeculate that the accelerated amyloid fibrillation in the microf-luidic system was caused by the continuous flow of A� solution,which should allow more frequent interactions between im-mobilized A� seeds and fresh A� monomers in the feedingsolution, thus facilitating the rapid formation of amyloid ag-gregates. When solid surfaces without immobilized A�42 mono-meric seeds were incubated in fresh A�42 solution, no fibrillaraggregate was observed on their surface, irrespective of systemsemployed (data not shown). The presence of seeds should triggerthe formation of amyloid fibrils by reducing the length of the initiallag phase for amyloid nucleation.29

Interestingly, we observed that the long axis of amyloid fibrilsformed in the microchannels were aligned parallel to the flowdirection of the A� feeding solution (the lower panel in Figure3), whereas amyloid fibrils grown in the bulk system exhibited arandom orientation (the upper panel in Figure 3). The orientationalordering of amyloid fibrils in the microfluidic system might becaused by the shear stress applied by the continuous flow offeeding solution. Shearing processes such as shaking, mechanicalagitation, or sonication were reported to influence the morphologyand formation rate of amyloid aggregates.9,10 For example, Hillet al. reported that shear flow enhances the formation of amyloidaggregates and influences the conformational change and theorientational ordering of amyloidogenic peptides.10 Thus, it

(28) LeVine, H. Protein Sci. 1993, 2, 404–410.(29) Baskakov, I. V.; Legname, G.; Baldwin, M. A.; Prusiner, S. B.; Cohen, F. E.

J. Biol. Chem. 2002, 277, 21140–21148.

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suggests that the shear flow of the feeding solution in themicrofluidic system is responsible not only for the accelerationof the A� aggregation but also for the alignment of amyloid fibrils.

In the current microfluidic system, a 10 µM A�42 solution wasinjected into the microchannel at a flow rate of 5 µL/h, so theamount of A�42 peptides used was approximately 4.8 µg per eachmicrochannel having a cross-sectional dimension of 100 µm × 100µm when incubating for 24 h. Note that the incubation time (i.e.,injection time) and thus the amount of A� used could be reducedby at least 2 times because fluorescence intensity was strongenough to measure even after 12 h of incubation as shown inFigure 2A. We expect that we could further decrease theconsumption of A� peptide and other reagents by a factor of n2

by reducing the dimension of microchannels by n times whenthe flux of the feeding solution is kept constant (e.g., 24 ng ofA�42 consumption per each microchannel having a dimensionof 10 µm × 10 µm). With a drastic reduction of microchanneldimension, however, problems like channel clogging may occurdue to the amyloid aggregation. This possibility needs to beinvestigated in future. The highly accelerated fibrillation in the

microfluidic system in comparison to that in the bulk systemsuggests that we could further decrease in the consumptionof expensive reagents, such as A� peptides, by optimizingexperimental conditions (e.g., reducing the A� concentration).

High-Throughput Screening of Small-Molecule InhibitorsUsing a Microfluidic System. On the basis of our results, weapplied the microfluidic system to the screening of small-moleculeinhibitors against A� aggregation. An attractive strategy to treator prevent amyloidosis is the discovery of drug candidates thatcan prevent the aggregation of amyloidogenic peptides,6,30,31 butit has been quite difficult to effectively screen antiamyloid agentsmainly because of the lack of efficient analytical tools.16-20 Forexample, an ELISA-based parallel screening method was recentlysuggested by Inbar et al.,18 but the method is suitable forscreening molecules that can bind to fully grown A� fibrils but is

(30) Mason, J. M.; Kokkoni, N.; Stott, K.; Doig, A. J. Curr. Opin. Struct. Biol.2003, 13, 526–532.

(31) Feng, B. Y.; Toyama, B. H.; Wille, H.; Colby, D. W.; Collins, S. R.; May,B. C. H.; Prusiner, S. B.; Weissman, J.; Shoichet, B. K. Nat. Chem. Biol.2008, 4, 197–199.

Figure 2. Formation of amyloid aggregates within microchannels under a continuous-flow condition. (A) Formation of A� aggregates insidemicrochannels was characterized by ThT-induced fluorescence microscopy and ex situ AFM after incubating microchannels with A�42 feedingsolution for 0, 3, 12, and 24 h. (B) Fluorescence micrographs shown in panel A were further processed with ImageJ software to obtain theintensity profile of each microchannel.

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unsuitable for those affecting the dynamic aggregation processstarting from monomers. Considering that both A� oligomers andfibrils are cytotoxic,4,6,32,33 it is important to screen moleculesinhibiting the formation of oligomers or fibrils. In this regard, themicrofluidic system is much more versatile because it utilizes theformation of amyloid aggregates from the very beginning. In orderto demonstrate the feasibility of our microfluidic system for high-throughput screening of inhibitors against A� aggregation, wetested 12 different small molecules listed in Figure 1B. Thesemolecules can be classified into four groups: (1) natural osmolytesknown to stabilize native structures of peptides and proteins(ectoine,34 hydroxyectoine,34 and trehalose35), (2) structuralanalogues of Congo red binding specifically to amyloid aggregates(Congo red,36 chrysamine G,37 curcumin,38 and rosmarinic acid38),

(3) conventional drug molecules reported to lower the risk forAD when used for a long-term (ibuprofen39,40 (nonsteroidal anti-inflammatory drug, NSAID) and simvastatin41 (cardiovasculardrug)), and (4) other molecules reported to interfere with theaggregation of A� (melatonin,42 nicotine,43 and hemin44).

In this work, we compared the inhibitory effect of the smallmolecules against A� aggregation by ThT-induced fluorescencemicroscopy after a continuous flow of both A� monomers (10 µM)mixed with each small molecule (100 µM) through microchannelsfor 24 h (Figure 4). In order to evaluate and compare the inhibitoryeffect more quantitatively and precisely, fluorescence micrographwas further processed with ImageJ software and Origin softwareto calculate the total area below the intensity profile (Figure 5).As a control experiment, microchannels were also incubated withA� feeding solution under the continuous-flow condition in the

(32) Walsh, D. M.; Hartley, D. M.; Kusumoto, Y.; Fezoui, Y. F.; Condron, M. M.;Lomakin, A.; Benedek, G. B.; Selkoe, D. J.; Teplow, D. B. J. Biol. Chem.1999, 274, 25945–25952.

(33) Girigoswami, K.; Ku, S. H.; Ryu, J.; Park, C. B. Biomaterials 2008, 29,2813–2819.

(34) Kanapathipillai, M.; Lentzen, G.; Sierks, M.; Park, C. B. FEBS Lett. 2005,579, 4775–4780.

(35) Liu, R.; Barkhordarian, H.; Emadi, S.; Park, C. B.; Sierks, M. R. Neurobiol.Aging 2005, 20, 74–81.

(36) Frid, P.; Anisimov, S. V.; Popovic, N. Brain Res. Rev. 2007, 53, 135–160.(37) Ishii, K.; Klunk, W. E.; Arawaka, S.; Debnath, M. L.; Furiya, Y.; Sahara, N.;

Shoji, S.; Tamaoka, A.; Pettegrew, J. W.; Mori, H. Neurosci. Lett. 2002,333, 5–8.

(38) Ono, K.; Hasegawa, K.; Naiki, H.; Yamada, M. J. Neurosci. Res. 2004, 75,742–750.

(39) Weggen, S.; Eriksen, J. L.; Das, P.; Sagi, S. A.; Wang, R.; Pietrzik, C. U.;Findlay, K. A.; Smith, T. E.; Murphy, M. P.; Bulter, T.; Kang, D. E.; Marquez-Sterling, N.; Golde, T. E.; Koo, E. H. Nature 2001, 414, 212–216.

(40) Hirohata, M.; Ono, K.; Naiki, H.; Yamada, M. Neuropharmacology 2005,49, 1088–1099.

(41) Fassbender, K.; Simons, M.; Bergmann, C.; Stroick, M.; Lutjohann, D.;Keller, P.; Runz, H.; Kuhl, S.; Bertsch, T.; von Bergmann, K.; Hennerici,M.; Beyreuther, K.; Hartmann, T. Proc. Natl. Acad. Soc. U.S.A. 2001, 98,5856–5861.

(42) Pappolla, M.; Bozner, P.; Soto, C.; Shao, H.; Robakis, N. K.; Zagorski, M.;Frangione, B.; Ghiso, J. J. Biol. Chem. 1998, 273, 7185–7188.

(43) Ono, K.; Hasegawa, K.; Yamada, M.; Naiki, H. Biol. Psychiatry 2002, 52,880–886.

(44) Atamna, H.; Boyle, K. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 3381–3386.

Figure 3. AFM micrographs showing the difference between A� aggregation in bulk and microfluidic systems. The bulk (upper panel) andmicrofluidic (lower panel) systems were incubated with A� solution for 24 h under no-flow and continuous-flow conditions, respectively. AFMmicrographs clearly show that A� aggregation is highly accelerated in the microfluidic system compared to that in the bulk system. An arrow inthe right-side image in the lower panel depicts the flow direction of A� feeding solution.

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presence of human serum albumin (HSA) (0.2 mM) or NaCl (100mM), which are major components of human blood plasma. Wefound that HSA and NaCl exhibited a negligible effect on theaggregation of A� as shown in Figure 5. According to our results,ibuprofen exhibited the highest degree of inhibition (∼70-75%)among 12 tested small molecules, whereas the degree of inhibitioncaused by trehalose and nicotine was quite low (∼30% and lessthan 20%, respectively). Molecular osmolytes, ectoine and hy-droxyectoine, showed a moderate inhibitory effect, and ectoineexhibited a higher inhibition effect than hydroxyectoine at thesame concentration level as reported previously.34 When we

compared the effect of Congo red and its analogues, curcuminexhibited the best inhibition efficiency (∼70%). Interestingly,simvastatin, which is known to decrease the A� level in vivo bylowering the production of A� from APP,41 was also highlyeffective for inhibiting the aggregation of A�, suggesting itspossible role as a therapeutic against Alzheimer’s disease. Notethat heme cannot be used as a drug for Alzheimer’s diseasedespite its high inhibitory effect because the heme-A� complexhas a peroxidase activity that was suggested to be responsiblefor the oxidative damage in the AD brain.44 In summary, theinhibitory effect of the small molecules was almost consistent withprevious results. This indicates that our microfluidic system issuitable for simultaneous screening of diverse derivatives foridentifying unknown drug candidates. Furthermore, the microf-luidic system enabled the evaluation of the effect of smallmolecules irrespective of their fluorescence property because thebackground signal from unbound fluorophore could be minimizedby flushing the microchannel with a fresh solution. For example,when we performed A� aggregation in a bulk solution, it wasdifficult to analyze the effect of curcumin because of its ownfluorescence that overlaps with the ThT fluorescence (data notshown).

Effect of Metal Ions on Formation of Amyloid Aggregateswithin Microchannels. Metal ions, such as Fe3+, Al3+, Cu2+, andZn2+, are believed to play key roles in the abnormal aggregationof A� peptides as well as in the progression of AD.8,11-13 Sofar, ample evidence has been accumulated indicating a closerelationship between metal ions and AD. For example, a high levelof metal ions (Zn2+, ∼1100 µM; Cu2+, ∼400 µM; Fe3+, ∼1000µM) was found in the rims and cores of amyloid plaques

Figure 4. Fluorescence micrograph showing the effect of small molecules on the aggregation of A� within microchannels. Each microchannelwas incubated for 24 h with a continuously injected A� solution containing a molecule of interest, listed in Figure 1B. HSA and NaCl werechosen as negative controls. The fluorescence micrograph was further processed with ImageJ software to quantify the relative intensity profileof the florescence from ThT binding.

Figure 5. Degree of inhibition by small molecules against A�aggregation. The degree of inhibition was calculated in terms of thedecreased ThT fluorescence intensity in the presence of each smallmolecule relative to that of A�42-alone microchannel.

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compared to other brain tissues.11-13 In order to demonstratethat the microfluidic system can be also used to efficientlyinvestigate the effect of metal ions on A� aggregation in vitro, westudied the effect of four different metal ions on the aggregationof A�42 peptide using the microfluidic system. Here we used aTris-HCl buffer (20 mM) containing low-affinity metal-bindingsmall ligands (e.g., histidine for Zn2+, glycine for Cu2+, andsodium citrate for Fe3+) to prevent the precipitation of metalions.45 As shown in Figure 6, the degree of A� aggregation wasdifferentially affected by the type of metal ion; both fluorescenceand ex situ AFM analysis showed that the fibrillation of A� wasfacilitated in the presence of Al3+ or Fe3+ ions, but Cu2+ andZn2+ ions caused the formation of amorphous aggregatesinstead of fibrillar ones. These observations indicate that Al3+

or Fe3+ ions may play a significant role in the formation of fibrilswith a cross-�-sheet structure, but the interaction of Cu2+ orZn2+ ions with A�42 can prevent the conformational change ofA�42 to a fibrillar form, rich in cross-�-sheet structure. Herewe have shown that our microfluidic system can also be usedto study the effect of individual metal ions on the aggregationof A�, and the results obtained from the microfluidic system

confirmed previous research results. Considering that thereare various environmental factors that are closely related toeach other that affect amyloid aggregation, thus making themdifficult to study, our microfluidic system should be useful toexamine their combinatorial effect in a high-throughput manner.

CONCLUSION

We have developed a microfluidics-based assay system forhigh-throughput analysis of A� aggregation using a self-assemblyof monomeric A� peptide within microchannels. In the microflu-idic system, A� aggregation was highly accelerated by thecontinuous microflow of A� feeding solution, enabling rapidanalysis and a lower consumption of reagents compared to theconventional bulk system. We applied the microfluidic system tothe screening of diverse small molecules against A� aggregationin a high-throughput manner and found that the inhibitory effectof the molecules was almost consistent with results previouslyreported. In addition, we evaluated the effect of different metalions on the self-assembly of A� by using simultaneous fluorescentdetection and ex situ AFM analysis. We expect that the microf-luidic-based assay system could be extrapolated to a simultaneousscreening of therapeutic candidates prior to their in vivo evaluationand to the study of various environmental factors on the aggrega-

(45) Huang, X.; Atwood, C. S.; Hartshorn, M. A.; Multhaup, G.; Goldstein, L. E.;Scarpa, R. C.; Cuajungco, M. P.; Gray, D. N.; Lim, J.; Moir, R. D.; Tanzi,R. E.; Bush, A. I. Biochemistry 1999, 38, 7609–7616.

Figure 6. Effect of metal ions on the aggregation of A� in the microfluidic system. Microchannels were incubated for 24 h with the continuouslyinjected A�42 solution containing a metal ion of interest (e.g., Fe3+, Al3+, Zn2+, or Cu2+). A� aggregation was analyzed by ThT-induced fluorescencemicroscopy and ex situ AFM.

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tion of amyloidogenic peptides and proteins associated with manyneurodegenerative diseases.

ACKNOWLEDGMENTThis research was supported by Grants from the BioGreen 21

Program (20070301034038) and the Korea Science and Engineer-ing Foundation (KOSEF) National Research Laboratory (NRL)program (R0A-2008-000-20041-0). This research was also partially

supported by a Grant from the Eco-Technopia 21 project (010-081-036) from the Ministry of Environment, Republic of Korea.

Received for review December 19, 2008. AcceptedFebruary 12, 2009.

AC802701Z

2759Analytical Chemistry, Vol. 81, No. 7, April 1, 2009