Noninvasive Real-time Monitoring of Amyloid-� Fibrillization via Simultaneous Label-freeDielectric Relaxation Spectroscopy and Dark-Field Imaging

Yeonho Choi,† Soongweon Hong,† Taewook Kang,‡ and Luke P. Lee*,†

Biomolecular Nanotechnology Center, Berkeley Sensor and Actuator Center, Department of Bioengineering,UniVersity of California at Berkeley, Berkeley, California 94720, and Department of Chemical andBiomolecular Engineering, Sogang UniVersity, Seoul, 121-742, Korea

ReceiVed: May 4, 2009; ReVised Manuscript ReceiVed: May 26, 2009

Accumulated evidence suggests that amyloid-� has a critical role in chronic neurodegenerative disorderssuch as Alzheimer’s disease. Therefore, understanding of amyloid-� fibrillization is a focus of interest for thedevelopment of innovative therapeutic and diagnostic applications. The fibrillization of amyloid-� has similargrowth characteristics of polymeric nanoparticles and current monitoring methods show only qualitative orstatic information. Here we describe a noninvasive real-time monitoring of nanoscale amyloid-� fibrillizationby simultaneous Dielectric Relaxation Spectroscopy (DRS) and label-free dark-field imaging. First, thehydrodynamic radius is characterized by DRS, which can reflect the averaged radius of fibrilized amyloid-�,and we observe an increase from 19 to 21 nm during 48 h. Scattering intensity from dark-field imagingallowed us to visualize and quantify the fibrillization with respect to the incubation time of amyloid-�.Consequently, real-time observation and quantification of changes in both hydrodynamic radii and opticalproperties (i.e., scattering intensity) were performed simultaneously. Such a dual-mode technique may provevaluable for elucidating the mechanism of amyloid fibrillization and ultimately for designing possible diagnosticmethods.

Since the precise role of Amyloid-� (A�) aggregates incausing neurodegenerative diseases such as Alzheimer’s Disease(AD) is still unknown, systematic elucidation of oligomerizationand aggregation of A� peptides could provide a clue to unveilingthe conformation and dynamics of amyloid proteins as well asa definitive molecular basis for the clinical laboratory diagnosisof AD.1-11 In this regard, it is critical to develop characterizationmethods to track the structural evolution of A� oligomerizationor fibrillation from soluble A� peptide. Direct visualization ofA� fibrillation has been realized via transmission electronmicroscopy and atomic force microscopy, which have limitationsin real-time monitoring and quantification of A� aggregation.10-12

Similarly, fluorescence and circular dichroism measurementshave also been limited to qualitative analysis.13-16 Quartz crystalmicrobalance gives only quantitative information since itexploits a mass change of A� aggregate during aggregation.11,17

In the present work, we fabricated a transparent submicrometergap electrode as a dual-response sensor platform exhibiting adielectric and an optical response for combined optical dark-fieldimaging and dielectric relaxation spectroscopy (DRS) measurementof A� fibrillization noninvasively. In general, complementarymeasurement modalities provide more information and better crosschecks for a precise characterization of biological and chemicalevents than either method alone can yield.18-20 In addition to this,our dual mode detection technology provides several uniqueadvantages in the characterization of A� fibrillization. First, optical

and electrical measurements can be performed in aqueous solutionwhere A� can sustain and progress its fibrillization under naturalconditions. Second, in situ monitoring to characterize the fibrilli-zation progress can be carried out via simple dual mode measure-ments. Third, based on the DRS result, the dynamic dipolemoment’s change can be characterized during its fibrilizationprocess. The calculated hydrodynamic radius offers quantifiedinformation for the fibrilized status of A�.21,22 Fourth, the opticallymeasured scattering intensity change can be correlated with thehydrodynamic radius result.

The underlying concept of our real-time dual-mode detectionfor A� fibrillization is illustrated schematically in Figure 1.Conformational changes of A� can be resolved by optical dark-field imaging and electrical DRS system. In the DRS measurement,an alternating voltage is applied across a sample and the sinusoidalcurrent as a response is obtained as a function of frequency. Onthe basis of this measurement result, the various electrical propertiessuch as complex impedance, inductance, permittivity, conductance,and phase angle can be calculated. Among these obtained values,the complex permittivity can be related with properties determinedby the charge response in a system. Traditionally, a macroscalecapacitor and relatively high frequency (in general, >1 MHz) areused in DRS.18 However, the dielectric properties at low frequency(smaller than 1 MHz) are highly related with the overall effectivedipole moment change of detected molecules, because the resultsat low frequencies are primarily determined by the polarization ofoscillating counterions over the entire structure of A� and canindicate the overall structural change (i.e., the progress of thefibrillization). Moreover, as A� peptides are aggregated and formbigger oligomers or plaques, the scattering intensity increases in

* To whom correspondence should be addressed. E-mail: lplee@berkeley.edu. Phone: (510)642-5855.

† University of California at Berkeley.‡ Sogang University.

A

10.1021/jp904157j CCC: $40.75 XXXX American Chemical Society

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optical dark-field imaging measurements, and this optically resolvedin situ dark-field imaging can generate additional characterizationdata of A� fibrillization in our system.

As shown in Supporting Information Figure 2, a transparentsubmicrometer gap electrode for a dual-mode response is fabricatedby using two polished indium-tin oxide (ITO)-coated glass slides(CG-51N-1115, Delta Technologies) as transparent electrodes and50 nm polystyrene (PS) beads (Polyscience Inc.) as a dielectricspacer between the two glasses slides. In a typical experiment, 50nm PS bead solutions in methanol (13 µL, 30 nM) is depositedonto two polydimethylsiloxane (PDMS)-confined areas (2.5 mm× 12.5 mm each) on bottom ITO-coated glass. After drying thesolution, the PDMS block is removed, followed by placing anotherITO-coated glass on top of the bottom ITO glass. In order totransport and confine a solution of interest through the spacebetween the two ITO-coated glasses, 100 µm fluidic tubes areconnected at both sides. For the prevention of leakage and theattachment between two glasses, the open structure of the flowchannel is blocked by a dielectric material (Muscant, AcproductsInc.). During this process, external force (∼30 N) is applied inorder to prevent an increase in the gap size upon drying of thedielectric material. Finally, electrical wires are connected to bothITO-coated surfaces for DRS measurement. The gap size estimatedby a simple capacitance equation (C ) εA/d, where C is capacitance[F], ε is permittivity [F/m], A is area [m2], and d is gap distance[m]) gives ca. 770 ( 63 nm.

A� (Amyloid-�42 peptide, Sigma-Aldrich) is dissolved in deion-ized water at a concentration of 10 µM in order to minimize theionic concentration of the sample solution. After loading the A�

solution into our submicrometer gap electrode, the scattering imagesand DRS signal are collected for 48 at 4 h intervals by using adark-field microscopy system with a true-color imaging charge-coupled device camera and dielectric analyzer (Novocontrol)(Figure 1b).

In DRS measurement, the resonance peak in the imaginary partof the permittivity is shifted from 1900 to 850 Hz and the peakintensity increased 67% (Figure 2a). On the other hand, in the caseof the deionized water as a negative control experiment, the peakis not changed and the intensity is increased by only 7%(Supporting Information Figure 1). Assuming that the overalleffective dipole moment of the aggregated A� is relatively smallcompared with soluble A� peptide due to the size, this change indipole moment during the fibrilization can induce longer momen-tary delay with respect to the alternating electrical field, and theresonance peak shift to the lower frequency as well as intensitygrowth in permittivity are observed.

Moreover, the effective hydrodynamic radius is calculated tofurther characterize the oligomerization and aggregation of A�based on the DRS result. First, the Cole-Cole equation as adielectric relaxation model is applied to obtain the relaxationfrequency24,25

where

Figure 1. The schematics of the quantification for amyloid-� fibrilization by the optical and dielectric analysis system. (a) Amyloid-� polymerizationsteps in neurons. (b) Schematic diagram of experimental setup and the quantification of amyloid-� fibrillization by optical dark-field imaging and correspondingdielectric relaxation spectroscopy (DRS).

ε* ) ε∞ + ∆ε1 + i(ωτ0)

1-R (1)

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ε* is the complex dielectric constant, f is the frequency, f* and τ0

are the relaxation frequency and time, respectively, ∆ε is theincrement of the dielectric constant, ε0 and ε∞ are the dielectricconstant at low and infinite frequency, respectively, and R is theempirical parameter that can be derived from the best fit to theexperimental data. Since the Cole-Cole equation generates an arcof circle in which radius is r and center is (a, b) in the ε* complexplane, the DRS signal can be fitted into a form of

Then, using eqs 1-3, ε0, ε∞, and R can be derived as

Finally, based on the calculated result, the relaxation timecan be obtained as

where N is the number of the data points. From this relaxationfrequency, the effective hydrodynamic radius r which representsthe status of the fibrilized A� can be calculated using the equationunder the assumption that the shape of A� is sphere24,25

where η is the viscosity of the solvent. Since the calculated effectivehydrodynamic radius is directly related with the motion of gyration,the fibrilized A� structure can be quantified with this radius. Figure2b shows the calculated hydrodynamic radius change as a functionof incubation time. It indicates systematic increase from 19.1 to20.8 nm with increasing time. By stark contrast, the control showsthat any change in hydrodynamic radius without A� except theincrease in a standard deviation from 0.514 to 2.15% was notobserved. The regression coefficient in the hydrodynamic radiusversus the incubation time is higher than 0.97 for 4 days ofmeasurements.

In optical dark-field imaging, the scattered intensity by a singlesmall particle from an unpolarized light input of wavelength λ andintensity I0 is given based on the equation of Rayleigh scattering

where R is the distance to the particle, θ is the scattering angle, nis the refractive index of the particle, and d is the diameter of theamyloid-� particle.

Therefore, even small changes in the size of fibrilized A�can induce huge different scattering intensity, becausescattering intensity is proportional to the sixth order of thediameter of fibrilized A�. Upon fibrilization, soluble A�peptide gradually transforms into large-size insoluble oligo-mers or plaque. Therefore, as shown in Supporting Informa-tion Figure 3, bright dots after 16 h are clearly resolved in

Figure 2. DRS measurement of amyloid-� fibrillization. (a) Therepresentative in situ complex permittivity (ε′′) and (b) calculatedeffective hydrodynamic radius (RH) change as a function of incubationtime (t). The concentration of amyloid-�42 is 10 µM.

ε* ) ε′ - iε′′, ω ) 2πf, τ0 ) 2πf*, ∆ε ) ε0 - ε∞(2)

(ε′ - a)2 + (ε′′ - b)2 ) r2 (3)

ε0 ) √r2 - b2 + a, ε∞ ) √r2 - b2 - a

R ) -2 sin-1(b

r )π

(4)

τ0 ) 1N ∑

k)1

N1

2πfk((ε0′ - ε∞′ )2 + εk′′

(εk′ - ε∞′ )2 + εk′′)1/[2(1-R)]

(5)

Figure 3. Quantification of amyloid-� fibrilization by optical dark-field imaging. Total scattering intensity (IScat) changes as a function ofincubation time (t).

Figure 4. Hydrodynamic radius changes as a function of total Rayleighscattering intensity.

r ) ( kBT

8π2ηf*)1/3

(6)

IScat ) I01 + cos2 θ

2R2 (2πλ )4(n2 - 1

n2 + 2)2(dAmyloid

2 )6

(7)

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the dark-field image, and the number of dots increases withrespect to the incubation time. Consistently, total scatteringintensities in Figure 3 also correlate well with the dark-fieldimages. These results suggest that the fibrilization processof A� can be resolved optically via the dark-field imaging.As shown in Figure 4, DRS results (i.e., the calculatedhydrodynamic radii) are in a good agreement with the dark-field images showing a linear increase in the scatteringintensities.

In conclusion, noninvasive monitoring of nanoscale A�fibrilization by simultaneous label-free optical dark-fieldimaging and DRS is successfully performed with transparentelectrodes. Oligomerization and aggregation of A� causeschanges in hydrodynamic radius and scattering intensity,which are observed by DRS and dark-field imaging systems.Therefore our dual-mode electrode prepared via a simple andcost-effective method can give two independent sets ofqualitative and quantitative information at the same time. Wefurther expect our optofludic DRS and dark-field imaging-based dual-mode detection to impact on a wide range ofresearch areas in neurodegenerative disease such as Alzhe-imer’s disease pharmacology and molecular diagnostics.

Acknowledgment. This work was supported by the Na-tional Science Foundation (Award Number CBET-0239333)and the Center for Nanostructured Materials and Technology(CNMT) under the 21st Century Frontier Research Programsof the Korea government.

Supporting Information Available: Control experimentresults, fabrication steps for transparent submicrometer gapdevice, and representative time-resolved dark-field images.This material is available free of charge via the Internet athttp://pubs.acs.org.

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