Analytical Biochemistry 287, 261–271 (2000)doi:10.1006/abio.2000.4844, available online at http://www.idealibrary.com on

Implementation of Force Differentiationin the Immunoassay

Gil U. Lee,*,†,1 Steven Metzger,‡ Mohan Natesan,‡ Carolyn Yanavich,§ and Yves F. Dufrene*,2

*Chemistry Division, Naval Research Laboratory, Washington, DC 20375-5342; †School of Chemical Engineering,Purdue University, West Lafayette, Indiana 47907-1283; ‡Geo-Centers, Rockville, Maryland 20852-1633;and §Nova Research Incorporated, Alexandria, Virginia 22308

Received May 9, 2000

A technique has been developed to apply force to theantibody–antigen complex in a solid-phase immunoas-say. Force was applied to the immunochemical com-plex by labeling the secondary antibody with a mag-netically susceptible, micrometer-size particle andplacing the assay chamber in a magnetic field of de-fined magnitude and orientation. The force was strongenough to displace weakly bound particles but was notstrong enough to rupture the immunochemical com-plex. The number of particles bound to the surfaceafter applying the differentiation force was related tothe analyte concentration, thus an optical detectionscheme was developed for counting the number ofparticles on the surface. The sensitivity of the forcedifferentiation assay was demonstrated to be one totwo orders of magnitude higher than conventionalsolid-phase immunoassay techniques for model pro-tein, virus, and bacterial analytes, with 99% specific-ity. The enhanced sensitivity of this assay appears toresult from lowering the assay background throughthe identification of weakly adhesive, nonspecific in-teractions. © 2000 Academic Press

Force is increasingly being used as a parameter forprobing the function and structure of macromoleculesand their assemblies. The recent development of ultra-sensitive micropipette (1, 2), magnetic (3–5), optical (6,7), and microfabricated (8, 9) force transducers nowmake it possible to study inter- and intramolecularinteractions as force is applied to a molecule in a de-

1 To whom correspondence should be addressed. Fax: (765) 494-0805. E-mail: gl@atom.ecn.purdue.edu.

2 Current address: Unite de Chimie des Interfaces, Universite

Catholique de Louvain, Place Croix du Sud 2/18, B-1348 Louvain-la-Neuve, Belgium.

0003-2697/00 $35.00Copyright © 2000 by Academic PressAll rights of reproduction in any form reserved.

fined orientation. Studies of intermolecular interac-tions have resulted in a fundamental understanding ofthe mechanism of molecular recognition (10–13), rep-lication (14), cell growth (4), and motion (15, 16). In-tramolecular force studies have provided direct mea-surements of the mechanical properties ofpolynucleotides (17, 18), polysaccharides (19), andpolypeptides (20).

The measurements of specific intermolecular inter-actions in the streptavidin–biotin (8, 10) and proteinA–antibody (21) systems revealed that the force neces-sary to rupture a specific molecular interaction is afunction of the form of the interaction and the rate atwhich the bond is stressed. The fact that the ruptureforce is a characteristic of the bond energy suggeststhat force may be used to enhance the specificity of areceptor. If the rupture force of a specific molecularinteraction can be identified, for example, the specific-ity and sensitivity of the immunoassay could be en-hanced through the separation of specific interactionsfrom nonspecific interactions. This would dramaticallyreduce the primary source of signal background, falsepositives, and false negatives in the current generationof immunoassays.

One approach to sensing force in the immunoassayhas been to use the atomic force microscope (AFM)3 todetect specific molecular interactions (13, 22–25). Inthis assay, a functionalized probe is scanned over afunctionalized surface on which the analyte is bound(Fig. 1A). The power of this technique is that detailedinformation about the binding interaction can be de-

3 Abbreviations used: AFM, atomic force microscopy; FDA, forcedifferentiation assay; ELISA, enzyme-linked immunosorbent assay;PEI, poly(ethyleneimine); PEG, poly(ethylene glycol); SATA, N-suc-cinimidyl S-acetylthioacetate; SMCC, 4-(N-maleimidomethyl)-cyclo-

hexane-1-carboxylate; BSA, bovine serum albumin; PBS, phosphate-buffered saline; IEP, isoelectric point.

261

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262 LEE ET AL.

rived from force vs distance curves, and microfabri-cated probe arrays have recently been developed thatmake it possible to measure hundreds of distinct inter-actions with a single cantilever (26). However, apply-ing this technique to an assay is practically limited bythe fragile nature of the functionalized surface (27) andthe inherent complexity and cost of the AFM.

Here we describe the force differentiation assay(FDA), which is capable of simultaneously applyingforce to an array of millions of analyte–antibody inter-actions over 1 mm2 areas. As in conventional solid-phase assays, the immunochemical complex sand-wiches the analyte between two antibodies (Fig. 1B).The first antibody is used to immobilize the analyte atthe surface and separate it from the sample matrix.The second antibody is labeled with a magneticallysusceptible microscopic particle, which serves the dualrole of force transducer and label. Using magneticfields of defined amplitude and orientation, 0.1-pico-newton (pN) forces can be transduced to the antibody–antigen complex. This force is large enough to displaceweakly bound particles (the buoyant weight of a parti-cles is 0.003 pN) but is not large enough to displacespecifically bound particles, which may be attached to

FIG. 1. Approaches to implementing force differentiation in immu-oassays. (A) Atomic force microscopy: A microfabricated AFMrobe-cantilever (1) functionalized with antibodies (2) is used toetect the analyte (3) bound at a functionalized surface (4). (B) Forceifferentiation assay (FDA): Magnetic microparticles (5) are bound tohe analyte captured on the surface.

the surface by forces large as 40 pN. The concentrationof analyte is directly related the number of particles

that remain on the surface, as weakly bound particlesare quickly removed from the vicinity of the surface bythe magnetic force. The position of the particles can bedetected either optically, as described here, or throughthe force applied to the particles, as described else-where (28, 29). In this paper, we describe the physicalbasis of magnetic force differentiation, the surfacechemistries developed to implement FDA for an arrayof analytes, and an optical detection scheme, and com-pare the sensitivity and specificity of FDA to the en-zyme linked immunosorbant assays (ELISA).

MATERIALS AND METHODS

Chemicals and Reagents

Poly(ethyleneimine) (PEI) with a mean molecularweight of 300,000 was provided by BASF Chemicals(Polymin SNA, BASF, Rensselaer, NY). Two polyeth-ylene glycol (PEG) derivatives (a-biotin, v-N-hydroxy-succinimidyl ester of poly(ethylene glycol)-carbonateMW 3400 (NHS–PEG–biotin) and a-vinyl sulfone,v-N-hydroxysuccinimidyl ester of poly(ethylene gly-col)-propionic acid MW 3400 (NHS–PEG–VS)) wereprocured from Shearwater Polymers (Shearwater,Huntsville, AL). N-succinimidyl S-acetylthioacetate(SATA), succinimidyl 4-(N-maleimidomethyl)-cyclo-hexane-1-carboxylate (SMCC), and streptavidin werepurchased from Pierce (Pierce, Rockford, IL). FractionV bovine serum albumin (BSA), Tween-20, and allother chemicals were obtained from Sigma (SigmaChemical Company, St.Louis, MO). The polyclonal an-tibodies against ovalbumin, Bacillus globigii and MS2,were the kind gift of Jennifer Aldrich and ThomasO’Brien (Tetracore, LLC, Rockville, MD). Affinity-pu-rified antibodies raised against rabbit IgG in goat (goatanti-rabbit IgG) and raised against goat IgG in rabbit(rabbit anti-goat IgG) were purchased from Kirkegaardand Perry Laboratories (KPL, Inc. Gaithersburg, MD).Sera-Mag magnetic microparticles were obtained fromSeradyne (Seradyne, Inc., Indianapolis, IN).

Surface Chemistries

Figures 2 and 3 schematically illustrate the chemis-tries used to graft antibodies to polystyrene wells andmagnetic particles. The polystyrene surfaces werefunctionalized with dense biotin–PEG monolayers us-ing an approach similar to that described by Metzger etal. (28). Polystyrene 384-well microtiter plates wereobtained from Costar (Corning Costar Corp, Cam-bridge, MA) and cut into smaller 6 3 6-well plates.Each well in the miniplate was oxidized with a 4:1(w/w) KMnO4:H2SO4 solution for 30 s (Fig. 2A) followedby copious rinsing with water. Unless otherwise noted,

all reactions were carried out at room temperature andthe water was prepared with a Milli-Q purification

263FORCE DIFFERENTIATION IN IMMUNOASSAY

system (Millipore, Bedford, MA). PEI was adsorbedonto the oxidized wells by incubation with 5% PEI in 50mM Na2CO3, pH 8.2, for 2 h (Fig. 2B). Biotin–PEGmonolayers were grafted to the PEI surface (Fig. 2D)using 20 mg/ml NHS–PEG–biotin in 50 mM Na2CO3,pH 8.2, for 2 h at 37°C. The functionalized wells weredried under a stream of nitrogen and stored in a drystate until used.

The Sera-Mag particles consist of a magnetite(Fe3O4) core encapsulated in a carboxyl functionalizedpolymer and are nominally 0.85 mm in diameter. The

FIG. 2. Chemical scheme used to immobilize antibodies on poly-styrene surfaces. (A–C) The polystyrene surface is oxidized and thenfunctionalized with high molecular weight poly(ethyleneimine). (D)An a-biotin, v-N-hydroxysuccinimidyl ester of poly(ethylene glycol)-carbonate is grafted to the amine functionalized PEI surface. (E) Thebiotin–PEG surface is then functionalized with antibodies using anantibody–streptavidin conjugate.

particles were coated with 3% PEI in 50 mM carbonatebuffer, pH 8.2, for 1 h (Fig. 3B), magnetically separated

from the PEI solution, and thoroughly washed withcarbonate buffer. Particle aggregates were resus-pended with a microtip ultrasonic disrupter (BransonUltrasonics Corporation, Danbury, CT). The particleswere functionalized with vinyl-sulfone PEG monolay-

FIG. 3. Chemical scheme used to immobilize antibodies on Sera-Mag magnetic particles. (A–C) The particles are functionalized withvinyl sulfone groups using a two-step PEI and a-vinyl sulfone, v-N-hydroxysuccinimidyl ester of poly(ethylene glycol)-propionic acid

chemistry. (D) Thiolated antibodies are subsequently conjugated tothe PEG monolayer.

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264 LEE ET AL.

ers by reacting 40 mg/ml NHS–PEG–VS in 50 mMNa2CO3, pH 8.2, with the PEI-coated particles for 45min (Fig. 3C). The goat anti-rabbit or rabbit anti-goatIgG were activated with sulfhydryl groups using SATAin a 10:1 molar ratio in 50 mM Na2HPO4/NaH2PO4, 1mM EDTA, pH 7.5, buffer for 30 min. The sulfhydrylgroups on the SATA-antibody were deprotected in 50mM hydroxylamine–HCl, 2.5 mM EDTA, 62.5 mMNa2HPO4/NaH2PO4, pH 7.5, buffer for 2 h. Excess

ATA and deacylation buffer were removed by ultra-ltration (Microcon 30, Millipore). Approximately.3 3 1010 Sera-Mag–PEG–VS particles were reacted

with ;700 mg of the SATA activated antibody in 50M Na2HPO4/NaH2PO4, 1 mM EDTA, pH 7.2, buffer

or 3 h (Fig. 3D). The particles were washed twice andesuspended in 50 mM Na2HPO4/NaH2PO4, 0.15 MaCl (PBS) containing 1% BSA.

FM and Zeta Potential Measurements

All AFM measurements were made under aqueousonditions at room temperature (20–25°C) using aommercial instrument (Nanoscope III, Digital Instru-ents, Santa Barbara, CA). Thermal oxide sharpenedicrofabricated silicon nitride cantilevers (Thermo Mi-

roscopes, Mountain View, CA) with a nominal probeadius of 25 nm were used. The AFM probes wereither ozone cleaned (UVO-cleaner, No. 42 Jetlight Co,rvine, CA) or functionalized with a PEG–PEI mono-ayer using the chemistry described by Metzger et al.28). The sensitivity of the AFM detector was estimatedsing the slope of the retraction force curve in theegion of probe–sample contact. For quantitative inter-retation of the force–distance curves, the nonlinearesponse (displacement/applied voltage) of the piezo-lectric actuator of the scanner was corrected using aapacitance-based displacement detector (Capacitec,oston, MA). The cantilever spring constants, ranging

rom 0.01 to 0.6 N/m, were determined using microma-hined reference cantilevers of precisely controlledpring constants (29).The surface electrical properties of the particles were

haracterized after each chemical step with electro-horetic mobility measurements using a Pen Kemazer-Zee-Meter, Model 500 (Pen Kem, Bedford Hills,Y). Freshly prepared particles were resuspended in 1M KNO3 at a concentration of about 108 particles/ml.

The electrophoretic mobility was measured as a func-tion of pH, adjusted with HNO3 or KOH solutions. Thezeta potential z was deduced from the electrophoreticmobility m using the Helmholtz-Smoluchowski equa-tion, i.e., z 5 12.85 m. This equation implies that thesolid plus stagnant layer together behave electrokineti-cally as a solid particle; particles are nonconducting;

particle surfaces are smooth and homogeneous; andthe viscosity, dielectric constant, and ion conductivities

in the diffuse part of the double layer are equal to theirbulk values.

Force Differentiation Assay

The ovalbumin, MS2, and B. globigii assays wereperformed in an indirect sandwich format (Fig. 4). Thedried biotin–PEG wells were rehydrated by washingseveral times with distilled water and once with PBS.The wells were then activated with antibody by incu-bation with the capture antibody–streptavidin conju-gate (Fig. 2E) involving a 1-h incubation of 30 ml ofeagent followed by three washes with 0.05% Tween-20n PBS (PBST). The streptavidin-antibody conjugatesere prepared by crosslinking streptavidin to rabbitnti-B. globigii, goat anti-ovalbumin, and goat anti-

MS2 antibodies using the thiol-maleimide chemistrydescribed previously (28). Serial dilution of the conju-gate on the PEG–biotin surface was used to maximizeanalyte binding and minimize the background for eachconjugate. The sample was incubated with the well,and the appropriate sandwiching antibody was incu-bated with the wells to assemble the immunochemicalsandwich. Finally, anti-rabbit IgG-Sera-Mag or anti-goat IgG-Sera-Mag particles were added to the wells ata concentration and volume that brought the totalnumber of particles in each well to 5 3 107. The wellswere covered with a 35 3 50 mm2 coverslip (FisherScientific Company, Pittsburgh, PA) and incubated forexactly 20 min.

FIG. 4. Indirect, sandwich immunochemistry used in FDA. Theanalyte (3) is bound to a biotin–PEG–PEI polystyrene surface (1)functionalized with high-affinity, analyte-sensitive antibody–streptavidin conjugate (2). The bound analyte is identified with asecondary antibody (4) in a sandwich configuration. The sandwich isthen detected with an anti-secondary antibody, PEG–PEI-coatedmagnetic particle (5).

The total number of particles bound to each well wasmeasured with an optical reader before and after ap-

265FORCE DIFFERENTIATION IN IMMUNOASSAY

plying the magnetic field to the well. The reader wasassembled from an Axiovert 100TV inverted opticalmicroscope (Carl Zeiss Inc., Thornwood, NY) equippedwith a 63 X/0.9 NA water immersion objective operatedin bright-field, transmission mode. Images were ac-quired with a black-and-white 768 3 493-pixel-resolu-tion CCD camera (DAGE-MTI Inc., Michigan City, IN)with analog black level, gain, and stretch control.These images were digitized at videorate with an 8-bitresolution image acquisition card (Data TranslationsInc., Marlboro, MA). A magnetic field was then appliedto the plate with a 25 3 25 3 12.5 mm sintered, grade35 Nd2Fe14B block magnet (Magnet Sales and Mfg. Co.,Culvert City, CA). The magnet was approached towithin 11 mm of the surface of the well in a definedtrajectory. The differentiation force was applied to theparticles for 1 min, after which the magnet was re-tracted from the surface through the same trajectory.These conditions were chosen to maximize the differ-entiation force and remove weakly bound beads fromthe vicinity of the solid–liquid interface.

RESULTS

Implementation of force differentiation in an immu-noassay in the complex sample matrices encounteredin most bioanalytical environments requires control ofboth the nonspecific and the specific forces. In thissection, we describe the surface chemistry that we de-veloped to control the particle–surface interactions,and the performance of the FDA for three model ana-lytes.

Characterization of the PEG–PEI Chemistries

Atomic force microscopy was used to characterizethe physical properties of PEG–PEI monolayersgrafted to polystyrene surfaces (chemical schemeoutlined in Figs. 2A–2D). Force measurements weremade on PEG–PEI monolayers with bare siliconoxynitride probes and probes functionalized withPEG–PEI monolayers. Heterogeneity in the surfacechemistry was characterized by measuring forcecurves in a 16 3 16 array over several 1 mm2 areas ofthe surface. Measurements made with bare probeson the PEG films showed signs of variations in sur-face chemistry. The majority of force curves showedno signs of adhesion (Fig. 5A). However, some of theforce curves showed forces that are characteristic ofintramacromolecular interactions. Examination ofindividual force curves (Fig. 5B) reveal multiple un-binding events; moreover the form of adhesion ischaracteristic of the elongation of individual poly-mers (18). The ratio of adhesive to nonadhesive force

measurements was found to be probe dependent.Moreover, the population of force curves showing

signs of adhesion was observed to increase with themaximum load applied to the surface. Force mea-surements made on the PEG–PEI monolayer withPEG probes did not show signs of adhesion and werestrictly of the form seen in Fig. 5A.

The electrical properties of the magnetic particleswere measured with zeta potential measurements ateach stage of the surface chemistry used to functional-ize them with antibodies (Fig. 3). Figure 6 shows thezeta potential of the uncoated, PEI-coated, PEG–PEI-coated, and antibody–PEG–PEI-coated particles as afunction of pH. The uncoated particles clearly differfrom the other samples, the isoelectric point (IEP) be-ing around pH 4, which is consistent with the presenceof carboxyl functional groups. The IEP of the otherparticle surface chemistries varied from pH 7.5 to pH8.5, with the zeta potential decreasing in order PEI .

FIG. 5. Force–distance curves measured between (A) PEG-coatedand (B) bare silicon oxynitride probes and PEG–PEI monolayers inwater. The AFM does not measure absolute probe–sample separa-tion distance, but rather relative changes in the probe–sample sep-aration. The surface separation distance was calculated by subtract-ing the cantilever deflection from the probe–sample separation. Thepoint of contact was defined as the surface separation distance at thehighest load.

PEG–PEI . antibody–PEG–PEI particles.

266 LEE ET AL.

Force Differentiation Assay

Figure 7 illustrates the results of a typical forcedifferentiation assay for ovalbumin. Images in the leftcolumn were collected in the wells before force differ-entiation and reveal 200–300 particles in the 126 3 96mm2 image. Images in wells containing 0, 0.1, and 10ng/ml of the analyte ovalbumin are presented in thefirst, second, and third rows, respectively. Images inthe right column were taken in wells after force differ-entiation. They show that the fraction of particlesbound to the surface after application of the magnetforce increased with the concentration of ovalbumin inthe sample. These images reveal that individual parti-cles can be clearly identified with simple bright fieldoptics, and the background of the blank sample wasvery low.

Figure 8 shows the results of the ovalbumin, B. glo-bigii, and MS2 assays. The data are expressed as per-centage of particles bound on the surface after theexposure to the magnetic force for a given analyteconcentration, with the standard deviation calculatedfrom three measurements made in each well. Thesebinding curves demonstrate that the dynamic range ofthe assay was 0.1–10 ng/ml for ovalbumin, 104–108

colony-forming units per ml (CFU/ml) for B. globigii,and 104–107 plaque-forming units per ml (PFU/ml) forMS2. As expected from the indirect sandwich immuno-chemistry, the assay produced positive results athigher levels of analyte concentration, although theconcentration of analyte could no longer be accuratelymeasured.

We define the sensitivity of the assay as the concen-tration of analyte at which the percentage of particlesbound to the surface equals the background in thenegative control plus three times the average standarddeviation observed in the assay. The background for

FIG. 6. Zeta potential measured as a function of pH for “bare”Seradyne particles, PEI-coated particles, PEI–PEG-coated particles,and PEI–PEG–IgG-coated particles.

FDA ranged from 2–8%, with background variationswithin a set of PEG surfaces no greater than 2–4%.

Using these conditions, the FDA sensitivity was 0.1ng/ml ovalbumin, 104 CFU/ml B. globigi, and 105

PFU/ml MS2. This sensitivity was compared to ELISAoptimized using the same antibody reagents and PEGfilms. The ELISA sensitivity was 1 ng/ml ovalbumin,105 CFU/ml B. globigii, and 107 PFU/ml MS2, which isone to two orders of magnitude less sensitive thanFDA.

Specificity of the assay was evaluated in a blindlaboratory trial involving 320 samples that were ana-lyzed over a 10-day period. Ovalbumin was provided atconcentrations of 0.05, 0.1, 1, and 10 ng/ml, with 16replicates of each concentration. B. globigii was pro-vided at concentrations 102, 102.5, 103, 104, and 105

CFU/ml, with 16 replicates of each concentration. MS2was provided at concentrations of 104, 105, 106, and 107

PFU/ml, with 16 replicates of each concentration. Alsoincluded in the trial were 16 samples containing morethan one analyte, 80 samples containing various con-centrations of E. herbicola, and 16 PBS blanks. Of the320 samples only 3 samples were falsely identified;these included one ovalbumin sample and two B. glo-bigii samples. The resulting specificity of the FDA forovalbumin, B. globigii, and MS2 was 99.7, 99.4, and100%, respectively.

DISCUSSION

Physical Properties of the Particle and SubstrateSurfaces

Studies of specific molecular interaction forces usingsingle-molecule techniques indicate that strong inter-actions will produce adhesive forces as large as 150–750 pN (8–10, 18), while weaker antibody–antigen in-teractions will produce forces of 40 pN or less (11, 21).The nonspecific adhesive forces occurring between par-ticles coated with several proteins and polystyrene sur-faces have recently been measured by Sagvolden et al.(30, 31). The force required to displace a particle wasfound to be dependent on the rate of loading of theparticle and the physical properties of the protein coat,with the average rupture force varying from 90 to 277pN for 1-s displacement lifetimes. The fact that non-specific interactions between a protein and polystyrenecan be as large if not larger than specific molecularinteractions makes force differentiation impossible toimplement on surfaces typically used for solid-phaseimmunoassays.

To mitigate the effect of nonspecific protein–surfaceinteractions, we modified the surface chemistry of boththe substrate and the magnetic particle. Antibodieswere covalently immobilized onto the microtiter plateand particles through a 3400-MW PEG layer known tominimize the adsorption of proteins and cells to sur-

faces (Fig. 4) (32). The chemistries used to graft PEG tothese surfaces (Figs. 2 and 3) are based on the adsorp-

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267FORCE DIFFERENTIATION IN IMMUNOASSAY

tion of PEI to a negatively charged surface followed bya reaction with n-hydroxysuccinimide functionalized

EG (33, 34). Characterization of PEG–PEI films onilica with XPS and ellipsometry indicated that theEI forms a thin, conformal monolayer while the PEG

orms a dense, brush-like monolayer at high graftingoncentrations (28, 34). Functional radioisotope assaysnot presented) indicated that the PEG–PEI filmsormed on the oxidized polystyrene surfaces are similaro those formed on silica. Antibodies were attached tohe PEG films on the beads and surface using eitherntibody–streptavidin conjugate (28) or thiolated anti-ody, respectively. The biotin–PEG surface chemis-

FIG. 7. Optical micrographs from the ovalbumin force differentiatio(C and F) of ovalbumin. Images A–C and D–F are before and after amm2.

ries made it possible to prepare the surfaces in ad-ance, store them for long periods of time, and activate

them with a suitable antibody conjugate at a defineddensity. It would have been convenient to be able tofunctionalize the magnetic particles with the biotin–PEG chemistry, but we found that this combination ofchemistries led to strong adhesion of the particles tothe surface. We speculate that this adhesion resultsfrom crosslinking of the biotin monolayers though thestreptavidin of the antibody conjugate. Covalent link-age of the antibody directly to VS–PEG films produceddense PEG films with high antibody activity.

Electrophoretic mobility measurements were carriedout at each step of the surface-coating process to vali-date the efficiency of the different treatments and ac-

ssay results for 0 ng/ml (A and D), 0.1 ng/ml (B and E), and 10 ng/mlication of the magnetic field, respectively. Field of view is 126 3 96

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cess the physical state of the surface. The electricalproperties of bare particles (Fig. 6) appear to be con-

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268 LEE ET AL.

trolled by acidic groups, as shown by the low IEP (;pH). In contrast, the higher IEP values (pH in the rangef 7.5–8.5) of PEI-coated particles indicate that electri-al properties are primarily determined by surfacemine groups, which are indicative of the presence ofrimary, secondary, and tertiary amine groups of PEI.he small shift in IEP for PEG–PEI-coated particlesay reflect a decrease in the number of ionizable

mine groups, as a result of the reaction with NHS–EG. The IEP measured for the PEG–PEI and PEI-oated particles is very close to values found for PEG–EI films on quartz substrates (35). The lower absolutealues of zeta potentials observed over the whole pHange for PEG–PEI and IgG–PEG–PEI-coated parti-les, compared to PEI-coated particles, may be attrib-ted to an outward shift in the plane of shear. Burns etl. (35) also observed a decrease of electroosmotic mo-ility when comparing PEI-coated quartz substrataith PEG–PEI-coated quartz substrata, an effect that

ncreased with the PEG molecular weight. The facthat the zeta potential of IgG–PEG–PEI particles isven more reduced reflects an additional outward shift

FIG. 8. Typical binding curves obtained by FDA for the detection of(A) ovalbumin, (B) B. globigii, and (C) MS2. The percentage ofparticles bound after the magnet application is plotted against con-centration of the analyte. The readings are the mean of triplicatemeasurements with error bars representing the standard deviation.

n the plane of shear and a shift in IEP associated withhe antibody layer. It is interesting to compare the very

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ifferent electrical properties of the samples at pH 7.5,hich is the pH at which FDA is performed. Contrary

o COOH- and PEI-coated particles, which show signif-cant negative and positive net surface charge, respec-ively, the IgG–PEG–PEI particles are close to electri-al neutrality. This electrical neutrality will minimizeonspecific ionic interactions between the proteinsrafted to the particles and surfaces.The AFM measurements made on the PEG–PEIonolayers with PEG–PEI functionalized probes show

epulsive forces together with a lack of adhesion uponetraction (Fig. 5A). Since both surfaces were treatedith the polycation PEI, one may argue that the repul-

ion may result from electrostatic double-layer forcesetween the two positively charged surfaces. However,his interpretation seems unlikely since the AFM mea-urements were performed at pH 5.5, 7.5, and 8.2, ando significant change in surface force could be detectedFig. 6). The observed repulsion may therefore be at-ributed to steric repulsive forces that have been mea-ured between dense PEG layers grafted to mica (36,7). Steric repulsion is thought to be the physical basisor the resistance of PEG monolayer to protein adsorp-ion (36–38), although this is clearly a very complexnteraction. The adhesive interactions measured be-ween the PEG monolayer and the bare AFM probeFig. 5B) are indicative of a strong interaction betweenhe silicon oxynitride surface and the copolymer. Thelongated adhesive force reflects the stretching of PEI,s the observed 100-nm rupture length is significantlyonger than the length of a fully stretched 3400-MWinear poly(ethylene glycol) molecule. We note that thisbservation is consistent with the “bridging” of poly-ers suggested by studies of physically adsorbed poly-

tyrene on mica at low grafting densities (39–41). Thedhesive forces between PEG monolayers and dissim-lar surfaces were the basis for designing surface coat-ngs for both the surface of the microtiter plate andarticle with PEG films.

agnetic Force Differentiation

The uniformity of the force applied to the antibody–ntigen bonds formed between the array of particlesnd surface determines the force sensitivity of thisssay. In this section, we describe the role that thearticle and field-generating device play in producing aagnetic force and the transduction of that force to the

ntibody–antigen bond. The magnitude and orienta-ion of the magnetic force F applied to a particle is aharacteristic of the particle and the magnetic field,

F 5 M~B! v dB/dz, [1]

here M(B) is the magnetization of the particle, v ishe volume of the particle, and dB/dz is the magnetic

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269FORCE DIFFERENTIATION IN IMMUNOASSAY

field gradient at the particle. Microscopic magneticparticles have found applications in biotechnology incell separation, protein/nucleic acid purification, andelectrochemiluminescent/fluorogenic immunoassays(42). Commercial particles are typically designed to beweakly paramagnetic to avoid spontaneous coagula-tion and make them reusable for separation applica-tions. The Sera-Dyn particles were chosen for theiruniform size and surface chemistry. The measuredmagnetic susceptibility of these particles is 45 emu/cm3

at 5000 Oe, which is a factor of 5 higher than othercommercially available microparticles we considered.

The magnitude of the force tranduced to the anti-body–antigen bond is highly dependent on the orienta-tion of the magnetic field. Components of the force inthe plane of the surface FT will cause the particles toroll away from the antibody–antigen bond, producing apivot point through which torque is transduced to theantibody–antigen bond (Fig. 9) (43). The torque willamplify the in-plane force FT transduced to the anti-body–antigen bond,

T 5 FT Î a2~L 2 h!

, [2]

here T is tensile load, a is radius of the particles, h isthe bead-surface separation resulting from surfaceroughness, and L is bond length of the specific molec-ular assembly. For the particles and molecular recep-tor properties used in these experiments, a .. L or h;hus off-axis forces are amplified by as much as anrder of magnitude by torque. Clearly, careful orienta-

FIG. 9. Geometry of a single binder-analyte attachment to a sphereunder a tangential load. Here a is the radius of the particles, h is theeparation distance between the particles and plate, L is the bondength, T is the tension of the bond, N is the normal nonspecific force,nd F and t are the external force and torque exerted on the particle,espectively.

ion of the externally applied magnetic field is requiredo apply a uniform force to the binder–analyte complex.

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The magnetic field was generated with a single per-anent magnet placed at a known distance from the

ssay surface. A 25 3 25 3 12.5-mm rare-earth mag-et, magnetized along its minor axis, was used to max-

mize the field gradient normal to the assay surfacend minimize the off-axis field gradient. A grade 35d2Fe14B magnetic material was chosen for its high

residual flux density (12,300 G) and intrinsic coerciveforce (11,300 Oe). The force transduced to a particle ata known separation can be calculated using the mea-sured magnetic field, magnetic field gradient, and mag-netization of the particle with Eq. [1]. The field gener-ated by the magnet (Fig. 10) was characterized using aHall effect probe and finite-element modeling. The fi-nite-element modeling, confirmed by the magnetom-eter measurements, indicates that off-axis componentof force across the well at the 11-mm separation dis-tance is no greater than 2% of the total force. Themaximum force applied to the Sera-Mag particles withthe magnet was calculated to be 0.4 6 0.3 pN. Theuncertainty in the absolute force applied to the particleresults from the variation in the particle-to-particlemagnetization and the accuracy in placing the magneta specific height above the assay surface.

The force applied to the particles in this assay is notlarge enough to rupture the weak antibody–antigen orthe strong biotin–streptavidin bonds that form in theimmunochemical complex. However, it is strongenough to displace particles that settle to the surfaceunder their own buoyant weight or that are weaklybound through nonspecific interactions. These resultsare consistent with single molecule measurements of

FIG. 10. Magnetic field generated by a 25 3 25 3 12.5-mm Grade5 NdFB magnet that was measured with a Hall effect probe (GroupTeslameter, Auckland, New Zealand). The effect of modifying the

hape of the magnet was investigated by adding cone-shaped, high-ermittivity material to the magnet.

gtcsphsw

tpmiffitcaipbsb

fidnmor

270 LEE ET AL.

strong and weak specific biomolecular interactions (2,8, 9, 11, 12, 21).

Specificity and Sensitivity

The blind trial demonstrated the specificity of theforce differentiation assay is .99% for ovalbumin, B.lobigii, and MS2, which represent model protein, bac-erial, and virus analytes, respectively. This trial wasonducted with a large number of blank/cross-reactingamples and samples in which multiple analytes wereresent. In all cases in which ELISA was scored FDAad a superior specificity. We attribute the enhancedpecificity of the FDA to the force used to identifyeakly bound particles.The sensitivity of a solid-phase immunoassay is de-

ermined by many factors, including the mass-trans-ort conditions and the affinity of the receptor. Theass-transport conditions were characterized by vary-

ng the incubation time of the analyte with the surfaceor 10, 60, and 180 min. No difference could be identi-ed in the 60- and 180-min incubation periods, and theotal number of particles bound to the surface de-reased only slightly at the lowest concentrations ofnalyte for the 10-min incubation period. These resultsndicate the sensitivity of the assay is not mass trans-ort limited, but is limited by the on-rate of the anti-ody–antigen reaction. We attribute the enhanced sen-itivity of FDA over ELISA to a lowering of theackground of the assay.

CONCLUSIONS

We have demonstrated for the first time that forcecan be implemented as a variable in a solid-phaseimmunoassay, resulting in an assay one to two ordersof magnitude more sensitivity than ELISA. By graftingantibodies to dense poly(ethylene glycol) monolayers itwas possible to apply FDA to protein, virus, and bac-terial analytes, in complex matrices with .99% speci-

city. Future improvements in particle and magnetesign promise more accurate control over higher mag-itude forces, which could lead to further enhance-ents in sensitivity. However, the ultimate sensitivity

f this assay is limited by the affinity of the molecularecognition system used.

ACKNOWLEDGMENTS

The authors thank Geir Sagvolden, Tom O’Brien, Jennifer Aldrich,James van Alstine, Leslie Meyers, and Karolyn Hansen for valuablediscussions. We also acknowledge Valerie Browning and YasmineAdrianenses for their assistance with characterization of the mag-netic and electrical properties of the particles. The Office of Naval

Research supported this research, and Y.F.D. thanks the NationalFoundation for Scientific Research of Belgium for financial support.

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