J. Biomater. Sci. Polymer Edn, Vol. 15, No. 3, pp. 297–310 (2004)Ó VSP 2004.Also available online - www.vsppub.com

Flow-stress-induced discrimination of a K-ras pointmutation by sandwiched polymer microsphere-enhancedsurface plasmon resonance

YASUNOBU SATO, YUKA SATO, AYA OKUMURA, KOJI SUZUKIand HARUMA KAWAGUCHI¤

Department of Applied Chemistry, Faculty of Science and Technology, Keio University,3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan

Received 25 August 2003; revised 7 November 2003; accepted 7 November 2003

Abstract—The highly sensitive detection of a K-ras point mutation with the aid of DNA-carryingmicrospheres as a � ow-stress receptor is proposed at the surface of a surface plasmon resonance (SPR)biosensor. Single-stranded DNAs were immobilized onto epoxy-group-derivatizedgold surfaces andthe hybridization of DNA targets was monitored. The subsequent interaction with DNA-carryingmicospheres enhanced the SPR response. The increase of � ow rate during the event of dissociationchanged the amount of detachment of the DNA-carrying microspheres for the mismatched pair. Inaddition, the viscosity was changed by addition of glycerol to the buffer. The increase of shear stressfrom the � ow resulted in detachment of DNA-carrying microspheres hybridized with the mismatchedsequence and increased the ability to discriminate a point mutation. This is a new method whichnot only increases the lower detection limit of evanescent wave-based biosensors, but also the abilityto discriminate a point mutation which is a critical factor for ultrasensitive DNA detection in � owdevices.

Key words: K-ras codon 12; point mutation; surface plasmon resonance (SPR); polymer microsphere;sandwich method; � ow shear stress

INTRODUCTION

Determination of speci� c nucleic-acid sequences within a DNA sample of unknowncomposition is of great interest in all life sciences and is used for diagnosticand analytical purposes. In the area of diagnostics there is a demand of earlyand fast detection of cancer and various types of diseases; therefore, nucleic-acid ampli� cation and detection have become increasingly important [1–3]. The

¤To whom correspondence should be addressed. Tel.: (81-45) 566-1563. Fax: (81-45) 564-5095.E-mail: haruma@applc.keio.ac.jp.

298 Y. Sato et al.

detection of the speci� c G–G mismatch in K-ras codon 12, which is frequentlyfound in pancreatic cancer [4], is an important task required for clinical usage.Surface plasmon resonance (SPR) biosensors are promising tools for detectingbiological substances [5, 6]. There are now reports on detecting target DNA samplesthat are small in molecular weight and low in concentration by ‘sandwich’ methods,which use gold nanoparticles and other substances, such as proteins, which actas a second probe [7–9]. We have introduced the usage of microspheres withaf� nity [10] for highly sensitive DNA detection with SPR biosensors and ourinterest here is in introducing a novel detection method based on microspheresin a � ow detection system for point mutation discrimination. Micro-� uidic-basedchip devices offer new opportunities in the � eld of clinical usage where automationis required and the combination of microspheres with micro-� uidic technology isan area of interest [11]. From past research we have come to the � nding that thewashing procedure of the SPR device (BIAcore2000) detaches the DNA-carryingmicrospheres with the mismatched sequence. Swift et al. have reported on the effectof � ow on the adhesion of cells mediated by receptors [12] and Zhang et al. havereported the possibility of the usage of hydrodynamic � ow forces on microspheredetachment in DNA detection by using avidin-coated slides and microspheres [13].In this paper we discuss a similar approach in a more practical manner by usinga sandwich method and by focusing in the change of � ow rate and the viscosityof the medium which affects the shear rate that acts as a force on DNA-carryingmicrospheres. The microspheres with af� nity were prepared by the conjugationof DNA with polymer microspheres which were prepared by soap-free emulsionpolymerization. The results show that � ow stress can be used to improve the abilityto discriminate a point mutation at the surfaces of biosensors employing DNA-carrying microspheres.

MATERIALS AND METHODS

Materials

1,6-Hexanedithiol was purchased from Aldrich (USA), Streptavidin (SA) from ICNBiomedicals (USA), EZ-Link™ Biotin-PEO-Amine was from Pierce (USA), eth-ylene glycol diglycidyl ether (EGDE), Na2HPO4, NaH2PO4, NaCl for PBS bufferand NaOH for regeneration of sensor surfaces were from Wako (Japan). Ethanolspecial-grade was from Junsei (Japan) and non-ionic detergent tert-octylphenoxypoly(oxyethylene)ethanol (IGEPAL CA-630) from Sigma (USA). For preparationof polymer microspheres, styrene (St) special-grade reagent was distilled underreduced pressure (46±C, 21.5 mmHg), glycidyl-methacrylate (GMA) � rst-gradereagent was distilled under reduced pressure (33±C, 2 mmHg), divinylbenzene(DVB) was used without further puri� cation, 2,20-Azobis(2-methylpropionamidine)dihydrochloride (V-50) and mercapto acetic acid (MAA) were used as receivedfrom Wako (Japan). 1-Ethyl-3-(3-dimethyl-aminopropyl)carbodiimide hydrochlo-ride (EDC) was from Dojin (Japan), Seamless Cellulose Tubing (MWCO 1:4£104)

Flow-stress-induceddiscrimination of a K-ras point mutation 299

from Viskase and Oligreen ssDNA quantitation reagent kit from Molecular Probes(Leiden, Netherlands) were used as received. All oligonucleotides were chemicallysynthesized by JBios (Japan) and puri� ed by column prior to use. The biotinylatedDNA11 ligand which detects the point mutation of DNA analytes (50-Bio-GCCACG AGC TC-30) had a triethyleneglycol spacer (BiotinTEG Phosphoramidite)from Glen Research (USA) at the 50-end. The pre� x number of the DNA analytesindicates the length of the analyte and the suf� x the place of the mismatch fromthe 30-end. M is complementary to the DNA ligand, W has a one-base mismatchand neg is without a complementary sequence. 60-mer DNA analytes were used forSPR experiments. 60M8 (complementary): 50-GGA GAG AGG CCT GCT GAAAAT GAC TGA ATA TAA ACT TGT GGT AGT TGG AGC TcG TGG CGT-30,60W8 (one-base mismatch): 50-GGA GAG AGG CCT GCT GAA AAT GAC TGAATA TAA ACT TGT GGT AGT TGG AGC TgG TGG CGT-30, 60neg (negative):50-GGA GAG AGG CCT GCT GAA AAT GAC TGA ATA TAA ACT TGT GGTAGT GTT CTA GAT GTT ATG-30, DNA probe (DNA-NH2: 50-AGC AGG CCTCTC TCC TTT TTT TTT TTT TTT NH2-30), bound to microspheres, has a com-plementary sequence to 60M8, 60W8 and 60neg and hybridizes with the 15-baseend from the 50-end.

Preparation of sensor surface

The SPR experiments utilized Au sensor chips with a 3-nm layer of titanium oxideand a 48-nm layer of gold evaporated onto micro-cover glass slides (9:8£11:5 mm).The bare Au chip was cleaned with 25% H2O2/75% H2SO4 (piranha solution) for30 min to remove organic adsorbate impurities from the gold surface and rinsedwith ethanol and MilliQ water several times. Self-assembled monolayers (SAMs)consisting of thiol units at the surface were prepared by immersing the Au chip into5 mM 1,6-hexanedithiol in ethanol for 24 h at room temperature as reported [14].After thorough rinsing with ethanol and MilliQ water the SAM-coated chip wasimmersed in 1 ml EGDE at 37±C for 24 h to introduce epoxy groups to the surface,which is a technique also used in the coating of microsphere surfaces for minimizingnon-speci� c protein adsorption [15]. After thorough rinsing with MilliQ the surfacewas dried with N2 air and 200 ¹l of 10 mM Biotin-PEO-amine in sodium phosphatebuffer (pH 9.4) was deposited on the surface and incubated at 37±C for 24 h.After preparation of Biotin chips the chips were kept under N2 condition andstored in a refrigerator. Biotin chips were docked to the BIAcore 2000 system andpreconditioned with three consecutive 1-min injections of 10 mM NaOH before SAimmobilization.

Preparation of polymer microspheres

Basic polymer microspheres were prepared by soap-free emulsion copolymeriza-tion. St, GMA and DVB were copolymerized in a soap-free aqueous mediumusing V-50 as a polymerization initiator according to the method described pre-

300 Y. Sato et al.

viously [16, 17]. To introduce carboxyl groups to the surface of SG microspheresMAA was reacted with epoxy groups originated from GMA at 70±C, pH 10, ad-justed by addition of NaOH and reacted for 24 h. The resulting carboxylated SGmicrospheres were washed by centrifugation/decantation/redispersion in distilledwater three times and dialyzed by cellulose tubing by repeated change of water toremove unreacted MAA. The surface concentration of carboxyl groups was deter-mined by conductometric titration. The hydrodynamic size of microspheres wasmeasured by photon correlation spectroscopy (PCS), PARIII s from Ohtsuka Elec-tronics (Japan).

DNA conjugation to carboxylated SG microspheres

2.5 mg of carboxylated SG microspheres were washed three times with 400 ¹l ofPBS (150 mM NaCl in 10 mM Na2HPO4 NaH2PO4, pH 7.4) buffer and amino-functionalized DNA was conjugated using 100 ¹l of 50 mM EDC and 100 ¹l of20 ¹M DNA-NH2. The coupling reaction was carried out at 4±C for 24 h. Afterconjugation the DNA-carrying microspheres (SG-DNA) were centrifuged in a mi-crocentrifuge at 1:5 £ 104 rpm for 15 min and washed three times with PBS buffer.The amount of immobilized DNA probes onto SG microspheres was determinedfrom the difference between the initial and the residual oligonucleotide concentra-tion deduced from the supernatant and the wash solutions. Oligonucleotide quan-titation was performed by Oligreen ssDNA quantitation reagent, which is an ultra-sensitive � uorescent nucleic-acid stain for quantitating ssDNA in solution [18]. The� uorescence was recorded using a Fluorometer, Fluoroskan Ascent FL from Lab-systems (Finland). The SG-DNA was then suspended in 400 ¹l of SPR runningbuffer (150 mM NaCl in 10 mM Na2HPO4/NaH2PO4 (pH 7.4), 0.05 wt% IGEPAL)and prepared for SPR experiments.

SPR experiments and sandwich assay

A schematic diagram of the experiment is shown in Fig. 1A and the SPR � owcell in Fig. 1B. All ligand/analyte hybridization experiments were performed ona Biacore2000 biosensor and all experimental data were evaluated by Biaevaluation3.1 from Biacore (Sweden). SPR running buffer was chosen for SPR experimentsand was run until the sensorgram came to a steady state. 50 ¹l of 10 ppm SA inSPR buffer was injected and immobilized to the biotin sensor surface. Biotinylated11mer DNA ligand was dissolved in SPR running buffer at a concentration of100 nM and immobilization was carried out at 25±C at a � ow rate of 5 ¹l/minusing the Manual inject mode which makes it possible to stop a sample injectionat a desired time. Non-immobilized ligands were removed by washing the surfacewith 5 ¹l of 10 mM NaOH.

After immobilization of DNA ligands, 500 nM of complementary (60M8),mismatched (60W8) and non-complementary (60neg) 60mer DNA analytes (50 ¹l)were injected to the sensor surface as presented in Fig. 2 (Phase I) using the Kinject

Flow-stress-induceddiscrimination of a K-ras point mutation 301

Figure 1. Schematic representationof SPR experiments. (A) Hybridizationof DNA analytes to DNAligands (Phase I), signal enhancement by SG-DNA microspheres (Phase II) and change of � ow rateduring the dissociationphase (Phase III) in a DNA point mutation detection SPR system. (B) Diagramof the SPR � ow cell used for experiments.

302 Y. Sato et al.

command which is a method to minimize sample dispersion at the beginning andend of sample injection. All DNA hybridization experiments were carried outat a � ow rate of 5 ¹l/min. The SPR signal of response enhancement by SG-DNA microspheres was recorded immediately after the DNA analyte hybridizationexperiment (Fig. 2, Phase II). 50 ¹l of 0.625 wt% (2.5 mg in 400 ¹l of runningbuffer) of SG-DNA microspheres was injected and hybridization was measured.During the dissociation phase the � ow rate was changed (Phase III) from 0 ¹l/minto 50 ¹l/min to examine the amount of detachment of SG-DNA microspheres.In order to regenerate the surface 10 mM NaOH aqueous solution was injectedtwice for 1 min at the end of each experiment. Response of enhancement bySG-DNA was taken as the difference between the SPR signals before and afterinteraction.

The effect of viscosity of the solution was examined by adding glycerol to thebuffers in which SG-DNA microspheres were suspended. In addition, the effect ofglycerol on the stability of DNA was examined by performing a separate experimentby adding glycerol to the buffers in which DNA was dissolved. The effect oftemperature was examined by increasing the temperature of the SPR system from25±C to 35±C.

Figure 2. Sensorgrams of the hybridization of 500 nM 60mer DNA analytes interacting withDNA ligands and response enhancement by SG-DNA microspheres at a concentration of 0.625 wt%at 25±C. Non-speci� c adsorption of SG-DNA microspheres to the sensor surface is shown as aninteraction with 60neg. All hybridization experiments were performed by injecting analytes and SG-DNA microspheres at a volume of 50 ¹l (10 min interaction) using a � ow rate of 5 ¹l/min in SPRrunning buffer.

Flow-stress-induceddiscrimination of a K-ras point mutation 303

RESULTS AND DISCUSSION

Preparation of sensor surface and immobilization of SA and DNA ligand

A surface of densely packed dithiol/gold self-assembled monolayers (SAMs) wasprepared and EGDE was reacted to give a surface with epoxy groups as shown inFig. 1. The assembling of SAMs and the reaction with EGDE, were con� rmedfrom non-speci� c adsorption tests of SA. When the surface was coated with EGDEthere was no adsorption of SA to the surface. This result shows the effect ofEGDE as a surface of low non-speci� c adsorption of proteins and an easy wayto prepare a sensor surface that could be used for biological substances, which hasbeen proposed using latex microspheres by our group previously [15]. Althoughimmobilization of thiolated DNA to a gold surface is a common way to prepareDNA chips, the above method decreases non-speci� c adsorption of proteins andreduces steric hindrance by giving a space between ligands for incoming analytes forhybridization. The amount of SA immobilized to the sensor surface was typicallyin the order of 1000 RU corresponding to 1.2 ng of SA in an area of 1.2 mm2. Inthis case one SA molecule occupies an area of 180 nm2 which shows a non-closelypacked structure at the surface. The DNA ligands were then immobilized to thesurface by manual injection and showed a small increase in response as 80 RU,which corresponds to one DNA ligand to one SA molecule which is estimated tobe 1:24 £ 1012 molecules/cm2 . This density of DNA ligand is lower compared tothat of reported ones which use covalent immobilization of thiolated DNAs to thesurface of SAM surfaces [7].

Preparation of SG microspheres and conjugation with DNA probes

Introduction of carboxyl groups by the reaction of MAA with the epoxy groupsoriginated from GMA was successful and from conductometric titration the car-boxyl groups were quantitated as 24.4 groups/nm2 . The polydispersity with regardto the hydrodynamic diameter evaluated by PCS revealed a narrow size distribu-tion, Dw=Dn D 1:02, with Dw D 199 nm as the weight average diameter andDn D 195 nm as the number average diameter. Carboxyl groups originated fromMAA were used as a reactive group with 30-amino-modi� ed DNA probe and wasconjugated by the water-soluble coupling agent EDC [19]. The amount of the DNAin the wash solutions was determined by Oligreen staining method which is capa-ble of quantitating ssDNAs. As a result on average, 500 DNA strands/microspherewere con� rmed to be bound.

Interaction of DNA/DNA and response enhancement by SG-DNA microspheres

Figure 2 represents an example of SPR experiments in which 500 nM of DNA an-alytes are interacted with the DNA-ligand-immobilized surface (Phase I), responseenhancement by SG-DNA microspheres (Phase II) and � ow-rate change during thedissociation phase (Phase III). Although it is dif� cult to discriminate the response of

304 Y. Sato et al.

the fully complementary analyte (60M8) from that of the mismatched one (60W8) inPhase I, the enhancement made available by the interaction with SG-DNA is shownin Phase II. It is clear that the SG-DNA microsphere acts as a response enhancerwhich is a result of the increase in dielectric constant at the near sensor surfacebased on the theory of evanescent wave based sensors. In addition, from Phase IIthe weak af� nity of W, due to a single mismatch which makes the DNA hybrid,unstable is revealed. The regeneration by 10 mM NaOH shows the dissociation ofDNA analytes and SG-DNA microspheres by alkalinity which destabilizes the hy-drogen bonds between the DNA strands. In addition, when using the same chip andsamples, reproduction of results was con� rmed, showing that the detachment of themicrospheres were not the desorption of SA molecules from the surface which wasanticipated by Zhang et al. [13]. Although SA was bound by avidin/biotin interac-tion at the surface, our system showed a practical application of the usage of � owwith microspheres for point mutation discrimination.

The effect of � ow rate on DNA-carrying microspheres

Phase III, where the � ow rate was changed, is enlarged in Fig. 3 and the y-axisis normalized for comparison. This result indicates that the change in � ow ratehas a different effect on the stability of microspheres in a sandwiched geometry

Figure 3. Magni� cation and comparison of the event of dissociation (Phase III) between 60M8 and60W8 where the � ow rate is changed. The � ow rate of the dissociationphase is changed consecutivelyas follows: 5 ! 0 ! 10 ! 0 ! 25 ! 0 ! 50 ! 0 ! 5, and the y-axis is normalized forcomparison. The concentration of 60mer DNA analyte was 500 nM and response enhancement wasmade by the interaction of 0.625 wt% SG-DNA microspheres at 25±C.

Flow-stress-induceddiscrimination of a K-ras point mutation 305

which are hybridized to the analytes. For the complementary strand (60M8) theincrease in the � ow rate from 0 ¹l/min results in a small increase in response andthen shows a gradual decrease. This small increase could be explained by theapproach of the SG-DNA microspheres to the sensor surface by the increase in� ow shear stress. There are reports on the deformation of cells at the surface whichalso increase the response at the surfaces of evanescent wave-based biosensors [20].The result could be explained by the occupation of the evanescent � eld whichexponentially decreases from the sensor surface resulting in a change in intensityof the response. On the other hand, with the mismatched strand (60W8), theincrease in the � ow results in an immediate decrease in response, correspondingto a detachment of SG-DNA microspheres from the sensor surface. This could beunderstood by the decrease of the strength of binding force between the ligandand analyte where a mismatch exists. There are several reports on this area wheremechanical pulling is attained between double-strand DNAs [21–23]. It could bepredicted that the DNA with a mismatch has a weaker binding force to withstandthe pulling compared to that of the complementary sequence. For comparison ofthe ability to discriminate a point mutation, SPR response values of SG-DNA wereobtained from the sensorgrams at point A and point B in Fig. 3 and the ability todiscriminate a point mutation was calculated using equation (1).

Ability to discriminate a point mutation (%) DÁ

1 ¡ RU of mismatched strand WRU of complementary strand M

!£ 100 (1)

The ability at point A is 64% and after the increase of � ow rate the ability in-creased to 81%, showing a 17% increase in the ability to discriminate a point muta-tion. SG-DNA microspheres within a � ow detection system in a sandwich geometrynot only increases the response but also increases the ability to discriminate a pointmutation providing a better method for all or none based DNA sensors which areneeded in practice.

Effect of viscosity and temperature on the hybridization of DNA-carryingmicrospheres

Figure 4 provides SPR sensorgrams of SG-DNA interaction with various concentra-tions of glycerol. Note that no glycerol was added to the event of DNA hybridiza-tion (Phase I) to assure the same amount of DNA hybridized at the surface so thatthe effect of glycerol on the enhancement of SG-DNA signals could be examined.Figure 4A shows the whole sensorgram from DNA analyte hybridization to SG-DNA interaction. The response of SG-DNA shows a sharp increase at the point ofinjection, followed by a sharp fall. This is because of the differences in the dielec-tric constants between the sample buffer containing glycerol and the SPR runningbuffer. Although it is dif� cult to analyze the responses in Fig. 4A, 3 min after disso-ciation was taken as the response of enhancement in Fig. 4B and 4C and plotted inFig. 5A as a function of glycerol concentration. The addition of glycerol decreasesthe amount of DNA-analyte-mediated SG-DNA microspheres at the sensor surface

306 Y. Sato et al.

(A)

(B)

(C)

Figure 4. (A) Sensorgrams of 60mer DNA analytes interacting with DNA ligands at a concentrationof 500 nM and response enhancement by 0.625 wt% SG-DNA microspheres at 25±C in variousconcentrations of glycerol at a � ow rate of 5 ¹l/min. The arrows represent the point taken as theamount of hybridization. (B) and (C) show the magni� cation of (A) from 1000 s to 2400 s of theevent of SG-DNA interaction with 60M8 (complementary) and 60W8 (mismatched), respectively.

Flow-stress-induceddiscrimination of a K-ras point mutation 307

(A)

(B)

Figure 5. SPR response signals of hybridizationof 60mer DNA analytes at a concentrationof 500 nMand responseenhancementby SG-DNA microspheresat a concentrationof 0.625 wt% at (A) 25±C and(B) 35±C in various concentrations of glycerol. All experiments were performed by injecting DNAanalytes and SG-DNA microspheres at a volume of 50 ¹l (10 min interaction) with a dissociationtime of 7 min using a � ow rate of 5 ¹l/min. DNA hybridization, dependent on the concentration ofglycerol, was performed as separate experiments and the ability to discriminate a point mutation wascalculated and plotted with the ability of SG-DNA microspheres on the second y-axis. The error barindicates the standard deviation (n D 4).

308 Y. Sato et al.

and increases the ability to discriminate a point mutation. Two reasons could ex-plain this phenomenon. One factor is the difference in viscosity of the buffer. Theaddition of glycerol results in an increase in viscosity, which acts as a force to themicrospheres. This could be easily understood from the Stokes relation which givesan idea of how strongly the surrounding � uid resists the motion of the particle, de-pending on the shape and size of particle and the viscosity of � uid. The other effectis the destabilization of the DNA double helix by glycerol, which is a result fromthe increase of the electrostatic interactions between the phosphate groups due to thedecrease in dielectric constant of the medium [24]. However, the ability to discrim-inate a point mutation for DNA hybridization dependent on glycerol concentrationrepresented in Fig. 5A shows that the addition of glycerol causes neither a decreasein the melting temperature, nor an increase in the ability. Therefore, the effect ofviscosity will be the most favorable explanation for the increase in the ability todiscriminate a point mutation in this experiment.

Figure 5B shows a similar experiment performed at 35±C. As shown in Fig. 5B,a decrease in response is con� rmed with the increase of glycerol concentration.This is attributed to the change in viscosity of the medium. However, the abilityto discriminate a point mutation is high in Fig. 5B without the addition of glycerolcompared to that of Fig. 5A. The explanation for the increase in ability is that thedecrease in the af� nity between the mismatched pairs was promoted during PhaseI because the temperature was raised. This could be understood by comparing theabilities of DNA hybridization at 35±C and 25±C without the addition of glycerol,which is the exact same condition where SG-DNA microspheres were interacted.The melting temperature of the mismatched pair is known to be lower than that ofthe complementary pair (38±C from nearest neighbor calculations [25]), which isclose to the experiment temperature 35±C and the decrease of melting temperaturesfor tethered DNAs are reported previously [26]. Another difference in Fig. 5B isthat the response of SG-DNA hybridization with the complementary strand showsa higher value compared to that of Fig. 5A. This is a result of the Brownian motionwhere the microspheres increase their collision with the surface, resulting in anincrease in probability to react with DNA analytes at the surface. Overall, thereis a good chance in increasing the ability to discriminate a point mutation byincreasing the viscosity of the medium which increases the shear stress that actson microspheres.

CONCLUSIONS

This work demonstrates a successful approach of increasing the ability to discrimi-nate a point mutation by the usage of 11mer DNA ligands with the combination ofDNA-carrying microspheres. We found out that the sandwich method with the ef-fect of � ow shear stress increased the ability to discriminate a point mutation. Thisimprovement was attributed to the shear stress from the � ow to the microsphereswhich was con� rmed with the mismatched pair. Glycerol also had an effect on the

Flow-stress-induceddiscrimination of a K-ras point mutation 309

ability which is a result of the increase in viscosity and the destabilization of theDNA hybrids. In addition, an increase in temperature resulted in an increase in in-teraction of microspheres with the sensor surface and an increase in the ability todiscriminate a point mutation by the difference of melting temperature. In this paperDNA-carrying SG microspheres were introduced in a new way, showing that theyare promising tools for SPR response signal enhancers and also for increasing theability to discriminate a point mutation. Furthermore, this method could be appliedfor other biological substances with af� nity and a possibility of using this methodto examine the binding forces between biological molecules.

Acknowledgements

This work was supported by the Grant-in-Aid for the 21st Century COE program‘KEIO Life Conjugated Chemistry’, the project on ‘Molecules, Supra-Moleculesand Supra-Structured Materials’ from the Ministry of Education, Culture, Sports,Science and Technology, Japan and the Sasakawa Scienti� c Research Grant fromthe Japan Science Society.

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