scfv gold description

A High-Affinity Gold-Binding Camel Antibody: Antibody Engineeringfor One-Pot Functionalization of Gold Nanoparticles as BiointerfaceMoleculesTakamitsu Hattori,† Mitsuo Umetsu,*,†,‡ Takeshi Nakanishi,† Satoko Sawai,† Shinsuke Kikuchi,†

Ryutaro Asano,† and Izumi Kumagai*,†

†Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba 6-6-11, Aramaki, Aoba-ku,Sendai 980-8579, Japan‡Center for Interdisciplinary Research, Tohoku University, Sendai, Aoba 6-3, Aramaki, Aoba-ku, Sendai 980-8578, Japan

*S Supporting Information

ABSTRACT: Antibodies, with their high affinity andspecificity, are widely utilized in the field of proteinengineering, medicinal chemistry, and nanotechnology appli-cations, and our recent studies have demonstrated therecognition and binding of antibody for the surface oninorganic material. In this study, we generated a high-affinitygold-binding antibody fragment by a combination of peptide-grafting and phage-display techniques and showed theavailability of the material-binding fragment for one-potfunctionalization of nanoparticles as interface molecules.After a gold-binding peptide sequence was grafted into oneof the complementarity determining regions of a single variable domain of a heavy-chain camel antibody, a combinatorial libraryapproach raised by 20 times the affinity of the peptide-grafted fragment. The high-affinity gold-binding fragment (E32)spontaneously adsorbed on gold nanoparticles, and consequently the nanoparticles formed a stable dispersion in a high-ionic-strength solution. Multivalent and bispecific antibodies constructed on the E32 platform by means of fusion technologyfunctionalized gold nanoparticles in one pot, and these functionalized nanoparticles could be used to obtain surface plasmonresonance scattering images of cancer cells and to spontaneously link two different nanomaterials. Here, we propose the bispecificantibodies as convenient interface molecules in the nanosized world.

! INTRODUCTION

The downsizing of inorganic materials can impart uniqueelectrical, photonic, and optical properties to the materials.Inorganic nanomaterials have been used as probes for biologicalsensing and imaging,1,2 and they can also serve as buildingblocks in bottom-up construction of nanodevices.3,4 Forexample, semiconductor nanomaterials called quantum dotsexhibit size-dependent fluorescence and are widely used forcellular imaging,5 and superparamagnetic iron oxide particleshave been used for signal amplification in magnetic resonanceimaging.6 Homogeneous inorganic nanocrystals with definedshape and surface characteristics self-assemble into square andhexagonal packing,7 and two types of nanoparticles withdifferent sizes and shapes have been shown to assemble into abinary superlattice.8 For both biological and nanotechnologicalapplications, nanoparticles must form stable dispersions insolution and have surfaces designed to interact with targetssuch as proteins, cells, or nanoparticles. However, the methodsfor preparing nanoparticles with these two characteristics varywidely depending on the materials, and thus a trial and errorprocess is required.

One method for preparing nanoparticles with the desiredproperties involves the use of material-binding peptides. Duringthe past decade, several peptides with affinity for nonbiologicalmaterials, such as metals, metal oxides, and semiconductors,have been identified by means of combinatorial libraryapproaches.9,10 Such material-binding peptides have beenused for bottom-up fabrication of bionanotechnology applica-tions, such as patterning and assembly of proteins andnanomaterials,11!13 biofunctionalization of nanoparticles,14,15

and synthesis of crystalline metal nanoparticles.16!18 However,the binding affinities of peptides are often not high enough forsuch applications.19,20 Recently, we focused on antibodies asalternative material-binding molecules because they have higheraffinities and specificities than peptides.21,22 We preparedseveral antibody fragments with high affinity for the surface ofinorganic nanomaterials;20,23,24 the fragments strongly bind tothe surface of specific inorganic nanoparticles, and the resulting

Received: June 15, 2012Revised: August 5, 2012Published: August 8, 2012

Article

pubs.acs.org/bc

© 2012 American Chemical Society 1934 dx.doi.org/10.1021/bc300316p | Bioconjugate Chem. 2012, 23, 1934!1944

nanoparticles can be as stably dispersed as proteins, even inhigh-salt buffer.24

In this study, we demonstrate the potential of material-binding antibodies as interface molecules that can stablydisperse nanoparticles in high-ionic-strength solutions andsimultaneously bind to targets. Gold nanomaterials are amongthe most well-studied nanomaterials. Gold nanostructuresscatter and absorb visible and near-infrared light at a plasmonresonance wavelength that depends on the size and shape of thestructure, and these characteristics have been used forbiosensing,25 imaging,26 and optical nanocircuitry.27 Here, wegenerated antibody fragments with high affinity and specificityfor gold surfaces by means of a method we refer to as the“construction of antibodies by integrating grafting andevolution technology” (CAnIGET, Scheme 1),20 in which the

single variable domain of the heavy chain of a heavy chaincamel antibody (VHH fragment) was functionalized by acombination of peptide grafting and in vitro selection. Thegenerated high-affinity gold-binding VHH fragment (desig-nated E32) strongly adsorbed on gold nanoparticles, whichcould then be stably dispersed in a high-ionic-strength solution.In addition, we designed and constructed two bispecificantibodies by joining two different VHH fragments. A bispecificantibody comprising a VHH fragment with affinity for goldnanoparticles and a VHH fragment with affinity for theepidermal growth factor receptor (EGFR) on cancer cellsenabled us to obtain surface plasmon resonance (SPR)scattering images of the cells; and a bispecific antibodyconstructed from gold-binding and ZnO-binding VHH frag-ments allowed us to spontaneously link two differentnanomaterials. Here, we propose a new methodology for stabledispersion of nanoparticles together with functionalization inone pot by using high-affinity material-binding antibodyfragments.

! EXPERIMENTAL PROCEDURESConstruction of the Expression Vector for VHH

Fragments with a Gold-Binding Peptide in Comple-mentarity Determining Regions (CDRs). The DNAsequence coding VHHGBP1, a VHH fragment with a previouslyreported gold-binding peptide (GBP: LKAHLPPSRLPS)28 inthe CDR 1 loop, was synthesized by means of overlap extensionPCR from a pRA-wtVHH-FLAG plasmid.20 The gene frag-ments were inserted into NcoI!SacII restriction sites of pRA-FLAG vectors to produce the pRA-VHHGBP1-FLAG plasmid.

Construction of a Phage Library and Selection of VHHFragments with High Affinity for Gold Surfaces.Construction of phage library and selection of VHH fragmentswith high affinity for gold surfaces were performed by means ofthe CAnIGET method.20 Briefly, a gene encoding the VHHGBP1fragment with a randomized sequence in the CDR3 loop wasamplified from the pRA-VHHGBP1-FLAG plasmid and insertedinto the phagemid vector pTZ-PsFv2.29 For the selection ofVHH fragments with high affinity for gold surfaces,approximately 109 phages were mixed with 1 mg of goldpowder (particle size <10 μm, Sigma-Aldrich, Tokyo, Japan) ina 10 mM phosphate solution (pH 7.5) containing 200 mMNaCl and 0.05% Tween 20. After the powder was washed withthe phosphate solution five times, residual phages bound to thegold surface were eluted with 500 mM phosphate solution (pH7.5) containing 200 mM NaCl. This panning procedure wasperformed four times, during which the phosphate concen-tration of the washing solution was increased from 30 to 50mM (second wash, 30 mM; third and fourth washes, 50 mM).The amino acid sequences of 100 randomly chosen clones wereanalyzed. The selected VHH genes were inserted into theNcoI!SacII restriction sites in the pRA-FLAG vector forexpression in Escherichia (E.) coli.

Construction of Expression Vectors for BiotinylatedVHH and Bispecific VHH Dimers. To construct bispecificantibodies with affinity for gold and EGFR, and with affinity forgold and ZnO, the gene for E32 VHH was fused at the C-terminus of Ia1 VHH (which has affinity for EGFR30) or 4F2VHH (which has affinity for ZnO20) via a llama IgG2 upperhinge-linker (EPKIPQPQPKPQPQPQPQPQPKPQPKPEP).31

The genes of the dimeric VHH fragments (Ia1-E32 and 4F2-E32) were generated by means of overlap extension PCR withLA-Taq DNA polymerase, and the gene fragments wereinserted into the NcoI!SacII sites of pRA-FLAG vectors(pRA-Ia1VHH-LH-AuE32VHH-FLAG and pRA-4F2VHH-LH-AuE32VHH-FLAG).The DNA sequence coding one of the selected VHH genes

(E32 clone) with the IgA hinge linker (SPSTPPTPSPSTPP)and a biotin acceptor peptide (Avitag; GGLDNDIFEAG-KIEWH) at the C-terminus of VHH in that order (as reportedby Cloutier et al.32) was generated by overlap extension PCRfrom the pRA-FLAG vector containing the E32 VHH fragment(pRA-AuE32VHH-FLAG), and the amplified fragments wereinserted into the NcoI!SpeI restriction sites of the pRA-FLAGvector (pRA-AuE32VHH-Avitag).

Expression and Purification of VHH Fragments.Transformed E. coli BL21 (DE3) cells harboring the expressionplasmid encoding the VHH and dimeric VHH fragments wereincubated in 2 ! yeast extract tryptone medium containing 100g/mL ampicillin at 28 °C, and expression of antibody fragmentsunder the control of the T7 promoter was induced by adding 1mM isopropyl β-D-thiogalactopyranoside. VHH fragments were

Scheme 1. Construction of High-Affinity Material-BindingAntibody Fragments and Multivalent and BispecificAntibody Fragments

Bioconjugate Chemistry Article

dx.doi.org/10.1021/bc300316p | Bioconjugate Chem. 2012, 23, 1934!19441935

extracted from the periplasm of the harvested cells by osmoticshock and were purified by anion/cation exchange chromatog-raphy and gel filtration chromatography (Hi-Load 16/600Superdex 75 size exclusion column, GE Healthcare, LittleChalfont, UK) after treatment with ammonium sulfate. Thebispecific VHH dimers were first purified by means of affinitychromatography on an anti-FLAG M2 affinity gel column(Sigma, Tokyo, Japan) and then fractionated by gel filtrationchromatography.For the expression of biotinylated VHH fragments, E. coli

BL21 (DE3) cells were transformed by the plasmid ofpBIRAcm encoding biotin ligase (Avidity Inc., Aurora, CO,USA), and then the same cells were transformed by theplasmids of pRA-AuE32VHH-Avitag. The transformed E. colicells were incubated in 2 ! yeast extract tryptone mediumcontaining 100 μg/mL ampicillin and 34 μg/mL chloramphe-nicol at 28 °C. Expression of biotinylated VHH and biotinligase was induced by adding 1 mM isopropyl β-D-thiogalactopyranoside in the presence of 50 μM of D-biotin(Sigma, St. Louis, MO, USA). VHH fragments were extractedfrom the periplasm of the harvested cells by osmotic shock andpurified by means of affinity chromatography on a SoftLink SoftRelease Avidin Resin column (Promega, Madison, WI, USA)and gel filtration chromatography. The fractionated biotinylatedVHH was collected after the presence of biotin was confirmedin the proteins by means of Western blotting usingstreptavidin!horseradish peroxidase (GE Healthcare).Analysis of Binding Affinities and Specificity of VHH

Fragments for Gold Surfaces. Gold powder (6 mg) wasadded to 300 μL of 10 mM phosphate solution (pH 7.5; 200mM NaCl, 0.05% Tween 20) containing VHH, and thesolution was incubated for 30 min at 4 °C. After the solutionwas centrifuged at 20000g for 10 min, the amount of unboundVHH in the supernatant was measured by means of thebicinchoninic acid method with a Micro BCA Protein AssayReagent Kit (Pierce Biotechnology, Rockford, IL, USA). Theamount of protein adsorbed on the gold powder was calculatedby subtracting the amount of unbound protein from the totalamount of VHH used.For analysis of the specificity of the VHH fragments for gold

surfaces, commercially available gold, platinum, palladium, andsilver plates (Nilaco, Tokyo, Japan) were soaked for 30 min in10 mM phosphate solution (pH 7.5; 200 mM NaCl, 0.1%Tween 20) containing the VHH fragments with a FLAG tag.The plates were washed three times with the same phosphate

solution without VHH and then soaked in a solution of mouseanti-FLAG M2 antibody (pH 7.5; 10 mM phosphate, 200 mMNaCl, 0.1% Tween 20). The plates were washed again threetimes, and the chemiluminescence from peroxidase conjugatedto the anti-mouse antibody was observed on the plates.

Suppression of Gold Nanoparticle Aggregation byGold-Binding VHH Fragments. A solution of gold nano-particles (size 20 nm; Sigma-Aldrich, Tokyo, Japan) wasdialyzed against 10 mM phosphate solution (pH 7.5). To thegold nanoparticle solution, bovine serum albumin (BSA, Sigma-Aldrich) or E32 VHH was added such that the concentrationsof the nanoparticles and the protein were 0.1 nM and 40 nM,respectively, at various NaCl concentrations. The absorptionspectra of the gold nanoparticle solutions were measured with aU-3000 spectrophotometer (Hitachi, Tokyo, Japan) every 30min for 360 min, and the difference between the molarextinction coefficients at 523 and 630 nm (Δε523!630) wastraced.

SPR Scattering Imaging of Carcinoma Cells Over-expressing EGFR. Gold nanoparticle solutions (size 41.6 nm;Tanaka Kikinzoku Kogyo, Tokyo, Japan) were dialyzed against10 mM phosphate solution (pH 7.5), and Ia1-E32 VHH dimerswere added to the 0.2 nM solution of gold nanoparticles tobring the dimer concentration to 1 μM. The solution of thenanoparticles and dimers was added to 1.0 ! 104 Chinesehamster ovary cells and A431 cancerous cells. The cells wereincubated for 15 min and washed with 10 mM phosphatesolution (pH 7.5). SPR scattering images of the washed cellswere obtained with an inverted Olympus IX81 microscope(Olympus, Tokyo, Japan) with a dark field condenser (U-DCW). A UPLFLN 100!O2 objective lens was used to collectonly the scattered light from the samples.

Assembly of Gold Nanoparticles from Gold-BindingVHH Tetramers. A 36 μM solution of the E32 VHHfragments with the biotin tag at the C-terminus was mixedwith 9 μM streptavidin to form E32 VHH tetramers. Thetetramerization of E32 VHH was confirmed by gel filtrationchromatography (Superdex 200 10/300 size exclusion column,GE Healthcare, Little Chalfont, U.K.). VHH tetramers atvarious concentrations were mixed with 0.1 nM goldnanoparticles in a 10 mM phosphate solution (pH 7.5). TheΔε523!630 values were measured every 30 min for 540 min toobserve the aggregation of the gold nanoparticles.

Linkage of Gold and ZnO Nanoparticles via BispecificVHH Dimers. A 10 mM phosphate solution (1 mL, pH 7.5;

Table 1. Amino Acid Sequences of CDR Loops of GBP-Grafted VHH Fragmentsb

aα and β in CDR3 of randomized VHHGBP1 were randomized to R or H and R, G, L, or V, respectively. bThe sequence of the GBP is underlined.The numbering of the amino acids of cAbBCII10 VHH follows the Kabat numbering system.33 The amino acids of the GBP-grafted CDR1 andrandomized CDR3 are numbered as 1-1 to 1-18 and 3-1 to 3-16, respectively.20



200 mM NaCl) containing 40 μg of ZnO particles (!100 nm;Hosokawa Micron Inc., Hirakata, Japan) was sonicated for 1min. E32 VHH, 4F2 VHH, and 4F2-E32 dimers were added tothe suspension at various concentrations, and then 20 nm goldnanoparticles were added at a final concentration of 0.2 nM.The solutions were centrifuged at 9500g for 5 s, and theabsorption spectra of the supernatants were measured.In addition, solutions of 40"400 μg/mL ZnO particles and 1

μM 4F2-E32 dimeric VHH fragments were allowed to stand for3 h without centrifugation after the addition of 0.2 nM goldnanoparticles, for observation of spontaneous precipitation ofgold and ZnO nanoparticles. In addition, after the goldnanoparticles were added, the solution was dried on collodion-coated 400 mesh copper grids for transmission electronmicroscopy with a LEO 912 AB OMEGA electron microscope(Carl Zeiss, Oberkochen, Germany) operating 100 kV.

! RESULTSGeneration of High-Affinity Gold-Binding VHH Frag-

ments. In CAnIGET method,20 material-binding peptide is

replaced with one of three CDRs in VHH fragment andanother CDR was randomized to select high-affinity VHHfragment by means of phage display approach. To generateVHH fragments with high affinity for gold surfaces, we replacedthe CDR1 sequence in the VHH fragment of camel anti-BcII β-lactamase antibody cAbBCII1033 with the sequence of apreviously reported gold-binding peptide (GBP) (Table 1).28

The CDR3 loop in the resulting GBP-grafted VHH fragment(VHHGBP1) was randomized with the αββα repeating sequenceformat, where the α and β residues were randomized to Arg orHis and to Arg, Gly, Leu, or Val, respectively; and the

randomized VHH fragments were displayed on filamentousbacteriophage M13. The phages were mixed with gold powder,and then the powder was washed with 50"200 mM phosphatesolutions containing 0.05% Tween 20 to remove nonspecificallybound phages. Residual phages on the powder were eluted witha highly concentrated phosphate solution (500 mM), whichinhibits protein adsorption on inorganic materials.20,34 Afterfour rounds of selection, we randomly picked 100 colonies andthen compiled statistics for the frequency of amino acidresidues at each position in CDR3 before selection and afterfour rounds of selection by analyzing the sequences of thechosen clones (Figure S1 in the Supporting Information). Thestatistical analysis showed the concentrations of specific aminoacids at each position resulting from the molecular evolutionalprocess. Several colonies had the same clones (E3, E9, E12, andE32), each of which had the same arginine-rich sequence(RRVRGG, Table 2). We attempted to estimate the gold-binding affinity of these four clones. However, only the E32

Table 2. Sequences of VHH Fragments after Four Rounds of Selection against Gold Particlesa

aSelected sequences are underlined. The amino acids of CDR3 in the selected VHH fragments are numbered as 3-1 to 3-16.20

Figure 1. Adsorption isotherms for cAbBCII10 VHH (open circles),VHHGBP1 (filled circles), and E32 VHH (open squares) against 6 mgof gold particles in 10 mM phosphate solution (pH 7.5; 200 mMNaCl, 0.05% Tween 20).

Figure 2. Specificity of VHHGBP1 and E32 VHH for material surfaces.Untreated gold, platinum, palladium, and silver plates (A) were soakedin a solution containing cAbBCII10 (B), VHHGBP1 (C), or E32 VHH(D), and then the residual VHH fragments with a FLAG tag at the Cterminus on the plates were detected by the chemiluminescence fromanti-mouse IgG antibody horseradish peroxidise.


dx.doi.org/10.1021/bc300316p | Bioconjugate Chem. 2012, 23, 1934"19441937

clone was adequately expressed in E. coli, which was used forthe binding assay; therefore, we measured the adsorptionisotherms of the VHHGBP1 and E32 VHH fragments (Figure 1).Only small amounts of the cAbBCII10 VHH fragment wereadsorbed on the gold particles (open circles in Figure 1),whereas the VHHGBP1 and E32 VHH fragments were boundwith dissociation equilibrium constants (KD) of 3 μM (closecircles in Figure 1) and 150 nM (open squares in Figure 1),respectively: the affinity of E32 VHH was about 20 times thatof VHHGBP1. The grafting of material-binding peptide in CDR1 functionalized the VHH fragment and the optimization ofCDR3 sequence using phage display method promoted theaffinity of the peptide-grafted VHH.In addition, we also analyzed the affinity of VHHGBP1 and

E32 VHH fragments for other inorganic materials. We soakedgold, palladium, platinum, and silver plates in a solutioncontaining VHH fragments bearing a FLAG tag at the Ctermini. After washing the plates, we detected residual VHHfragments on the plates by measuring the chemiluminescencefrom horseradish peroxidase conjugated with anti-mouse Fc

antibody via anti-FLAG M2 mouse antibody. Selectiveadsorption of the VHHGBP1 and E32 VHH fragments on thegold surface was clearly observed, although E32 VHH alsobound weakly to palladium (Figure 2). These results indicatethat the binding functionalities of VHHGBP1 and E32 VHHwere selective for gold surfaces.

Suppression of Gold Nanoparticle Aggregation underHigh-Ionic-Strength Conditions by E32 VHH Fragments.We used the E32 VHH fragments to suppress gold nanoparticleaggregation in aqueous solution as follows. E32 VHHfragments (40 nM) were added to 10 mM phosphate (pH7.5) solution containing 0.1 nM gold nanoparticles (size: 20nm, Figure 3A). We chose the 1:400 molar ratio of goldnanoparticles to E32 VHH because at least 400 molecules ofE32 VHH are required for complete coverage of the surface of20 nm gold nanoparticles. A solution of 20 nm goldnanoparticles generally has a plasmon absorption at around520 nm; however, the nanoparticles aggregate easily as the ionicstrength is increased, and as a result, the plasmon absorption isred-shifted from the red to the purple region. Here, we

Figure 3. Suppression of gold nanoparticle aggregation by E32 VHH. (A) Experimental scheme for the analysis of aggregation suppression by VHHfragments. (B) Absorption spectra of 0.1 nM gold nanoparticles in 10 mM phosphate solution (pH 7.5) at NaCl concentrations of 0 M (bold solidline), 50 mM (solid line), 200 mM (dotted line), 500 mM (dashed line), and 1000 mM (dashed and dotted line), without protein (i), with 40 nMBSA (ii), and with 40 nM E32 VHH (iii). (C) Time dependence of the plasmon absorption at 520 nm from 0 to 360 min without protein (i), with40 nM BSA (ii), and with 40 nM E32 VHH (iii) at NaCl concentrations of 0, 50, 200, 500, and 1000 mM.



observed that the plasmon absorption band of 0.1 nM goldnanoparticles in 10 mM NaCl (data not shown) was red-shiftedrelative to the band in the absence of NaCl, and the banddisappeared when the NaCl concentration was increased to 50mM in the absence of detergents (Figure 3Bi). Bovine serumalbumin (BSA) is often used to inhibit nanoparticleaggregation,35,36 and the molecular weight (BSA: !60 kDa)is larger than that of VHH (!15 kDa); however, 40 nM BSAonly slightly suppressed the aggregation of gold nanoparticlesin 50 mM NaCl solution (Figure 3Bii). In contrast, the additionof 40 nM E32 VHH markedly suppressed aggregation even in1000 mM NaCl solution (Figure 3Biii). Evaluation of the timedependence of the plasmon absorption at 520 nm at variousNaCl concentrations indicated that E32 VHH suppressed theaggregation of gold nanoparticles for 6 h (Figure 3C). Althoughthe plasmon absorption intensity decreased slightly with time atNaCl concentrations exceeding 200 mM even in the presenceof E32 VHH, E32 VHH markedly suppressed gold nanoparticleaggregation at all the tested NaCl concentrations.

One-Pot Biofunctionalization of Gold Nanoparticles.Gold nanoparticles can resonantly scatter visible light whentheir surface plasmon oscillation is excited.26 Here, wefunctionalized gold nanoparticles with bispecific VHH dimerswith affinity for both EGFR and gold surfaces, for use in cellularimaging by means of SPR scattering. One advantage of workingwith antibodies is the ability to use various fusion technologies.E32 VHH was fused at the C-terminus of Ia1 VHH with affinityfor EGFR30 via a llama IgG2 upper hinge-linker,31 andbispecific antibody with affinity for gold and EGFR (Ia1-E32VHH) was constructed. In the absence of the Ia1-E32 VHHdimer, no SPR scattering was detected on A431 cancerous cellsthat overexpress EGFR (Figure 4A). In contrast, in thepresence of the Ia1-E32 VHH dimer, clear SPR scattering bygold nanoparticles was detected on the cancerous cells (Figure4B). In addition, we added gold nanoparticles functionalizedwith Ia1-E32 VHH dimers to Chinese hamster ovary cells onwhich no EGFRs were expressed; in this case, no SPRscattering from gold nanoparticles was observed even in thepresence of the Ia1-E32 VHH dimer (Figure 4C). These resultsindicate that the dimer interlinked the gold nanoparticles andthe EGFR-displaying cancerous cells. The dimer simultaneouslystabilized and biofunctionalized the gold nanoparticles.

Construction of Biointerfaces between Nanomateri-als. To demonstrate that material-binding camel antibodyfragments could serve as biointerfaces between nanomaterials,we constructed E32 VHH tetramers by taking advantage of thebiotin"avidin interaction (Figure 5A). E32 VHH fragments (36μM) with the biotin acceptor peptide fused at the C-terminiwere mixed with 9 μM streptavidin, and the resulting E32 VHHtetramers were fractionated by size exclusion chromatography(Figure S2 in the Supporting Information). Then the tetramerswere added at various concentrations to a 0.1 nM solution ofgold nanoparticles in the absence of NaCl. When the tetramerconcentration was <20 nM, the plasmon intensity at 520 nmdecreased, but no red-shift was observed (Figure 5Bi,ii).However, when the tetramer concentration was >40 nM, theplasmon band was red-shifted (Figure 5Biii,iv), and theplasmon absorption intensity decreased rapidly with time(Figure 5C). These results indicate that the tetramers acted asinterface molecules between the gold nanoparticles and that thenanoparticles spontaneously aggregated via the tetramers.To use a material-binding VHH as a biointerface between

different nanomaterials, we also designed bispecific VHHdimers from E32 VHH and a ZnO-binding VHH (4F2 VHH)20

by fusing E32 VHH at the C-terminus of 4F2 VHH via a hinge-linker. When we centrifuged a suspension containing 0.2 nMgold nanoparticles (20 nm) and 40 μg/mL ZnO particles(!100 nm) with the E32 VHH or 4F2 VHH monomer, onlyZnO particles precipitated; as a result, the supernatant had thesame plasmon absorption intensity before centrifugation (openand closed circles in Figure 6). In contrast, when bispecific 4F2-E32 VHH dimers were added to the mixture of gold and ZnOparticles, the plasmon absorption intensity decreased withincreasing dimer concentration (open squares in Figure 6): theplasmon absorption intensity of the gold nanoparticlesdecreased at a dimer concentration of 40 nM, and completeprecipitation of gold and ZnO nanoparticles was observedwhen the dimer concentration exceeded 500 nM. These resultsindicate that the 4F2-E32 VHH dimers linked the gold andZnO particles and induced the precipitation of gold nano-particles with ZnO.

Figure 4. Micrograph (i) and surface plasmon resonance (SPR)scattering image (ii) of A431carcinoma cells with 0.2 nM goldnanoparticles (A), A431carcinoma cells with 1 μM VHH dimer and0.2 nM gold nanoparticles (B), and CHO noncarcinoma cells with 1μM VHH dimer and 0.2 nM gold nanoparticles (C). SPR scatteringimages were obtained by using a dark-field condenser. Gold colorshows SPR scattering of gold particles. Scale bars = 10 μm.



We also observed spontaneous precipitation of gold andZnO particles in the presence of bispecific 4F2-E32 VHHdimers. We observed no change in a gold nanoparticle solutionwhen ZnO particles were added in the absence of the bispecificdimers; whereas in the presence of dimers (1 μM), the additionof ZnO particles resulted in an immediate change in color ofthe solution from red to purple, and gold and ZnO particlesspontaneously precipitated (Figure 7Ai). Spontaneous precip-itation in the presence of bispecific VHH dimers was alsoobserved when the amount of added ZnO particles wasincreased to 400 μg, and the particle aggregation rate wasaccelerated (Figure 7Aii).The ZnO suspensions just after gold nanoparticles were

added were dried on collodion-coated grids for transmissionelectron microscopy (Figure 7B). No gold nanoparticles wereobserved on the ZnO particles in the absence of the VHH

dimer, whereas many gold nanoparticles were observed on theZnO particles in the presence of the VHH dimer. These resultsindicate that the bispecific 4F2-E32 VHH dimers linked theZnO and gold nanoparticles and induced spontaneousprecipitation of both types of nanoparticles.

! DISCUSSIONGeneration of VHH Fragments with High Affinity and

Specificity for Gold Surfaces. Antibodies are naturallyoccurring proteins with excellent molecular recognition in theimmune system, and they have been widely used in medical,sensing, and imaging applications.37,38 A few antibodies that canbind inorganic material surfaces have been identified,24,39,40 butthe number of material-binding antibodies is far lower than thenumber of material-binding peptides because of the lowimmunogenic potential of inorganic materials. Even when in

Figure 5. Functionalization of gold nanoparticles with a VHH tetramer. (A) Experimental scheme for functionalization by the VHH tetramer. (B)Time dependence of the absorption spectra of 0.1 nM gold nanoparticles in 10 mM phosphate (pH 7.5) in the presence of 10 nM (i), 20 nM (ii), 40nM (iii), and 80 nM (iv) VHH tetramer in the absence of NaCl. The spectra were collected every 30 min for 540 min. (C) Aggregation behavior ofgold nanoparticles upon addition of the VHH tetramer at various concentrations.



vitro selection methods are used, the fact that antibodyfragments have higher molecular weights than peptides limitsthe library diversity. Previously, Barbas et al. generated thehuman antibody fragments with affinity for integrins by acombination of peptide grafting and in vitro selection: thesequence of the integrin-binding tripeptide (Arg-Gly-Asp) wasgrafted in a CDR, and the conformation of the grafted CDRwas optimized by in vitro selection.41 On the basis of theirconcept (a combination of peptide grafting and in vitroselection), we, recently, proposed the CAnIGET approach andconveniently generated the antibody fragments with highaffinity for ZnO, CoO, and Al2O3 surfaces.20,23 This methodinvolves the application of the variable domain of a camelVHH, which is a simple single domain, as a scaffold for peptidegrafting. The VHH domain is functionalized by replacing oneCDR loop with a material-binding peptide and randomizinganother CDR loop and selecting high-affinity fragments.In this study, we grafted a previously reported gold-binding

peptide sequence of GBP into the CDR1 loop of cAbBCII10and selected high-affinity VHH fragments from a library inwhich the CDR3 loop of VHHGBP1 was randomized with anαββα motif (Arg or His at the α position, and Arg, Gly, Leu, orVal at the β position). After the selection of high-affinity clones,a specific amino acid was selected at each position.Interestingly, Arg was predominantly selected at the α positions(Figure S1 in the Supporting Information), and four clones(E3, E9, E12, and E32) found in several colonies among the100 colonies that were chosen had the same Arg-rich alignmentsequence (RRVRGG, Table 2). A His residue has been foundin many previously reported material-binding peptides, but astudy of the use of homopolypeptides for the adsorption ongold surfaces suggested that Arg and Thr residues have anaffinity for gold surfaces.42 This suggestion supports thepreferred selection of Arg in CDR3 that we observed in thisstudy.Both VHHGBP1 and E32 VHH had specificity for gold

surfaces (Figure 2C,D). This result supports our previoussuggestion that the specificity of a selected VHH for inorganicmaterials depends on the peptide grafted into CDR1.20

Recently, we identified a fragment of variable region (Fv) ofa human antibody, A14P-b2, with affinity and specificity forgold surfaces from a phage-displayed library of a humanantibody library.24 The fragment was used for specificpatterning of proteins and for an immunoassay on a gold-patterned substrate.24 However, the fact that the human Fv is a

heterodimer composed of the variable regions of the heavychain (VH) and light chain (VL) complicates the design ofbispecific molecules, such as diabodies and tandem single-chainFvs (scFvs);43 consequently, the preparation of bispecificantibodies in bacteria is often difficult. Previously, we used onlythe VH domain of A14P-b2 to generate a bispecific molecule byfusing the VH at the terminus of an antilysozyme scFv, and wedemonstrated the use of the VH-scFv molecule for animmunoassay.19,44 However, isolated VH domains tend toaggregate.45 In the current study, we generated a gold-bindingVHH fragment, which is a simple single domain and is stableeven without the light chain fragment, and we expected thatbispecific molecules generated with the VHH would be morestable.

One-Pot Functionalization of Gold Nanoparticles byMeans of Gold-Binding VHH Fragments. Gold nano-particles strongly absorb and scatter visible and near-infraredlight by means of plasmon resonance.2,25,26 The opticalproperties of nanoparticles depend on their size and colloidalstability, and they can be used as probes for sensing25 andimaging26 applications. The colloidal stability of gold nano-particles is determined by the balance between van der Waalsand electrostatic interactions.46,47 Bare gold nanoparticlesaggregate in response to small changes in ionic strength,owing to a decrease in electrostatic repulsion between particlesthat occurs upon binding of counterions to the particle surfaces.Surface modification with surfactants, polymers, or biomole-cules can suppress the aggregation.48

Chemical coupling between gold and thiol groups has beenoften used to conjugate organic molecules to gold nanoparticlesurfaces.48 Proteins can be conjugated to the nanoparticles ifthe organic molecule linker has an appropriate functional groupat the terminus opposite to the thiol group. However, the seriesof reactions required to generate such biofunctionalized goldnanoparticles is complicated, and it often reduces the activity ofthe immobilized proteins because the method cannot offercontrol of protein orientation at the surface and get arounddenaturation of proteins.49 Here, we performed one-potfunctionalization of gold nanoparticles by using the high-affinity, high-specificity interaction of material-binding antibodyfragments with material surfaces. We obtained high-affinity E32VHH fragments that spontaneously bound to gold nano-particles and suppressed nanoparticle aggregation even in ahigh-ionic-strength solution. In addition, the simple single-domain structure of the VHH enabled us to design bispecificdimers by fusing different VHH fragments at the N-terminus ofE32 VHH. For example, an anti-EGFR VHH was orientation-ally immobilized on gold nanoparticles in a single step.In general, to obtain biofunctional nanoparticles for sensing

and imaging, IgG-type antibodies with molecular weights of!150 kDa (15 nm) are conjugated to gold nanoparticles.Conjugating several antibodies on a single nanoparticle isgenerally difficult. However, because the VHH dimers used inthis study (!30 kDa) were much smaller than IgG typeantibodies, the antigen-binding domains could be moreefficiently and densely conjugated on the gold nanoparticles.The resulting functionalized nanoparticles can be expected tobe useful for diagnostic applications such as gold labeling50,51

and photothermal therapy involving gold nanorods.52

High-Affinity VHH Fragments as Interface Molecules.Although nanomaterials that have strictly controlled shapes anddisperse completely in solution can self-assemble by means oftwo-dimensional crystallization,7,8 interface linkers that act as a

Figure 6. Absorption intensity of the plasmon band at 520 nm in thesupernatant of a mixture of gold and ZnO nanoparticles at variousconcentrations (0.1, 0.5, 1.0, 1.25, and 1.5 μM) of E32 VHH (opencircles), 4F2 VHH (filled circles), and VHH dimer (open squares)after centrifugation.



glue between different nanomaterials can facilitate complicatedbottom-up assembly of nanodevices. Biomolecules such asnucleic acids and polypeptides are among the most attractivelinkers because of their high specificity. For example, DNA!DNA,53 biotin!avidin,54,55 and antigen!antibody56 interac-tions have been used to mediate self-assembly of nanomaterialsby covalently conjugating the biomolecules on nanoparticles. Inthis study, we generated biomolecules with direct affinity forthe surface of inorganic nanoparticles, which obviated the needfor the fabrication of nanoparticle surfaces with specificfunctional groups for conjugating biomolecules. Many peptideswith affinity for inorganic material surfaces have been identifiedby means of combinatorial approaches, and such peptides havebeen used for the patterning of nanoparticles and proteins onfilms.13,57,58 However, there have been fewer reports on the use

of material-binding peptides to link nanoparticles because thebinding affinity of such peptides is not sufficient to overcomeelectrostatic interactions between nanoparticles or theBrownian motion of nanoparticles.47,48 Here, we generatedhigh-affinity gold-binding antibody fragments, and we usedsome previously reported structural formats of variousmultivalent and multispecific antibodies43 to create tetravalentantibodies from gold-binding VHH fragments and bispecificVHH dimers by fusing a ZnO-binding VHH with a gold-binding VHH. These multivalent and multispecific antibodyfragments spontaneously linked two different types of nano-particles (gold!gold, gold!ZnO). Our results demonstrate thepotential of high-affinity multivalent and bispecific antibodies toact as glues between nanomaterials.

Figure 7. (A) Photographs showing spontaneous precipitation before the addition of 1 μM VHH dimer (1) and 60 min (2), 120 min (3), and 180min (4) after addition of the dimer. (B) Transmission electron microscopy images of the mixture of gold nanoparticles (0.2 nM) and ZnOnanoparticles: 40 μg/mL (i) and 400 μg/mL (ii).



In conclusion, we generated antibody fragments with highaffinity and specificity for gold nanoparticle surfaces by a one-pot procedure involving integration of peptide-grafting andphage-display techniques. The resulting antibody fragmentsuppressed the aggregation of gold nanoparticles even in a high-ionic-strength solution, and multivalent and bispecific antibodyfragments based on the gold-binding VHH could be used toselectively contact gold nanoparticles with cells displaying theEGFR receptor, as well as to spontaneously link two differentnanomaterials (gold!gold, gold!ZnO). We describe theutilization of high affinitive material-binding antibody fragmentsfor one-pot functionalization of gold nanoparticles.

! ASSOCIATED CONTENT*S Supporting InformationAdditional figures for the frequencies of amino acids inrandomized CDR3 loop of VHHGBP1 and the size-exclusionchromatography for E32 tetramer. This material is available freeof charge via the Internet at http://pubs.acs.org.

! AUTHOR INFORMATIONCorresponding Author*Tel and fax: +81-22-795-7276. E-mail: [email protected] authors declare no competing financial interest.

! ACKNOWLEDGMENTSThis work was supported by a Scientific Research Grant fromthe Ministry of Education, Science, Sports, and Culture ofJapan (M.U. and I.K.), by Precursory Research for EmbryonicScience and Technology from the Japan Science andTechnology Agency (JST, M.U.), and JSPS research fellowshipsfor young scientists (T.H.).

! ABBREVIATIONSBSA, bovine serum albumin; CAnIGET, construction ofantibodies by integrating grafting and evolution technology;CDR, complementarity determining regions; EGFR, epidermalgrowth factor receptor; GBP, gold-binding peptide; SPR,surface plasmon resonance; VHH, variable domain of theheavy chain of a heavy chain camel antibody

! REFERENCES(1) Chan, W. C. W., and Nie, S. (1998) Quantum Dot Bioconjugatesfor Ultrasensitive Nonisotopic Detection. Science 281, 2016!2018.(2) Cao, Y., Jin, R., and Mirkin, C. (2002) Nanoparticles with Ramanspectroscopic fingerprints for DNA and RNA detection. Science 297,1536!1540.(3) Joo, S. H., Choi, S. J., Oh, I., Kwak, J., Liu, Z., Terasaki, O., andRyoo, R. (2001) Ordered nanoporous arrays of carbon supportinghigh dispersions of platinum nanoparticles. Nature 412, 169!172.(4) Caruso, F., Caruso, R. A., and Mohwald, H. (1998) Nano-engineering of inorganic and hybrid hollow spheres by colloidaltemplating. Science 282, 1111!1114.(5) Gao, X., Cui, Y., Levenson, R. M., Chung, L. W. K., and Nie, S.(2004) In vivo cancer targeting and imaging with semiconductorquantum dots. Nat. Biotechnol. 22, 969!976.(6) Mornet, S., Vasseur, S., Grasset, F., and Duguet, E. (2004)Magnetic nanoparticle design for medical diagnosis and therapy. J.Mater. Chem. 14, 2161!2175.(7) Zhang, J., Ohara, S., Umetsu, M., Naka, T., Hatakeyama, Y., andAdschiri, T. (2007) Colloidal Ceria Nanocrystals: A Tailor-madeCrystal Morphology in Supercritical Water. Adv. Mater. 19, 203!206.

(8) Shevchenko, E. V., Talapin, D. V., Kotov, N. A., O’Brien, S., andMurray, C. B. (2006) Structural diversity in binary nanoparticlesuperlattices. Nature 439, 55!59.(9) Whaley, S. R., English, D. S., Hu, E. L., Barbara, P. E., andBelcher, A. M. (2000) Selection of peptides with semiconductorbinding specificity for directed nanocrystal assembly. Nature 405,665!668.(10) Sarikaya, M., Tamerler, C., Jen, A. K-Y., Schulten, K., andBaneyx, F. (2003) Molecular biomimetics: nanotechnology throughbiology. Nat. Mater. 2, 577!585.(11) Naik, R. R., Stringer, S. J., Agarwal, G., Jones, S. E., and Stone,M. O. (2002) Biomimetic synthesis and patterning of silvernanoparticles. Nat. Mater. 1, 169!172.(12) Mao, C., Solis, D. J., Reiss, B. D., Kottmann, S. T., Sweeney, R.Y., Hayhurst, A., Georgiou, G., Iverson, B., and Belcher, A. M. (2004)Virus-based toolkit for the directed synthesis of magnetic andsemiconducting nanowires. Science 303, 213!217.(13) Shimada, Y., Suzuki, M., Sugiyama, M., Kumagai, I., and Umetsu,M. (2011) Bioassisted capture and release of nanoparticles on nano-lithographed ZnO films. Nanotechnology 22, 275302(1!6).(14) Park, T. J., Lee, S. Y., Lee, S. J., Park, J. P., Yang, K. S., Lee, K. B.,Ko, S., Park, J. B., Kim, T., Kim, S. K., Shin, Y. B., Chung, B. H., Ku, S.J., Kim do, H., and Choi, I. S. (2006) Protein nanopatterns andbiosensors using gold binding polypeptide as a fusion partner. Anal.Chem. 78, 7197!7205.(15) Zhou, W., Schwartz, D. T., and Baneyx, F. (2010) Single-potbiofabrication of zinc sulfide immuno-quantum dots. J. Am. Chem. Soc.132, 4731!4738.(16) Umetsu, M., Mizuta, M., Tsumoto, K., Ohara, S., Takami, S.,Watanabe, H., Kumagai, I., and Adschiri, T. (2005) Bioassisted room-temperature immobilization and mineralization of zinc oxide - thestructural ordering of ZnO nanoparticles into a flower-typemorphology. Adv. Mater. 17, 2571!2575.(17) Kramer, R. M., Li, C., Carter, D. C., Stone, M. O., and Naik, R.R. (2004) Engineered Protein Cages for Nanomaterial Synthesis. J.Am. Chem. Soc. 126, 13282!13286.(18) Chiu, C.-Y., Li, Y., Ruan, L., Ye, X., Murray, C. B., and Huang, Y.(2011) Platinum nanocrystals selectively shaped using facet-specificpeptide sequences. Nat. Chem. 3, 393!399.(19) Ibii, T., Kaieda, M., Hatakeyama, S., Shiotuka, H., Kumagai, I.,Watanabe, H., Umetsu, M., and Imamura, T. (2010) Directimmobilization of gold-binding antibody fragments for immunosensorapplications. Anal. Chem. 82, 4229!4235.(20) Hattori, T., Umetsu, M., Nakanishi, T., Togashi, T., Yokoo, N.,Abe, H., Ohara, S., Adschiri, T., and Kumagai, I. (2010) High affinityanti-inorganic material antibody generation by integrating graft andevolution technologies. Potential of antibodies as biointerfacemolecules. J. Biol. Chem. 285, 7784!7793.(21) Bogan, A. A., and Thorn, K. S. (1998) Anatomy of hot spots inprotein interfaces. J. Mol. Biol. 280, 1!9.(22) Li, Y., Li, H., Yang, F., Smith-Gill, S. J., and Mariuzza, R. A.(2003) X-ray snapshots of the maturation of an antibody response to aprotein antigen. Nat. Struct. Biol. 10, 482!488.(23) Hattori, T., Umetsu, M., Nakanishi, T., Tsumoto, K., Ohara, S.,Abe, H., Naito, M., Asano, R., Adschiri, T., and Kumagai, I. (2008)Grafting of material-binding function into antibodies: functionalizationby peptide grafting. Biochem. Biophys. Res. Commun. 365, 751!757.(24) Watanabe, H., Nakanishi, T., Umetsu, M., and Kumagai, I.(2008) Human anti-gold antibodies: Biofunctionalization of goldnanoparticles and surfaces with anti-gold antibodies. J. Biol. Chem. 283,36031!38.(25) Mirkin, C. A., Letsinger, R. L., Mucic, R. C., and Storhoff, J. J.(1996) A DNA-based method for rationally assembling nanoparticlesinto macroscopic materials. Nature 382, 607!609.(26) El-Sayed, I. H., Huang, X., and El-Sayed, M. A. (2005) Surfaceplasmon resonance scattering and absorption of anti-EGFR antibodyconjugated gold nanoparticles in cancer diagnostics: applications inoral cancer. Nano Lett. 5, 829!834.



(27) Huang, J.-S., Callegari, V., Geisler, P., Bruning, C., JKern, J.,Prangsma, J. C., Wu., X., Feichtner, T., Ziegler, J., Weinmann, P.,Kamp, M., Forchel, A., Biagioni, P., Sennhauser, U., and Hecht, B.(2010) Atomically flat single-crystalline gold nanostructures forplasmonic nanocircuitry. Nat. Commun. 1, 150(1!8).(28) Nam, K. T., Kim, D. W., Yoo, P. J., Chiang, C. Y., Meethong, N.,Hammond, P. T., Chiang, Y. M., and Belcher, A. M. (2006) Virus-enabled synthesis and assembly of nanowires for lithium ion batteryelectrodes. Science 312, 885!888.(29) Maenaka, K., Furuta, M., Tsumoto, K., Watanabe, K., Ueda, Y.,and Kumagai, I. (1996) A stable phage-display system using aphagemid vector: phage display of hen egg-white lysozyme (HEL),Escherichia coli alkaline, phosphatase, and anti-HEL monoclonalantibody, HyHEL10. Biochem. Biophys. Res. Commun. 218, 682!687.(30) Roovers, R. C., Laeremans, T., Huang, L., De Taeye, S., Verkleij,A. J., Revets, H., De Haard, H. J., and Van Bergen En Henegouwen, P.M. P. (2007) Efficient inhibition of EGFR signalling and of tumourgrowth by antagonistic anti-EGFR nanobodies. Cancer Immunol.Immunother. 56, 303!317.(31) Conrath, K., Lauwereys, M., Wyns, L., and Muyldermans, S.(2001) Camel single-domain antibodies as modular building units inbispecific and bivalent antibody constructs. J. Biol. Chem. 276, 7346!7350.(32) Cloutier, S. M., Couty, S., Terskikh, A., Marguerat, L., Crivelli,V., Pugnieres, M., Mani, J., Leisinger, H., Mach, J., and Deperthes, D.(2000) Streptabody, a high avidity molecule made by tetramerizationof in vivo biotinylated, phage display-selected scFv fragments onstreptavidin. Mol. Immunol. 37, 1067!1077.(33) Saerens, D., Pellis, M., Loris, R., Pardon, E., Dumoulin, M.,Matagne, A., Wyns, L., Muyldermans, S., and Conrath, K. (2005)Identification of a universal VHH framework to graft non-canonicalantigen-binding loops of camel single-domain antibodies. J. Mol. Biol.352, 597!607.(34) Yokoo, N., Togashi, T., Umetsu, M., Tsumoto, K., Hattori, T.,Nakanishi, T., Ohara, S., Takami, S., Naka, T., Abe, H., Kumagai, I.,and Adschiri, T. (2010) Direct and selective immobilization ofproteins by means of an inorganic material-binding peptide: discussionon functionalization in the elongation to material-binding peptide. J.Phys. Chem. B 114, 480!486.(35) Kramer, S., Xie, H., Gaff, J., Williamson, J. R., Tkachenko, A. G.,Nouri, N., Feldheim, D. A., and Feldheim, D. L. (2004) Preparation ofprotein gradients through the controlled deposition of protein-nanoparticle conjugates onto functionalized surfaces. J. Am. Chem.Soc. 126, 5388!5395.(36) Burt, J. L., Gutierrez-Wing, C., Miki-Yoshida, M., and Jose-Yacaman, M. (2004) Noble-metal nanoparticles directly conjugated toglobular proteins. Langmuir 20, 11778!11783.(37) Adams, G. P., and Weiner, L. M. (2005) Monoclonal antibodytherapy of cancer. Nat. Biotechnol. 23, 1147!1157.(38) Sharkey, R. M., Cardillo, T. M., Rossi, E. A., Chang, C. H.,Karacay, H., McBride, W. J., Hansen, H. J., Horak, I. D., andGoldenberg, D. M. (2005) Signal amplification in molecular imagingby pretargeting a multivalent, bispecific antibody. Nat. Med. 11, 1250!1255.(39) Barbas, C. F., Rosenblum, J., and Lerner, R. (1993) Directselection of antibodies that coordinate metals from semisyntheticcombinatorial libraries. Proc. Natl. Acad. Sci. U.S.A. 90, 6385!6389.(40) Schnirman, A. A., Zahavi, E., Yeger, H., Rosenfeld, R., Benhar, I.,Reiter, Y., and Sivan, U. (2006) Antibody molecules discriminatebetween crystalline facets of a gallium arsenide semiconductor. NanoLett. 6, 1870!1874.(41) Barbas, C. F., Languino, L. R., and Smith, J. W. (1993) High-affinity self-reactive human antibodies by design and selection:Targeting the integrin ligand binding site. Proc. Natl. Acad. Sci.U.S.A. 90, 10003!10007.(42) Peelle, B. R., Krauland, E. M., Wittrup, K. D., and Belcher, A. M.(2005) Design criteria for engineering inorganic material-specificpeptides. Langmuir 21, 6929!6933.

(43) Holliger, P., and Hudson, P. J. (2005) Engineered antibodyfragments and the rise of single domains. Nat. Biotechnol. 23, 1126!1136.(44) Watanabe, H., Kanazaki, K., Nakanishi, T., Shiotsuka, H.,Hatakeyama, S., Kaieda, M., Imamura, T., Umetsu, M., and Kumagai, I.(2011) Biomimetic engineering of modular bispecific antibodies forbiomolecule immobilization. Langmuir 27, 9656!9661.(45) Ward, E. S., Gussow, D., Griffiths, A. D., Jones, P. T., andWinter, G. (1989) Binding activities of a repertoire of singleimmunoglobulin variable domains secreted from Escherichia coli.Nature 341, 544!546.(46) Derjaguin, B. V., and Landau, L. (1941) Theory of the stabilityof strongly charged lyophobic sols and of the adhesion of stronglycharged particles in solutions of electrolytes. Acta Physicochim. URSS14, 633!662.(47) Verwey, E. J. W. (1947) Theory of the stability of lyophobiccolloids. J. Phys. Colloid Chem. 51, 631!636.(48) Daniel, M. C., and Astruc, D. (2004) Gold nanoparticles:assembly, supramolecular chemistry, quantum-size-related properties,and applications toward biology, catalysis, and nanotechnology. Chem.Rev. 104, 293!346.(49) Norde, W. (1986) Adsorption of proteins from solution at thesolid-liquid interface. Adv. Colloid Interface Sci. 25, 267!340.(50) Lucocq, J. (1994) Quantitation of gold labelling and antigens inimmunolabelled ultrathin sections. J. Anat. 184, 1!13.(51) Mayhew, T. M., Muhlfeld, C., Vanhecke, D., and Ochs, M.(2009) A review of recent methods for efficiently quantifyingimmunogold and other nanoparticles using TEM sections throughcells, tissues and organs. Ann. Anat. 191, 153!170.(52) El-Sayed, I. H., Huang, X., and El-Sayed, M. A. (2006) Selectivelaser photo-thermal therapy of epithelial carcinoma using anti-EGFRantibody conjugated gold nanoparticles. Cancer Lett. 239, 129!135.(53) Jones, M. R., Macfarlane, R. J., Lee, B., Zhang, J., Young, K. L.,Senesi, A. J., and Mirkin, C. A. (2010) DNA-nanoparticle superlatticesformed from anisotropic building blocks. Nat. Mater. 9, 913!917.(54) Connolly, S., Fitzmaurice, D. S., and Connolly, D. (1999)Fitzmaurice, Programmed assembly of gold nanocrystals in aqueoussolution. Adv. Mater. 11, 1202!1205.(55) Dimitrijevic, N. M., Saponjic, Z. V., Rabatic, B. M., and Rajh, T.(2005) Assembly and charge transfer in hybrid TiO2 architecturesusing biotin-avidin as a connector. J. Am. Chem. Soc. 127, 1344!1345.(56) Shenton, W., Davis, S. A., and Mann, S. (1999) Directed self-assembly of nanoparticles into macroscopic materials using antibody!antigen recognition. Adv. Mater. 11, 449!452.(57) Kacar, T., Ray, J., Gungormus, M., Oren, E. E., Tamerler, C., andSarikaya, M. (2009) Quartz binding peptides as molecular linkerstowards fabricating multifunctional micropatterned substrates. Adv.Mater. 21, 295!299.(58) Wei, J. H., Kacar, T., Tamerler, C., Sarikaya, M., David, S., andGinger, D. S. (2009) Nanopatterning peptides as bifunctional inks fortemplated assembly. Small 5, 689!693.



scfv gold description

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