Porous Protein-Based Nanoparticle Hydrogel for Protein Chips with Improved Sensitivity

Communication

Porous Protein-Based Nanoparticle Hydrogelfor Protein Chips with Improved Sensitivity

Hiroyuki Tanaka, Tatsushi Isojima, Minako Hanasaki, Yasuo Ifuku,Hisao Takeuchi, Haruma Kawaguchi, Toshifumi Shiroya*

We have discovered a novel method to prepare a protein-based hydrogel, that is, a ‘three-dimensional nanostructured protein hydrogel’ (3D NPH), which is composed of looselyinter-connected protein–polymer hybrid nanoparticles. The 3D NPH can be easily preparedby spotting a protein/polymer mixture on asubstrate. Surprisingly, gold nanoparticlescarrying protein molecules easily diffuse intothe 3D NPH through pores and spaces. Wehave shown that the protein chip made byour 3D NPH method has tremendouslyimproved sensitivity in detecting protein–protein interactions compared with that bydirect protein immobilization methods.

Introduction

Protein chips are important tools for high-throughput

analysis of biological events.[1–7] Recently, a number of

spots, each containing an immobilized protein, have been

placed on a chip surface. The number of spots, i.e., the

surface density of spots, has been increasing dramatically,

and this trend towards a high surface density results in the

reduction of immobilized protein amount per one spotted

area. As a general rule, however, a lower immobilized

H. Tanaka, T. Isojima, M. Hanasaki, H. Takeuchi, T. ShiroyaMitsubishi Chemical Group Science and Technology ResearchCenter, INC., 1000 Kamoshida-cho, Aoba-ku, Yokohama 227-8502,JapanFax: þ81-45-963-3741; E-mail: [email protected]. IfukuMitsubishi Chemical Medience Corporation, 1144 Ohwadashin-den, Yachiyo, Chiba 276-0046, JapanH. Tanaka, H. KawaguchiGraduate School of Science and Technology, Keio University,3-14-1, Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan

Macromol. Rapid Commun. 2008, 29, 1287–1292

� 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

protein amount has resulted in lower sensitivity in protein

chips. This trade-off between the surface density and the

sensitivity requires a new method to increase the binding

capacity per unit area and to improve the sensitivity of

protein chips for detecting protein–protein interactions.

One such method can be found in protein immobilization

using hydrogels: proteins are immobilized in a three-

dimensional (3D) matrix of a hydrogel. There have been

various reports about hydrogel surfaces. For example,

carboxymethyl dextran (CMD),[8,9] polyacrylamide

gel,[10–14] agarose thin film,[15] poly(acrylic acid)

brushes,[16] supramolecular hydrogel,[17] and others,[18–20]

have been used to immobilize proteins. By using these

kinds of hydrogel, the immobilized amount of ligand

proteins can be increased with a reduction in non-specific

protein adsorption to the surfaces.

However, the increase in the amount of immobilized

ligand proteins has not necessarily achieved an increase

in the amount of analyte proteins bound to ligand

proteins.[9,16] These results could be explained by mass

transfer limitation and a reduced accessibility of the

DOI: 10.1002/marc.200800090 1287

H. Tanaka et al.

1288

hydrogels for analyte proteins. To improve the accessibility

of analytes to the ligands immobilized in the hydrogel

matrices, highly porous hydrogels have been made.[14]

However, since the mechanical stabilities of hydrogels

could be incompatible with high porosities, there are

limitations to the porosity of hydrogels.

From a different view for practical use, preparation

processes are required to be easy and well suited to mass

production. For example, recently, a hydrogel composed of

multi-functionalized polymers and proteins were formed

in situ using a one-step process.[21]

Here we report a novel method to prepare a protein-

based hydrogel, i.e., a ‘three-dimensional nanostructured

protein hydrogel’ (3D NPH), which is composed of loosely

inter-connected protein–polymer hybrid nanoparticles.

The 3D NPH can be easily prepared by spotting a protein/

polymer mixture on substrates. The morphology, physical

properties, and bio-functions of the resulting 3D NPH are

studied. In particular, the use of the 3D NPH as a substrate

for an affinity assay is focused on and carefully examined.

Experimental Part

Polymerization and Characterization of

Poly(NAM-co-NAS)

Poly[(N-acryloylmorpholine)-co-(N-acryloxysuccinimide)] [poly-

(NAM-co-NAS)] was prepared by free-radical polymerization

of N-acryloylmorpholine (Sigma-Aldrich, MO, U.S.A.) and

N-acryloxysuccinimide (Acros Organics, NJ, U.S.A.) at an 80:20

molar ratio in anhydrous dioxane (Wako Pure Chemicals

Industries, Ltd., Osaka, Japan) as a solvent according to previous

reports.[22,23] The concentration of all of the monomers was

0.5 mol � L�1. The copolymerization was performed at 60 8C for 4 h

using azoisobutyronitrile (Wako Pure Chemicals Industries, Ltd.,

Osaka, Japan) as a polymerization initiator (1 mol-% of the

monomers). Poly(NAM-co-NAS) was precipitated into a large

volume of ethylic ether, recovered by filtration, and dried under

vacuum. The molecular weight was determined by size exclusion

chromatography (SEC) analysis: HLC-8220 GPC(L) (TOSOH, Japan),

using a TSK gel GMPW�1 column (TOSOH, Japan). The eluent

used was dimethylformamide (DMF) with 50� 10�3M LiBr (Wako

Pure Chemicals Industries, Ltd., Osaka, Japan) at a flow rate of

0.5 mL �min�1. The average copolymer composition was deter-

mined from 1H NMR spectra (Avance 500, Bruker, Germany) of the

copolymer according to a previous report.[22] Poly(NAM-co-NAS),

NAM/NAS¼ 65/35 (mol/mol), Mw ¼ 86 000) was prepared.

Preparation of 3D NPH on Substrates

An Au-coated plastic substrate was treated with a 10�10�3M

solution of 16-mercaptohexadecanoic acid (Sigma-Aldrich, MO,

U.S.A.) in ethanol (Junsei chemical, Tokyo, Japan) for 12 h. The

introduced carboxy groups on Au were activated to form

reactive succinimide esters using a solution of 0.1 mol � L�1

N-ethyl-N0-(3-dimethylaminopropyl) carbodiimide hydrochloride



(EDC, Dojindo corporate, Kumamoto, Japan) and 0.4 mol � L�1

N-hydroxysuccinimide (NHS, Pierce Biotechnology, Rockford,

U.S.A.) in water. After the activation reaction, one micro-liter of

a protein and poly(NAM-co-NAS) mixture (protein/polymer¼10/

1 (w/w) in buffer solution) was deposited by a pipette on the

substrates which have the succinimide groups, and subsequently

reacted by concentration of the mixture by a drying process. After

about 1 h reaction, the remaining succinimide groups were

masked using 1 M ethanolamine solution (pH 8.5) and the

substrates were washed with PBS 0.01 vol.-% Tween 20 (PBST)

buffer solutions five times.

Characterization of 3D NPH

The composition (protein/polymer weight ratio) of 3D NPH–IgG

was determined by amino acid analysis (amino acid analyzer

L8500, Hitachi, Japan) and pyrolysis gas chromatography mass

spectrometry (HP5973MSD, Agilent, USA) of polyacrylic acids after

the hydrolysis of 3D NPH–IgG by 6 N HCl. For the analysis, 100 mL

of 3D NPH–IgG ([protein]/[polymer]¼ 10/1, [protein]¼1.0 wt.-%)

was hydrolyzed by 6 N HCl into amino acids and polyacrylic acids,

the amounts of which were determined.

SEM measurements for the cross section of 3D NPH were carried

out using a JEOL JSM-6320F. 3D NPH–IgG was stained by osmium

tetroxide vapor at room temperature for 2 h and embedded in

Epon 812 epoxy resin (Polysciences, Inc., Pennsylvania, U.S.A.) at

60 8C for 48 h. The sample was etched by glow discharge at 400 V.

Atomic force microscopy (AFM) observations were performed

at room temperature under dry and wet conditions with a Multi

mode AFM (Digital Instrument USA) over a 5 mm�5 mm area in

tapping mode and the raw data was analyzed using the

Nanoscope III software package provided by the manufacturer.

Under dry conditions, the probe of SSS-NCH SUPERSHARPSILICON

(Nanoworld, L¼128 mm, R<5 nm) was used. The spring constant

was 25 N �m�1, and the frequency was 263 kHz. Under wet

(phosphate-buffered saline, PBS) conditions the probe of

OMCL-TR400PSA (OLYMPUS, L¼200 mm) was used. The spring

constant was 0.02 N �m�1, and the frequency was 7.62 kHz.

Transmission electron microscopy (TEM) measurements were

carried out using a JEOL LEM 1200 EX to examine a cross section of

3D NPH–IgG reacted with protein A–Au colloid particles. The

sample was fixed in 2 wt.-% glutaraldehyde in 0.1 M cacodylate

buffer (pH 7.4) at room temperature for 2 h; then it was

dehydrated in ethanol at room temperature for 30 min and

embedded in Epon 812 epoxy resin at 60 8C for 48 h. The sample

was etched by an ultramicrotome.

Cy3-Labeled Anti-Mouse IgG F(ab’)2 Binding

Experiments

Three kinds of 3D NPH (Streptavidin, MW 60 kDa, Wako Pure

Chemicals Industries, Ltd., Osaka, Japan, Protein A, MW 60 kDa,

Sigma-Aldrich, MO, U.S.A., and Mouse IgG, MW 150 kDa, LAMPIRE

Lab. PA, U.S.A.) were prepared using one micro-liter of a protein

and poly(NAM-co-NAS) mixture ([protein]/[polymer]¼10/1,

[protein]¼1.0 wt.-%) according to the method described above.

In the case of 2D surfaces, proteins were directly immobilized on a

substrate. The patterned 2D and 3D NPH surfaces were treated for

DOI: 10.1002/marc.200800090

Porous Protein-Based Nanoparticle Hydrogel for . . .

30 min with Cy3–anti-mouse IgG F(ab’)2 (10 mg �mL�1, MW 100

kDa, Sigma-Aldrich, MO, U.S.A.) prepared in phosphate-buffered

saline (PBS), at pH 7.4 after being blocked for 1 h with a 3 wt.-%

solution of bovine serum albumin (BSA, MW 60 kDa, Sigma-

Aldrich, MO, U.S.A.) prepared in PBS, at pH 7.4. All the 3D NPH and

2D surfaces were post-washed with PBST five times. The

fluorescence signals were detected using genepix 4000B (Axon

instrument). To examine the relation of the protein/polymer

concentrations in the feed to the analyte binding, three 3D

NPH–Mouse IgG solutions were prepared by changing the

protein/polymer total concentrations ([protein]/[polymer]¼10/

1, [protein]¼ 0.13, 0.25, and 0.50 wt.-%) in the feed. An Anti Biotin

IgG (MW 150 kDa, Sigma-Aldrich, MO, U.S.A.) solution of 3D NPH

was prepared using one micro-liter of protein and poly(NAM-co-

NAS) mixtures ([protein]/[polymer]¼ 10/1, [protein]¼ 1.0 wt.-%).

In the case of 2D surfaces, proteins were directly immobilized on a

substrate. The analyte concentration of Cy3–Biotin-HRP applied to

the 3D NPH–anti Biotin IgG and 2D surface was 10 mg �mL�1.

Cy3–Biotin-HRP was prepared with Biotin-HRP (MW 45 kDa,

Pierce Biotechnology, Rockford, U.S.A.) and Cy3 dye reagent (GE

Healthcare, Uppsala, Sweden).

Results and Discussion

Polymers contribute to the assembly protein molecules to

form nanoparticles, the assembly of nanoparticles to form

Figure 1. Schematic representation of the 3D NPH preparation proce



aggregates, and to connect the aggregates. The preparation

procedure of 3D NPH is shown in Figure 1. One micro-liter

of a protein/poly(NAM-co-NAS) mixture (protein/poly-

mer¼ 10/1 (w/w) in buffer solution) were deposited on

substrates which have succinimide groups, and amino

groups of the proteins reacted with the succinimide groups

of the poly(NAM-co-NAS) and/or substrates while the

mixed solution was concentrated through a drying

process. After 1 h of reaction, the remaining succinimide

groups were masked by 1 M ethanolamine solution (pH 8.5)

and the substrates were washed with buffer solutions five

times to remove unreacted proteins and polymers. This

preparation method of 3D NPH includes a gelation process

between proteins and the reactive polymers on substrates

upon concentrating the mixture. The hydrogel could be

made on the limited areas of the substrates by this

method. It has been pointed out that cross-contamination

and overflow during microdrop depositing may occur

because of an overall coverage of the substrate by a

hydrophilic hydrogel.[24] By using this 3D NPH method, we

can make a protein array with high surface densities

without cross-contamination among neighboring spots

and overflow.

The results of amino acid analysis and pyrolysis gas

chromatography mass spectrometry showed that 3D

ss and analyte interaction with 3D NPH.

www.mrc-journal.de 1289

H. Tanaka et al.

1290

NPH-IgG, which was prepared from a mixture of IgG/

poly(NAM-co-NAS) of 10/1(w/w) in the feed, had an IgG/

poly(NAM-co-NAS) ratio of 96/4 (w/w). This shows that the

composition of 3D NPH–IgG almost coincides with that in

the feed, and this means that the 3D NPH is a

protein-based hydrogel. Hereafter, in this study, the

composition of 3D NPH was fixed at 10/1 (w/w).

The surface concentration of IgG immobilized in the 3D

NPH–IgG was 26 mg � cm�2, and this value shows that

about 80 wt.-% of the amount of protein deposited on the

substrate remains as a hydrogel. The surface concentration

of the IgG monolayer in an end-on or side-on orientation

was reported as 1.85 and 0.27 mg � cm�2, respectively.[25]

Therefore, the surface concentration of 3D NPH–IgG is

equivalent to about 14–93 layers.

Figure 2. Structure analyses of the 3-D NPH. a) SEM image of a croscross-section of 3-NPH-IgG reacted with protein A Au colloid particles.Au colloid particles. d) AFM image of a surface of 3-D NPH-IgG in dry coNPH-IgG in wet condition; AFM image of a surface of 3-D NPH-strepimmobilized with gulteraldehyde.



Next, the structures of the 3D NPH were analyzed by

several microscopic observations such as scanning elec-

tron microscopy (SEM), TEM, and AFM. Figure 2(a) shows

an SEM image of a cross-section of 3D NPH–IgG which was

prepared by a 1 mL deposition of a protein/polymer

mixture ([protein]/[polymer]¼ 10/1, [protein]¼ 1.0 wt.-%)

in PBS, on a gold-coated plastic substrate, followed by

being embedded in epoxy and cut by a glow discharge. It is

clear from this image that the 3D NPH–IgG consists of

nanoparticles. These nanoparticles are presumed to be

conjugates composed of IgG and poly(NAM-co-NAS)

molecules.

Figure 2(b) and (c) show SEM and TEM images of the 3D

NPH–IgG after being stained with protein A–gold particles.

The rims surrounding the ellipsoidal aggregate are protein

s-section of 3-D NPH-IgG on the solid substrate. b) SEM image of ac) TEM image of a cross-section of 3-NPH-IgG reacted with protein Andition. e) The zoomed image of (d). f) AFM image of a surface of 3-D

tavidin, (g) and -BSA (h). i) AFM image of a surface of BSA hydrogel

DOI: 10.1002/marc.200800090

Porous Protein-Based Nanoparticle Hydrogel for . . .

Figure 3. a) Fluorescent image of three kinds of 3D NPH after probing withCy3–anti-Mouse IgG F(ab’)2 (Proteins: streptavidin, protein A, and Mouse IgG). b) Fluo-rescent image of the 3D NPH–Mouse IgG ([protein]/[polymer]¼ 10/1, [protein]¼0.13, 0.25,and 0.50 wt.-% in the feed) and of the 2D surface ([protein]¼0.13, 0.25, and 0.50 wt.-% inthe feed) after probing with Cy3–anti-Mouse IgG F(ab’)2. c) Line plot of fluorescentintensity vs. Mouse IgG concentrations in the feed. d) Fluorescent image of 3D NPH–antiBiotin IgG after probing with Cy3–Biotin-HRP.

A–gold particles (diameter: about 10

nm). These SEM and TEM images show

that actually 3D NPH consists of aggre-

gates that are micrometer in size, which

are composed of nanoparticles. The SEM

and TEM images reveal that protein

A–gold particles could diffuse into 3D

NPH–IgG and bind the Fc regions of the

IgG molecules immobilized in the 3D

NPH–IgG. This finding indicates that the

3D NPH–IgG has sufficient space in

water through which large molecules

such as analyte protein molecules can

diffuse. The protein A–gold particles

adsorbed on the vicinity of the aggre-

gate surfaces and did not penetrate into

them (Figure 2(c)). The degree of cross-

linking in the aggregates might be so

high that the protein A–gold particles

could not penetrate into the aggregates,

whereas the lower cross-linking degree

among the aggregates might enable the

protein A–gold particles to diffuse into

the 3D NPH–IgG through the voids between the aggre-

gates.

AFM images with a lower magnification of 3D NPH–IgG

surfaces are shown in Figure 2(d) and (f), which were

observed under dry and wet conditions, respectively.

Aggregates and voids are observed all over the 3D

NPH–IgG surface. The aggregates are about a micrometer

size, and there are voids around them. The aggregate sizes

almost coincided with the aggregate sizes shown in

Figure 2(c) and, on the surface of these aggregates,

nanoparticles could be recognized (Figure 2(e)). In water,

3D NPH–IgG swells and almost retains the original size of

the aggregates (Figure 2(f)). This finding indicates that 3D

NPH mainly swells between the aggregates. The reason

for this heterogeneous swelling behavior might also be

that the degree of the cross-linking in aggregates is higher

than that between the aggregates. We are currently trying

to control the size of the aggregates by changing the

preparation conditions such as polymer species, and

composition and concentration of the protein/polymer

mixtures in the feed.

Figure 2(g) and (h) show AFM images of 3D NPH surfaces

made by other kinds of proteins (streptavidin, albumin).

Nanoparticles and their aggregates could be also con-

firmed in these AFM images, whereas the distinctive

structure could not be recognized in the AFM image of the

albumin hydrogel made by glutaraldehyde (Figure 2(i)).

Since a large amount of ligand proteins could be

immobilized on the substrate and the analyte proteins

could diffuse into the 3D NPH smoothly, it is highly

expected that 3D NPH can be applied for protein arrays



with high sensitivity to detect specific protein–protein

interactions.

First, to examine specific and non-specific bindings to

the 3D NPH, the 3D NPH of several proteins (Streptavidin,

Protein A, Mouse IgG) were probed with Cy3–anti-Mouse

IgG F(ab’)2 (10 mg �mL�1) (Figure 3(a)) The increase in

fluorescence was only observed for the 3D NPH–Mouse

IgG, which indicates that specific protein–protein recogni-

tion occurred, and the analyte protein did not bind to the

other 3D NPH–Streptavidin and 3D NPH–Protein A

non-specifically.

Next we spotted four 3D NPH–Mouse IgG with different

protein/polymer total concentrations ([protein]/[poly-

mer]¼ 10/1, [protein]¼ 0.13, 0.25, and 0.50 wt.-%) in the

feed to test the capacity of the 3D NPH–Mouse IgG to bind

analyte proteins (Cy3–anti-Mouse IgG F(ab’)2). In addition,

to compare the analyte protein binding capacity, Mouse-

IgG molecules were immobilized directly on an Au

substrate that had succinimide groups (2D surface). The

fluorescence intensity of the 2-D-Mouse IgG was saturated

above 0.25 wt.%, however, the fluorescence intensity of the

3-D NPH–Mouse IgG increased almost linearly with

increasing the concentrations of the Mouse IgG/polymer

mixtures over the examined range (Figure 3(b)). The

fluorescence intensity of the 3D NPH–Mouse IgG

(0.50 wt.-%) was about 50-fold greater in fluorescence

signal than that of the Mouse IgG immobilized on a 2D

substrate (Figure 3(c)). This result showed that the binding

capacity of the 3D NPH could be controlled by the

concentrations of the protein/polymer mixtures in the

feed with good reproducibility. The significant increase

www.mrc-journal.de 1291

H. Tanaka et al.

1292

in the fluorescence intensity of the 3D NPH could be

attributed to the large amount of immobilized Mouse IgG

molecules and the unique porous structure of the 3D NPH.

In the case of conventional 3D methods using poly-

acrylamide or a poly(ethylene glycol) (PEG) cross-linker,

the 3D hydrogels were two to three fold greater in

fluorescence signal than the 2D surface.[10,13,21] Some

researchers controlled the porosity of polyacrylamide or

modified polyacrylamide gel to improve the diffusion of

analyte proteins into the hydrogel and completed the

assay within several hours.[10,26] In our case, owing to

the highly porous and hydrophilic matrix of 3D NPH,

analyte proteins can easily diffuse into the pores and

the analyte proteins can interact with ligand proteins

therein in assay times shorter than 30 min. It is difficult to

compare these results rigorously because of the difference

of the kind of proteins and hydrogels. However, we believe

that the 3D NPH method is superior to conventional 3D

methods and 2D methods in terms of the sensitivity and

speed of analysis.

Furthermore, to confirm whether the proteins immobi-

lized in 3D NPH retain their activities, we made 3D

NPH using anti-Biotin IgG ([protein]/[polymer]¼ 10/1,

[protein]¼ 1.0 wt.-%) in the feed and examined whether

they can capture Cy3–Biotin-HRP as the analyte protein

(Figure 3(d)). IgG molecules immobilized in the 3D NPH

maintained their activity to interact with the analytes, and

could capture a much larger amount of analyte compared

with the 2D surface.

Conclusion

We have demonstrated that a ‘3D nanostructured protein

hydrogel’ (3D NPH) composed of protein–polymer nano-

particles can be successfully prepared by condensing a

protein/polymer mixture on substrates. The 3D NPH could

immobilize a large mount of proteins and has novel porous

structures through which the analyte proteins may diffuse

into it. Therefore, the ability of the ligand proteins in the

3D NPH to capture the analyte proteins is remarkably high

compared with a 2D surface, and thus a highly sensitive

protein chip could be made by the 3D NPH method.

Acknowledgements: The authors thank Prof. Kenji Kubota ofGunma University and Dr. Hisashi Koga of Kazusa DNA



Research Institute for their helpful suggestions. They also thankDr. Takamasa Hanaichi for the TEM measurements.

Received: February 11, 2008 Revised: April 9, 2008; Accepted:April 14, 2008; DOI: 10.1002/marc.200800090

Keywords: biomaterials; hydrogels; nanoparticles; proteins

[1] G. MacBeath, S. L. Schreiber, Science 2000, 289, 1760.[2] A. Q. Emili, G. Cagney, Nat. Biotechnol. 2000, 18, 393.[3] H. Zhu, M. Snyder, Curr. Opin. Chem. Biol. 2001, 5, 40.[4] P. Mitchell, Nat. Biotechnol. 2002, 20, 225.[5] M. F. Templin, D. Stoll, M. Schrenk, P. C. Traub, C. F. Vohringer,

T. O. Joos, Trends Biotechnol. 2002, 20, 160.[6] H. Zhu, M. Snyder, Curr. Opin. Chem. Biol. 2003, 7, 55.[7] D. S. Wilson, S. Nock, Angew. Chem. Int. Ed. 2003, 42, 494.[8] S. Lofas, B. Johnsson, J. Chem. Soc., Chem. Commun. 1990, 21,

1526.[9] R. Polzius, Th. Schneider, F. F. Bier, U. Biltewski, W. Koscinski,

Biosens. Bioelectron. 1996, 11, 503.[10] P. Arenkov, A. Kukhtin, A. Gemmell, S. Voloschchuk, V.

Chupeeva, A. Mirzabekov, Anal. Biochem. 2000, 278, 123.[11] P. Angenendt, J. Glokler, D. Murphy, H. Lehrach, D. J. Cahill,

Anal. Biochem. 2002, 309, 253.[12] A. Y. Rubina, E. I. Dementieva, A. A. stomakhin, E. L. Darii, S. V.

Pan’kov, E. V. Konovalova, A. D. Mirzabekov, BioTechniques2003, 34, 1008.

[13] P. T. Charles, C. R. Taitt, E. R. Goldman, J. G. Rangasammy, D. A.Stenger, Langmuir 2004, 20, 270.

[14] S. B. Brueggemeier, D. Wu, S. J. Kron, S. P. Palecek, Biomacro-molecules 2005, 6, 2765.

[15] V. Afanassiev, V. Hanemann, S. Wolfl, Nucleic Acids Res. 1998,26, 5007.

[16] J. Dai, Z. Bao, L. Sun, S. U. Hong, G. L. Baker, M. L. Bruening,Langmuir 2006, 22, 4274.

[17] S. Kiyonaka, K. Sada, I. Yoshimura, S. Shinkai, N. Kato, I.Hamachi, Nat. Mater. 2004, 3, 58.

[18] I. Caelen, H. Gao, H. Sigrist, Langmuir 2002, 18, 2463.[19] M. S. Lord, M. H. Stenzel, A. Simmons, B. K. Milthorpe, Bio-

materials 2006, 27, 1341.[20] Y. Ito, M. Nogawa, M. Takeda, T. Shibuya, Biomaterials 2005,

26, 211.[21] M. M. Dominguez, M. Wathier, M. W. Grinstaff, S. E. Schaus,

Anal. Chem. 2007, 79, 1064.[22] F. M. Veronese, R. Largajolli, C. Visco, P. Ferruti, A. Miucci,

Appl. Biochem. Biotechnol. 1985, 11, 269.[23] F. D’Agosto, M.-T. Charreyre, F. Melis, B. Mandrand, C. Pichot,

J. Appl. Polym. Sci. 2003, 88, 1808.[24] Y. Zhou, O. Andersson, P. Lindberg, B. Liedberg, Microchim.

Acta. 2004, 147, 21.[25] A. Baszkin, D. J. Lyman, J. Biomed. Mater. Res. 1980, 14, 393.[26] A. Y. Rubina, A. Kolchinsky, A. Makarov, A. S. Zasedatelev,

Proteomics 2008, 8, 817.

DOI: 10.1002/marc.200800090

Porous Protein-Based Nanoparticle Hydrogel for Protein Chips with Improved Sensitivity

Documents

Transcript of Porous Protein-Based Nanoparticle Hydrogel for Protein Chips with Improved Sensitivity