Complementary Design for Pairing Between Two Types of

Jan 28, 2019 - Complementary Design for Pairing Between Two Types of Nanoparticles Mediated by a Bispecific Antibody: Bottom-up Formation of Porous ...
0 downloads 0 Views 1MB Size
Subscriber access provided by Iowa State University | Library

Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

Complementary Design for Pairing Between Two Types of Nanoparticles Mediated by a Bispecific Antibody: Bottomup Formation of Porous Materials from Nanoparticles Teppei Niide, Noriyoshi Manabe, Hikaru Nakazawa, Kazuto Akagi, Takamitsu Hattori, Izumi Kumagai, and Mitsuo Umetsu Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b03687 • Publication Date (Web): 28 Jan 2019 Downloaded from http://pubs.acs.org on January 29, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1

Complementary Design for Pairing Between Two

2

Types of Nanoparticles Mediated by a Bispecific

3

Antibody: Bottom-up Formation of Porous Materials

4

from Nanoparticles

5 6

Teppei Niide, † Noriyoshi Manabe, †, ‡ Hikaru Nakazawa, † Kazuto Akagi, § Takamitsu Hattori, † ,¶

7

Izumi Kumagai † and Mitsuo Umetsu *, †

8 9

† Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku

10

University, Aoba 6-6-11, Aramaki, Aoba-ku, Sendai 980-8579, Japan

11

§ Advanced Institute for Materials Research, Tohoku University, 2-1-1 Katahira, Aoba-ku,

12

Sendai 980-8577, Japan

13 14

KEYWORDS

15

Antibody, Mesoporous material, Nanocomposite, Protein engineering, Thin film

16

ACS Paragon Plus Environment

1

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 43

1

Recent advances in biotechnology have enabled the generation of antibodies with high affinity

2

for the surfaces of specific inorganic materials. Herein, we report the synthesis of functional

3

materials from multiple nanomaterials by using a small bispecific antibody recombinantly

4

constructed from gold-binding and ZnO-binding antibody fragments. The bispecific antibody

5

mediated spontaneous linkage of gold and ZnO nanoparticles to form a binary gold–ZnO

6

nanoparticle composite membrane. The relatively low melting point of the gold nanoparticles

7

and the solubility of ZnO in dilute acid solution then allowed for the bottom-up synthesis of a

8

nanoporous gold membrane by means of a low-energy, low-environmental-load protocol. The

9

nanoporous gold membrane showed high catalytic activity for the reduction of p-nitrophenol to

10

p-aminophenol by sodium borohydride. Here, we show the potential utility of nanoparticle

11

pairing mediated by bispecific antibodies for the bottom-up construction of nanostructured

12

materials from multiple nanomaterials.

13

ACS Paragon Plus Environment

2

Page 3 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1

INTRODUCTION

2

Advances in nanotechnology have enabled the generation of nanomaterials with unique

3

optical, magnetic, and electronic properties,1 and some of these nanomaterials can serve as

4

nanoscale building blocks for the bottom-up fabrication of organized composite structures with

5

functions that are difficult to achieve by means of top-down techniques.2–8 The functions of

6

materials assembled from nanomaterials are profoundly affected by the structure of the final

7

assembly. Nanomaterials with strictly controlled size and shape self-assemble into defined

8

organized structures,9–12 and external factors such as physical force fields or chemical additives

9

can be used to facilitate assembly into a desired dimensional geometry by controlling the

10

interactions between the nanomaterials.13–16 However, using only the properties of the

11

nanomaterials and external factors to control the organization of composite structures assembled

12

from multiple nanomaterials has limited utility because specific bonding between pairs of

13

nanomaterials, which is necessary for functionalization, is difficult to achieve.

14

One method for achieving specific pairing between nanomaterials involves the use of

15

biomolecules with high-affinity molecular-recognition ability, such as DNA17–25 and peptide

16

aptamers.25–29 Single-strand DNA that is chemically immobilized on nanomaterials mediates

17

reversible assembly and patterning via interaction with the complementary strand, and origami

18

sequence design enables the creation of artificial nanostructured templates on which DNA-

19

immobilized nanoparticles can be assembled.30–32 Peptide aptamers with affinity for material

20

surfaces can also specifically bind pairs of materials, and, in theory, they do not need to be

21

chemically immobilized on the materials to be bound. Peptide aptamers have been used for

22

bottom-up patterning and assembly of proteins and nanomaterials;33,34 however, there are few

23

reports on the use of peptide aptamers to link nanoparticles because the binding affinity of such

ACS Paragon Plus Environment

3

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 43

1

peptides is usually insufficient to overcome the electrostatic repulsion between nanomaterials

2

and the Brownian motion of nanoparticles.35,36

3

Recently, we generated several antibody fragments with high affinity for a specific inorganic

4

surface,37–39 and bispecific antibodies fabricated by fusing two different material-binding

5

antibody fragments have been used to link two different nanomaterials,39 showing the potential

6

utility of such antibody fragments for spontaneous assembly of nanomaterials. Building on our

7

previous work, in the present study we developed a bottom-up means of constructing functional

8

materials from multiple nanomaterials via material-binding antibodies. Specifically, we used a

9

recombinant bispecific molecule constructed from two camel heavy-chain antibodies (VHHs),

10

one with a high affinity for the surface of ZnO nanoparticles (4F2 VHH)38 and one with a high

11

affinity for the surface of gold nanoparticles (E32 VHH)39, to synthesize plasmonic precipitates

12

and nanoporous materials from ZnO and gold nanoparticles (Scheme 1). We chose to produce

13

nanoporous gold-based materials because such materials have been used as unsupported

14

heterogeneous catalysts and have been shown to have high selective catalytic activity for a wide

15

variety of chemical reactions.40–42 Examples of the catalytic applications of nanoporous gold-

16

based

17

electrochemical oxidation of methanol,45 gas- and liquid-phase O2 reduction,46 and semi-

18

hydrogenation of alkynes.47 In addition, these monolithic materials have attracted increased

19

attention from the viewpoint of green chemistry because they work under mild conditions (low

20

temperature and atmospheric pressure), have high durability and reusability, and are easily

21

separated from reaction mixtures

materials

include

gas-phase

CO

oxidation,43

liquid-phase

glucose-oxidation,44

22

Nanoporous gold materials are usually fabricated by alloying gold with other metals and then

23

dissolving the added metal with concentrated nitric acid,48,49 which has the disadvantages of

ACS Paragon Plus Environment

4

Page 5 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1

requiring a high energy input and leaving traces of the added metal in the resulting porous

2

material. In contrast, the method we describe herein allowed us to generate a nanoporous gold

3

membrane from relatively low-melting gold nanoparticles and soluble ZnO nanoparticles in

4

dilute acids, which required low energy input and had a low environmental load. The membrane

5

showed high catalytic activity for the reduction of p-nitrophenol to p-aminophenol by sodium

6

borohydride. Here, we show the potential of using both bispecific antibodies to design

7

complementary paired nanomaterials and binary nanomaterial complexes for building composite

8

materials from multiple nanomaterials.

ACS Paragon Plus Environment

5

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 43

1

EXPERIMENTAL PROCEDURES

2

Expression and Purification of the 4F2-E32 VHH Dimer. To prepare the bispecific

3

antibody, we used an expression vector encoding a dimer in which 4F2 VHH was fused to the N-

4

terminus of E32 VHH via a llama IgG2 upper hinge-linker; the expression vector has previously

5

been used to prepare bispecific antibodies with affinity for gold and ZnO.39 Escherichia coli

6

BL21 (DE3) (Life Technologies, Carlsbad, CA, USA) were transformed with the vector, and the

7

dimer was expressed and purified as described previously.39 Briefly, 2× YT medium containing

8

ampicillin

9

thiogalactopyranoside was added to induce the expression of antibody fragments. After

10

centrifugation, harvested cells were resuspended and sonicated to extract the expressed VHH

11

dimer. The extracted dimer was purified by means of affinity purification with an anti-FLAG-tag

12

resin column (Sigma-Aldrich, St. Louis, MO, USA) followed by size-exclusion chromatography

13

with a HiLoad Superdex 200-pg column 26/600 (GE Healthcare Bio-Sciences, Piscataway, NJ,

14

USA).

was

inoculated

with

transformed

E.

coli

cells,

and

isopropyl-β-d-

15 16

Use of the Bispecific Antibody for Preparation of Gold–ZnO Nanoparticles and

17

Nanoparticle Composite Membranes. Gold nanoparticles (20, 50, and 100 nm, Sigma-Aldrich)

18

and ZnO nanoparticles (100 nm, HakusuiTech Co., Osaka, Japan) were dispersed in separate 10

19

mM aliquots of sodium phosphate solution (pH 7.5); the concentrations of the 20, 50, and 100

20

nm gold nanoparticles were 500, 31.6, and 3.9 pM, respectively, and the ZnO nanoparticle

21

concentration ranged from 0.56 to 56.0 µg/mL. After sonication, the gold and ZnO nanoparticle

22

suspensions were mixed at various ratios in a vial, and then the 4F2-E32 VHH dimer was added

23

at a final concentration of 1 µM. The resulting solutions were incubated at 4 °C for 1 day, and

ACS Paragon Plus Environment

6

Page 7 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1

the precipitates that spontaneously formed in the presence of the VHH dimer were separated

2

from the supernatant and dried in a vacuum. To measure the absorption spectra of the

3

precipitates, we placed a glass plate at the bottom of a vial and poured the solution containing the

4

gold and ZnO nanoparticles into the vial before adding the VHH dimer solution. The absorption

5

spectra of the composite membranes that formed on the glass plates were measured by using

6

UV–vis spectroscopy (U-3000, Hitachi Science & Technology, Tokyo, Japan). In addition, we

7

used a field-emission scanning electron microscope (FE-SEM: S-4800, Hitachi Science &

8

Technology) operated at 15–25 kV to obtain images of the gold–ZnO nanoparticle composite

9

membranes.

10

The spatial arrangement of nanoparticles in the cross-section of a gold–ZnO nanoparticle

11

composite was observed by FE-SEM and transmission electron microscopy (TEM: JEM-2100F,

12

JEOL, Tokyo, Japan). To make a cross-sectional sample with ~100 nm thickness, two pieces of

13

substrate (3 × 5 mm2 each) were adhered together with epoxy resin (AR-R30, Nichiban Co.,

14

Tokyo, Japan) and incubated at room temperature for 24 h until the epoxy resin had cured. The

15

assembled substrate was then polished by using an ion milling system (PIPS Model 691, Gatan,

16

Pleasanton, CA).

17 18

Preparation of nanoporous gold membranes. Nanoporous gold membranes were prepared

19

from the gold–ZnO nanoparticle composite membranes obtained by using the VHH dimer.

20

Specifically, gold–ZnO nanoparticle membranes deposited on a glass plate were sintered at

21

various temperatures ranging from 200 to 400 °C for 36 h and were then soaked in a 100 mM

22

glycine–HCl solution (pH 3.5) at room temperature for 10 h to remove the Zn. The plates were

23

then washed three times with water and dried in a vacuum. The membranes that formed on the

ACS Paragon Plus Environment

7

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 43

1

plates were analyzed by means of UV–vis spectroscopy and FE-SEM. To prepare materials for

2

comparison, we concentrated a gold nanoparticle suspension 10-fold (to 5 nM) by means of

3

centrifugation and then dried the suspension on a glass plate in the presence or absence of ZnO

4

nanoparticles (5.6 µg/mL).

5 6

Catalytic reduction of p-nitrophenol in the presence of nanoporous gold membrane.

7

Phosphate buffer solutions of 20 nm gold nanoparticles and ZnO nanoparticles were mixed in a

8

vial at a ZnO/gold volume ratio of 1/1, and then the 4F2-E32 VHH dimer (1 µM in the same

9

buffer) was added to the mixture to precipitate the gold–ZnO nanoparticles. After the supernatant

10

was removed, the precipitates were dried in a vacuum and then subjected to sintering and

11

treatment with the glycine–HCl buffer in the vial, as described above. To prepare materials for

12

comparison, we concentrated a gold nanoparticle suspension 10-fold (to 5 nM) by means of

13

centrifugation and then dried the suspension in a vial in the presence or absence of ZnO

14

nanoparticles (5.6 µg/mL). The dried precipitates were also sintered and treated in the vial with

15

the glycine–HCl buffer. To each of the vials containing dried precipitates, we added 2.5 mL of a

16

solution containing 100 mM NaBH4 and 1 mM p-nitrophenol. Every 10 min, 10 µL of the

17

reaction solution was removed and diluted by 1/10, and the absorbance of p-nitrophenol at 400

18

nm was measured by means of UV–vis spectroscopy (U-3000).

19

ACS Paragon Plus Environment

8

Page 9 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1

RESULTS

2

Spontaneous assembly of gold–ZnO nanoparticles. When phosphate buffer solutions of 20

3

nm gold nanoparticles and approximately 100 nm ZnO nanoparticles were mixed at an equal

4

volume ratio and allowed to incubate for 1 day at 4 °C, no precipitate formed (Figure 1a-I) and

5

the supernatant showed plasmon absorption (Figure 1b-I). However, when the 4F2-E32 VHH

6

dimer, which was constructed from a ZnO-binding VHH fused to the N-terminus of a gold-

7

binding VHH via a rigid peptide linker, was added to the mixture of the two types of

8

nanoparticles, a red precipitate spontaneously formed (Figure 1a-II) and the supernatant showed

9

no plasmon absorption (Figure 1b-II). Aggregation of gold nanoparticles generally leads to a red-

10

shift of the plasmon absorption to purple and gray. However, the precipitate that formed in the

11

presence of the 4F2-E32 VHH dimer had red plasmon absorption, implying that the dimer linked

12

the gold and ZnO nanoparticles and that the linkages inhibited aggregation of the gold

13

nanoparticles.

14

Next, to study the influence of the ZnO/gold volume ratio on plasmon adsorption, we varied

15

the concentration of ZnO nanoparticles in the mixture. After the mixtures were dried on glass

16

plates, the resulting precipitates showed plasmon adsorption regardless of the ZnO/gold ratio

17

(Figure 1c-row1 and 1d; i–v). However, even when there was no ZnO in the mixture, a gold

18

nanoparticle precipitate formed in the presence of the 4F2-E32 VHH dimer (Figure 1c-row1 and

19

1d; 0), suggesting that 4F2 VHH (ZnO-binding VHH) in the dimer bound non-specifically to the

20

bare gold nanoparticle surface. This phenomenon is most likely the result of non-specific

21

interactions between bare nanoparticles and proteins, leading to the formation of a protein corona

22

on the surface of the gold nanoparticles.50,51 Although non-specific linking between the gold

23

nanoparticles was induced by the 4F2-E32 VHH dimer, the precipitate color and UV–vis spectra

ACS Paragon Plus Environment

9

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 43

1

varied depending on the ZnO/gold ratio. The extinction spectra derived from the gold

2

nanoparticles sharpened and were blue-shifted with increasing ZnO/gold ratio up to a ratio of 1/1

3

(Figure 1d: 0–iii). Whereas, further increases in the ratio broadened the band again, as well as

4

increasing the height of the baseline; however, the extinction spectra was observed around 520

5

nm (Figure 1d: iv, v). Considering that increasing the amount of ZnO likely led to interruption of

6

the transmission of incident light due to light scattering, it is possible that the plasmon absorption

7

band was also similarly obscured. According to these results, the ZnO might have prevented the

8

aggregation of the gold nanoparticles in the precipitates by specifically linking with gold

9

nanoparticle via the 4F2-E32 VHH dimer. When a 10 times concentrated (5 nM) gold

10

nanoparticle suspension without 4F2-E32 VHH dimer, which had obvious plasmon absorption

11

(Figure S1 in the Supporting Information), was dried on a glass plate, an uneven precipitate with

12

no plasmon absorption formed (Figure 1c-row1 and 1d; vi); loss of plasmon absorption was also

13

observed upon drying of a precipitate prepared from a concentrated gold nanoparticle suspension

14

that contained ZnO nanoparticles (Figure 1c-row 1 and 1d; vii). These results indicated that the

15

4F2-E32 VHH dimer induced assembly of gold–ZnO complexes with little loss of plasmon

16

resonance.

17

Gold–ZnO nanoparticle precipitates were also prepared by using gold nanoparticles with

18

diameters of 50 and 100 nm. Again, precipitates formed spontaneously in the presence of the

19

VHH dimer (Figure 1c-row 2 and -row 3; i–v); and the precipitates (Figure 1e and 1f), but not

20

the supernatants (Figure S2a and S2b in the Supporting Information), showed plasmon

21

absorption. The plasmon absorption bands of the precipitates on glass plates were sharper when

22

the ZnO/gold ratio was 0.5/1 and 1/1 than when other ratios were used (Figure 1e and 1f; ii and

23

iii), which was consistent with the results obtained when 20 nm gold nanoparticles were used.

ACS Paragon Plus Environment

10

Page 11 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1

Thus, controlling the ZnO/gold ratio was crucial for maintaining plasmon resonance in the gold–

2

ZnO nanoparticle precipitates.

3 4

Distribution of gold nanoparticles in the gold–ZnO nanoparticle composite membranes.

5

Scanning electron microscopy (SEM) was used to investigate the distribution of gold

6

nanoparticles within the nanoparticle precipitates that formed in the presence of the VHH dimer

7

(Figure 2). At ZnO/gold ratios of 0.5/1 and 1/1, the 20 nm gold nanoparticles were

8

homogenously dispersed throughout the precipitates (Figure 2-row1; ii and iii). These results are

9

consistent with the UV–vis spectra results shown in Figure 1d. Although in many parts of the

10

SEM images, the ZnO nanoparticles in the precipitate could not be observed clearly because of

11

the low contrast, the nanoparticles were visible in some of the more translucent areas of the

12

precipitate (Figure 2-row1; iv and v). We also found that 50 and 100 nm gold nanoparticles were

13

homogenously dispersed in the precipitates at a ZnO/gold ratio of 1/1 (Figure 2-row2 and -row3;

14

iii). However, at ZnO/gold ratios exceeding 1/1, no plasmon resonance was observed for the

15

precipitates (Figure 1e and 1f), even though the gold nanoparticles were not aggregated, as seen

16

in the SEM images (Figure 2-row2 and -row3; iv and v). Considering that increasing the amount

17

of ZnO likely led to interruption of the transmission of incident light due to light scattering,52–54

18

it is possible that the plasmon absorption band was also similarly obscured (Figure 1e and f; iv

19

and v).

20

To examine the interior of the nanoparticle precipitate, we prepared a partial cross-section

21

sample (thickness, ~100 nm) of gold–ZnO nanoparticle precipitate prepared with 20 nm gold

22

nanoparticles at a ZnO/gold ratio of 1/1, which was then observed by means of SEM,

23

transmission electron microscopy (TEM), and energy-dispersive X-ray (EDX) mapping (Figure

ACS Paragon Plus Environment

11

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 43

1

3). The cross-section SEM image showed that the ZnO and gold nanoparticles were widely

2

distributed throughout the precipitate (Figure 3a). The cross-section TEM image and EDX

3

mapping image showed that the precipitate contained both gold and ZnO nanoparticles and that

4

the two types of nanoparticles were bound in a complex (Figure 3b–d). Thus, we confirmed the

5

formation of a composite membrane derived from ZnO and gold nanoparticles.

6 7

Formation of nanoporous gold membranes. Next, we used gold–ZnO nanoparticle

8

composite membranes prepared with 20, 50, or 100 nm gold nanoparticles at a ZnO/gold ratio of

9

1/1 to produce nanoporous gold membranes by sintering the gold nanoparticles and dissolving

10

the ZnO with dilute acid. The melting point of bulk gold is 1064 °C, but nanoscale gold has a

11

lower melting point;55,56 and ZnO materials can be dissolved in dilute acid solution.57 First, we

12

heated the gold–ZnO membranes at various temperatures ranging from 200 to 400 °C to sinter

13

the gold nanoparticles, and we then dissolved the ZnO component in a glycine–HCl buffer

14

solution (pH 3.5). Figure 4 shows scanning electron micrographs and the elemental compositions

15

of the resulting materials.

16

For the membranes prepared with 20 nm gold nanoparticles, welding of the gold nanoparticles

17

was observed when the temperature exceeded 250 °C (Figure 4a-row1). Therefore, heating at

18

250 °C led to the formation of a nanoporous structure with a pore size of approximately 10 nm

19

(Figure 4b-i), but heating at higher temperatures led to the formation of larger vacancies (Figure

20

4a-row1). However, when the gold precipitate that formed at a ZnO/gold ratio of 0/1 in the

21

presence of 4F2-E32 VHH dimer was heated at 250 °C, SEM analysis revealed a heterogeneous

22

three-dimensional nanoporous structure (Figure S3a in the Supporting Information) that was

ACS Paragon Plus Environment

12

Page 13 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1

present even before sintering (Figure S3b in the Supporting Information), suggesting that 4F2-

2

E32 VHH dimer prevented excessive welding between gold nanoparticles in the precipitate.

3

For the membranes prepared with 50 and 100 nm gold nanoparticles, welding of the gold

4

nanoparticles was observed when the temperature exceeded 300 °C; in both cases, larger

5

vacancies were formed compared with when 20 nm gold nanoparticles were used (Figure 4a-

6

row2 and -row3). These results indicate that the pore size of the nanoporous structure can be

7

regulated by altering the size of the gold nanoparticles and the sintering temperature. That is, to

8

produce a porous network structure with a high surface area, small gold nanoparticles and low-

9

temperature sintering should be used. To examine the elemental composition of the nanoporous

10

gold membrane, we used EDX spectrometry (Figure 4b-ii). The spectrum of the membrane

11

formed at 250 °C from the 20 nm gold nanoparticles showed no ZnO signal, indicating that the

12

material was composed entirely of gold.

13 14

Catalytic performance of the nanoporous gold membranes. To evaluate the catalytic

15

activity of the nanoporous gold membrane, we measured the kinetics of the reduction of p-

16

nitrophenol to p-aminophenol by sodium borohydride (NaBH4). p-Nitrophenol has an intense

17

absorbance at 400 nm, and reduction to p-aminophenol leads to a blue-shift of the absorbance (to

18

300 nm), along with a decrease in its intensity. As control materials, we prepared a suspension

19

containing the same amount of 20 nm gold nanoparticles as was used for producing the

20

nanoporous membrane. The suspension was then dried with or without ZnO nanoparticles in the

21

absence of the VHH dimer, and the resulting residues were sintered at 250 °C and treated with

22

glycine–HCl buffer.

ACS Paragon Plus Environment

13

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 43

1

We analyzed the time dependence of the absorption spectrum of p-nitrophenol in the presence

2

of NaBH4 and the nanoporous gold membrane or one of the control materials (Figure 5). The

3

control material prepared from gold nanoparticles alone and that prepared from gold and ZnO

4

nanoparticles without the VHH dimer produced apparent reaction rates (kapp) of 0.080 × 10−3 and

5

0.227 × 10−3 s−1, respectively (Figure 5a, 5b, 5d-3, and 5d-4). In contrast, the nanoporous gold

6

membrane formed from the gold and ZnO nanoparticles in the presence of the VHH dimer

7

markedly enhanced the reaction rate (Figure 5c and 5d-6); specifically, kapp was 9 times that for

8

the material prepared from gold nanoparticles alone and 3 times that for the material prepared

9

from gold and ZnO nanoparticles without the VHH dimer (0.743 × 10−3 s−1). SEM showed that

10

the control material prepared from gold nanoparticles alone and that prepared from ZnO and gold

11

nanoparticles without the VHH dimer had no nanopores and relatively large nanopores,

12

respectively (Figure S4a and S4b in the Supporting Information), implying that the nanoporous

13

gold membrane formed in the presence of the VHH dimer had a larger surface area than did the

14

control materials.

15

We also measured the catalytic activity of a gold material prepared by simply drying gold

16

nanoparticles without subsequent sintering or treatment with glycine–HCl buffer, and we found

17

that the apparent reaction rate (0.245 × 10−3 s−1) was comparable with that for the control

18

material prepared from ZnO and gold nanoparticles without VHH dimer (Figure 5d-2 and 5d-4).

19

Because the gold nanoparticles retained their original shape in the material formed by simply

20

drying the nanoparticles (Figure S4c in the Supporting Information), the material showed high

21

catalytic activity. The reasons why the material prepared from gold nanoparticles alone did not

22

show the highest catalytic activity include the possible influence of molecules present on the

ACS Paragon Plus Environment

14

Page 15 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1

gold surface58–60 as well as differences in the crystal planes61 and surface area compared with the

2

other materials.

3

We also evaluated the catalytic activity of a nanoporous gold material prepared from the

4

precipitate that formed without ZnO nanoparticles in the presence of 4F2-E32 VHH dimer and

5

found that this material had a kapp value of 0.487 × 10−3 s−1 (Figure 5d-5), indicating that it had a

6

larger surface area and catalytic interface than the other control materials. The kapp value,

7

however, was approximately 2/3 times that for the nanoporous gold prepared from gold and ZnO

8

nanoparticles with the VHH dimer (0.743 × 10−3 s−1). This finding is likely the result of the gold

9

nanoparticles remaining separated from each other by ZnO nanoparticles during sintering, which

10

prevented the formation of large aggregates of the gold and increased the number of catalytically

11

active sites on the material surface. Thus we concluded that the nanoporous gold prepared from

12

the gold and ZnO nanoparticles in the presence of the VHH dimer offered high surface area led

13

to showing the high catalytic activity.

14

ACS Paragon Plus Environment

15

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 43

1

DISCUSSION

2

Pairing of nanomaterials by use of an interface molecule designed from complementary

3

antibody fragments. The bottom-up formation of superstructures from nanomaterials results in

4

functions that are difficult to obtain by means of top-down fabrication techniques. Van der Waals

5

and coulomb forces between nanomaterials are critical driving forces for their assembly, and

6

homogeneity in their size, shape, and surface properties leads to uniform interactions between

7

the materials and thus to the formation of well-ordered assemblies. However, only a limited

8

number of homogeneous nanomaterials have been synthesized, and intentional linking of two

9

specific nanomaterials has rarely been achieved, even with nanomaterials having strictly

10

controlled structures. Conjugation of organic molecules to the surface of nanomaterials can

11

homogenize their surface properties to induce uniform interactions between nanomaterials, and

12

conjugation between nanomaterials and molecules with recognition ability, such as DNA 17–24,62–

13

64

14

for molecules with recognition ability to be covalently conjugated to the nanomaterials, as well

15

as the conjugation process itself, can restrict the types of nanomaterials that can be used.

and proteins,65–67 can lead to interactions between specific nanomaterials. However, the need

16

In the present study, we used a small bispecific antibody comprising two material-binding

17

antibody fragments, E32 VHH and 4F2 VHH, which strongly bind to gold and ZnO surfaces,

18

respectively, without the need for surface modification. The strong affinity of the VHHs for their

19

respective nanomaterials resulted in spontaneous assembly and precipitation of gold–ZnO

20

nanoparticles, which could be deposited as thin films that exhibited plasmon absorption. A wide

21

variety of peptides with binding affinity for a specific material surface have been identified

22

through large-scale selection based on phage and cell surface display techniques, and material-

ACS Paragon Plus Environment

16

Page 17 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1

binding antibodies with high affinity can be generated from the peptides by means of our

2

CAnIGET method.38

3

Although the mechanisms of the binding of these antibodies to their respective material

4

surfaces are not clearly understood, the van’t Hoff plots in our previous reports suggest that

5

enthalpy change, not entropy change, is the major underlying factor in this binding.37,38 Indeed,

6

an important factor in the adsorption of proteins to material surfaces is usually the entropy

7

change that results from dehydration and desorption of the capping agents on the material surface

8

and the structural rearrangement of the adsorbed proteins.68,69 Therefore, we hypothesized that

9

the antibodies examined in the present study were held on the nanoparticle surface via selective

10

attachment of their complementary-determining region loop. When we used denaturing reagents

11

to detach the VHHs from the nanoparticles, 10 mM glycine–HCl buffer (pH 2.0) and 50 mM

12

NaOH caused complete dissolution of the ZnO–gold precipitates, resulting in a nanoparticle

13

dispersion with an extinction spectra at 520 nm (Figure S5 in the Supporting Information). In

14

contrast, adding distilled water or 1 M NaCl resulted in no dissolution of the precipitates. This

15

re-dispersion of the gold nanoparticles suggests that glycine–HCl buffer and NaOH solution

16

denatured the VHHs, which supports the idea that the ZnO and gold nanoparticles were linked

17

via 4F2-E32 VHH dimer.

18

With the setup used in the present study, we were able to harvest 4F2-E32 VHH dimer at an

19

average yield of 1.5 mg/L-culture of BL21 (DE3) strain, and 33.3 µg of dimer in 1 mL of

20

reaction suspension was needed to make a ZnO–gold membrane. Therefore, 4F2-E32 VHH

21

dimer harvested from 1 L of culture can be used to process 45 mL of reaction suspension.

22

Although our approach using bispecific material-binding antibodies is expensive in terms of

ACS Paragon Plus Environment

17

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 43

1

labor and time compared with conventional chemical and physical approaches, the antibody

2

fragments produced allow for the production of a wide array of superstructures.

3 4

Bottom-up formation of nanoporous gold materials. Porous materials with a large surface

5

area are critically important for various applications, including catalysis, filtration, trapping, and

6

sensing. Such materials can be formed by assembling atoms or nanomaterials into a desired

7

porous structure, or by supporting functional materials on a porous scaffold substrate. Dealloying

8

is a useful process for synthesizing highly functional porous gold materials.70 Specifically, gold

9

materials are alloyed with Ag, Al, or Cu metal, and the alloys are then soaked in concentrated

10

nitric acid to remove the added metal.48,49 However, because high temperature (>1000 °C) is

11

required for alloy formation, this is a high-energy process; in addition, traces of the metal

12

additive remain in the resulting porous materials. Gold nanoparticles have also been used for

13

synthesizing porous materials by means of a process involving deposition of the nanoparticles on

14

templates consisting of an organic colloidal crystal assembly.71,72 Heating at a relatively low

15

temperature (~300 °C) removes the organic template and sinters the gold nanoparticles to form a

16

macroporous structure. In the present study, we used gold nanoparticles, which can be melted at

17

a relatively low temperature (250–300 °C), and ZnO nanoparticles, which can be dissociated by

18

dilute acid; therefore, neither high-energy alloy formation nor the use of a concentrated acidic

19

solution was required. Furthermore, sintering of the gold nanoparticles and removal of the ZnO

20

led to the formation of a membrane with nanopores as small as those in the finest porous

21

structures reported previously; consequently, the membrane had high catalytic activity. We

22

expect that the nanoporous gold prepared in the present study can catalyze other chemical

23

reactions. For example, Wen et al. recently reported a gold aerogel prepared via dopamine-

ACS Paragon Plus Environment

18

Page 19 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1

induced destabilization of beta-cyclodextrin-functionalized gold nanoparticles and showed that

2

the aerogel catalyzed electrochemical oxidation of glucose and ethanol.73

3

Nanoporous gold-based materials have been extensively studied because of their catalytic

4

activities and high surface-to-volume ratios. Our present study contributes information useful for

5

tuning of the thickness and morphologies of nanoporous gold films without the introduction of

6

impurities. Compared with nanoporous gold without impurities, nanoporous gold prepared by

7

dealloying usually has higher chemical reactivity because metal atoms remaining from the

8

dealloying process accelerate catalytic reaction rates by suppressing surface reconstruction

9

dynamics and providing active sites for the reaction.61 Therefore, to maximize the catalytic

10

activity of nanoporous gold without impurities, further reduction of nanoparticle size and the

11

introduction of high-index facets should be explored.

12

ACS Paragon Plus Environment

19

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 43

1

CONCLUSIONS

2

In summary, we demonstrated pairing between two different types of nanoparticles mediated

3

by a bispecific material-binding antibody to form binary nanoparticles without the need for

4

surface modification. The binary nanoparticles showed plasmon resonance when assembled into

5

nanoparticle composite membranes. Sintering of the membranes and subsequent treatment with

6

glycine–HCl buffer led to the formation of a nanoporous gold membrane with high catalytic

7

activity for the reduction of p-nitrophenol by sodium hydride. Our results illustrate the utility of

8

high-affinity material-binding antibodies for the design of tools for mediating the pairing of

9

nanomaterials and subsequent formation of nanostructured hybrid materials.

10 11 12

ACS Paragon Plus Environment

20

Page 21 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1 2

Scheme 1. Synthesis of a nanoporous gold membrane from gold–ZnO nanoparticles via a

3

bispecific metal surface-binding antibody.

4

ACS Paragon Plus Environment

21

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 43

1 2

Figure 1. Precipitation of gold nanoparticles. (a) Photographs of phosphate buffer solutions of

3

gold nanoparticles (20 nm, 500 pM) and ZnO nanoparticles (~100 nm, 5.6 µg/mL) at a ZnO/gold

4

volume ratio of 1/1 after incubation for 1 day at 4 °C in the (I) absence or (II) presence of 1 µM

ACS Paragon Plus Environment

22

Page 23 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1

4F2-E32 VHH dimer. (b) Absorption spectra of (I) the gold and ZnO suspension after incubation

2

in the absence of the dimer and (II) the supernatant obtained after incubation in the presence of

3

the dimer. (c) Photographs of gold–ZnO precipitates formed on glass plates from gold

4

nanoparticles with diameters of 20, 50, and 100 nm and ZnO/gold volume ratios ranging from

5

0.1/1 to 10/1. (d–f) Absorption spectra of gold–ZnO precipitates formed from gold nanoparticles

6

with diameters of 20 nm (d), 50 nm (e), and 100 nm (f).

ACS Paragon Plus Environment

23

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 43

1 2

Figure 2. SEM images of gold–ZnO nanoparticle precipitates formed from gold nanoparticles

3

with diameters of 20, 50, and 100 nm at ZnO/gold volume ratios ranging from 0.1/1 to 10/1. The

4

images were obtained at acceleration voltages of 15–25 kV. Scale bars are 500 nm.

5

ACS Paragon Plus Environment

24

Page 25 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1 2

Figure 3. Cross-section micrographs of a gold–ZnO precipitate formed from 20 nm gold

3

nanoparticles. (a) SEM image, (b) TEM image, (c) magnified TEM image of the red-dashed

4

square in (b), and (d) EDX mapping image of the area shown in (b).

5

ACS Paragon Plus Environment

25

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 43

1 2

Figure 4. Structural analysis of the gold–ZnO membranes after heating and dissolving

3

treatments. (a) Scanning electron micrographs of membranes obtained after sintering the gold–

4

ZnO nanoparticle precipitates (prepared at a ZnO/gold ratio of 1/1) at various temperatures from

5

200 to 400 °C and then dissolving the ZnO in glycine–HCl buffer. All scale bars are 1 µm. (b)

6

Scanning electron micrographs (i) and EDX spectrum (ii) of the gold–ZnO membrane obtained

7

after sintering gold–ZnO precipitate fabricated with 20 nm gold nanoparticles at 250 °C and

8

dissolving the ZnO.

9

ACS Paragon Plus Environment

26

Page 27 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1 2

Figure 5. Temporal dependence of absorption spectra of a reaction mixture containing p-

3

nitrophenol, NaBH4, and (a) a control material prepared from gold nanoparticles, (b) a control

4

material prepared from gold and ZnO nanoparticles, and (c) a nanoporous gold membrane

5

formed from gold and ZnO nanoparticles in the presence of the 4F2-E32 VHH dimer; spectra are

6

shown at 10 min intervals. (d) Reaction time dependence of the log of absorbance at 400 nm: in

7

the absence of gold (1, black) and in the presence of a gold material prepared from gold

8

nanoparticles without sintering or acid treatment (2, orange), a gold material prepared from gold

9

nanoparticles with sintering and acid treatment (3, blue), a gold material prepared from gold and

10

ZnO nanoparticles (4, green), a gold material prepared with sintering and acid treatment from the

11

precipitate produced from gold nanoparticles in the presence of 4F2-E32 VHH dimer (5, purple),

12

and a nanoporous gold membrane prepared with sintering and acid treatment from gold and ZnO

13

nanoparticles in the presence of the 4F2-E32 VHH dimer (6, red). The catalytic reduction

ACS Paragon Plus Environment

27

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 43

1

reactions were carried out with 100 mM NaBH4, 1 mM p-nitrophenol, and ~122 µg of gold

2

catalyst (5 mL, prepared from 500 pmol/L 20 nm gold nanoparticles). The reaction solutions

3

were diluted by 1/10 prior to measurement of the absorption spectra.

ACS Paragon Plus Environment

28

Page 29 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1

ASSOCIATED CONTENT

2

Photographs and UV-vis spectra of phosphate buffer solutions containing gold nanoparticles

3

(diameters: 50 nm and 100 nm) and ZnO in the presence of 4F2-E32 VHH dimer; Photographs

4

of a concentrated colloidal suspension of 20 nm gold nanoparticles before and after drying; SEM

5

images of gold structures formed in the absence of 4F2-E32 VHH dimer

6

ACS Paragon Plus Environment

29

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

AUTHOR INFORMATION

2

Corresponding Author

3

*E-mail: [email protected]

4

Present Addresses

5

‡ Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, 4-4-1

6

Komatsushima, Aoba-ku, Sendai 981-8558, Japan

7

¶ Department of Biochemistry and Molecular Pharmacology, New York University School of

8

Medicine, New York, NY 10016, United States

9

Notes

10

Page 30 of 43

The authors declare no competing financial interest.

11

ACS Paragon Plus Environment

30

Page 31 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Langmuir

ACKNOWLEDGMENT

2

The authors thank Mr. Shunsuke Kayamori and Dr. Takamichi Miyazaki for his excellent

3

technical support for the preparation of the samples used to produce the cross-section

4

micrographs and in recording the electron micrographs. This work was supported by a Scientific

5

Research Grant from the Ministry of Education, Culture, Sports, Science and Technology of

6

Japan (to I.K.; 24000011, M.U.; 16H04570, 16K14483, H.N.; 16K18296, and T.N.; 18K14059)

7

and by the Precursory Research for Embryonic Science and Technology program of the Japan

8

Science and Technology Agency (to M.U.).

9

ACS Paragon Plus Environment

31

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 43

1

REFERENCES

2

(1)

3

Nanocrystals of Different Shapes. Chem. Rev. 2005, 105, 1025–1102.

4

(2)

5

T.; Schroedter, A.; Weller, H.; Yasuda, A. Optical and Electrical Properties of Three-

6

Dimensional Interlinked Gold Nanoparticle Assemblies. J. Am. Chem. Soc. 2004, 126, 3349–

7

3356.

8

(3)

9

Nanocrystals. Eur. J. Inorg. Chem. 2005, 3613–3623.

Burda, C.; Chen, X.; Narayanan, R.; El-sayed, M. A. Chemistry and Properties of

Wessels, J. M.; Nothofer, H.-G.; Ford, W. E.; von Wrochem, F.; Scholz, F.; Vossmeyer,

Shavel, A.; Gaponik, N.; Eychmüller, A. The Assembling of Semiconductor

10

(4)

Sreeprasad, T. S.; Samal, A. K.; Pradeep, T. One- , Two- , and Three-Dimensional

11

Superstructures of Gold Nanorods Induced by Dimercaptosuccinic Acid. Langmuir 2008, 24,

12

4589–4599.

13

(5)

14

Nanoparticles. ACS Nano 2010, 4, 3591–3605.

15

(6)

16

Somorjai, G. A.; Yang, P. Nanocrystal Bilayer for Tandem Catalysis. Nat. Chem. 2011, 3, 372–

17

376.

18

(7)

19

Acc. Chem. Res. 2012, 45, 1916–1926.

Grzelczak, M.; Vermant, J.; Furst, E. M.; Liz-Marzán, L. M. Directed Self-Assembly of

Yamada, Y.; Tsung, C.-K.; Huang, W.; Huo, Z.; Habas, S. E.; Soejima, T.; Aliaga, C. E.;

Wang, L.; Xu, L.; Kuang, H.; Xu, C.; Kotov, N. A. Dynamic Nanoparticle Assemblies.

ACS Paragon Plus Environment

32

Page 33 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1

(8)

Hamon, C.; Novikov, S.; Scarabelli, L.; Basabe-Desmonts, L.; Liz-Marzán, L. M.

2

Hierarchical Self-Assembly of Gold Nanoparticles into Patterned Plasmonic Nanostructures.

3

ACS Nano 2014, 8, 10694–10703.

4

(9)

5

Diversity in Binary Nanoparticle Superlattices. Nature 2006, 439, 55–59.

6

(10)

7

Hens, Z.; Manna, L. Self-Assembled Multilayers of Vertically Aligned Semiconductor Nanorods

8

on Device-Scale Areas. Adv. Mater. 2011, 23, 2205–2209.

9

(11)

Shevchenko, E. V.; Talapin, D. V.; Kotov, N. A.; O’Brien, S.; Murray, C. B. Structural

Zanella, M.; Gomes, R.; Povia, M.; Giannini, C.; Zhang, Y.; Riskin, A.; Van Bael, M.;

Miszta, K.; de Graaf, J.; Bertoni, G.; Dorfs, D.; Brescia, R.; Marras, S.; Ceseracciu, L.;

10

Cingolani, R.; van Roij, R.; Dijkstra, M.; Manna, L. Hierarchical Self-Assembly of Suspended

11

Branched Colloidal Nanocrystals into Superlattice Structures. Nat. Mater. 2011, 10, 872–876.

12

(12)

13

H.; Wang, Z.; Cao, Y. C. Self-Assembled Colloidal Superparticles from Nanorods. Science 2012,

14

338, 358–363.

15

(13)

16

Controlling Mesoscopic Organizations of Magnetic Nanocrystals. Nat. Mater. 2004, 3, 121–125.

17

(14)

18

Particles by Electric Fields. Soft Matter 2006, 2, 738–750.

Wang, T.; Zhuang, J.; Lynch, J.; Chen, O.; Wang, Z.; Wang, X.; Lamontagne, D.; Wu,

Lalatonne, Y.; Richardi, J.; Pileni, M. P. Van Der Waals versus Dipolar Forces

Velev, O. D.; Bhatt, K. H. On-Chip Micromanipulation and Assembly of Colloidal

ACS Paragon Plus Environment

33

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 43

1

(15)

Li, F.; Yoo, W. C.; Beernink, M. B.; Stein, A. Site-Specific Functionalization of

2

Anisotropic Nanoparticles: From Colloidal Atoms to Colloidal Molecules. J. Am. Chem. Soc.

3

2009, 131, 18548–18555.

4

(16)

5

onto Thermosensitive Magnetic Core-Shell Microgels for Thermally Tunable and Magnetically

6

Recyclable Catalysis. Small, 2015, 11, 2807–2816.

7

(17)

8

Rationally Assembling Nanoparticles into Macroscopic Materials. Nature 1996, 382, 607–609.

9

(18)

Liu, G.; Wang, D. ; Zhou, F.; Liu, W. Electrostatic Self-Assembly of Au Nanoparticles

Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. A DNA-Based Method for

Nykypanchuk, D.; Maye, M. M.; van der Lelie, D.; Gang, O. DNA-Guided

10

Crystallization of Colloidal Nanoparticles. Nature 2008, 451, 549–552.

11

(19)

12

DNA-Programmable Nanoparticle Crystallization. Nature 2008, 451, 553–556.

13

(20)

14

Nanoparticle Self-Similar Chain Structure Organized by DNA Origami. J. Am. Chem. Soc. 2010,

15

132, 3248–3249.

16

(21)

17

between Nanoparticle Superlattices via the Reprogramming of DNA-Mediated Interactions. Nat.

18

Mater. 2015, 14, 840–847.

Park, S. Y.; Lytton-Jean, A. K. R.; Lee, B.; Weigand, S.; Schatz, G. C.; Mirkin, C. A.

Ding, B.; Deng, Z.; Yan, H.; Cabrini, S.; Zuckermann, R. N.; Bokor, J. Gold

Zhang, Y.; Pal, S.; Srinivasan, B.; Vo, T.; Kumar, S.; Gang, O. Selective Transformations

ACS Paragon Plus Environment

34

Page 35 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1

(22)

Wang, Y.; Wang, Y.; Zheng, X.; Ducrot, É.; Lee, M.-G.; Yi, G.-R.; Weck, M.; Pine, D. J.

2

Synthetic Strategies Toward DNA-Coated Colloids that Crystallize. J. Am. Chem. Soc. 2015,

3

137, 10760–10766.

4

(23)

5

Plasmonic Devices: High-Yield Arrangement of Gold Nanoparticles on DNA Origami

6

Templates. ACS Nano 2016, 10, 5374–5382.

7

(24)

8

with DNA Origami Nanoflowers. ACS Nano 2016, 10, 7303–7306.

9

(25)

Gür, F. N.; Schwarz, F. W.; Ye, J.; Diez, S.; Schmidt, T. L. Toward Self-Assembled

Schreiber, R.; Santiago, I.; Ardavan, A.; Turberfield, A. J. Ordering Gold Nanoparticles

Brown, S. Metal-Recognition by Repeating Polypeptides. Nat. Biotechnol. 1997, 15,

10

269–272.

11

(26)

12

Peptides with Semiconductor Binding Specificity for Directed Nanocrystal Assembly. Nature

13

2000, 405, 665–668.

14

(27)

15

Titanium. J. Am. Chem. Soc. 2003, 125, 14234–14235.

16

(28)

17

I.; Adschiri, T. Bioassisted Room-Temperature Immobilization and Mineralization of Zinc

18

Oxide—The Structural Ordering of ZnO Nanoparticles into a Flower-Type Morphology. Adv.

19

Mater. 2005, 17, 2571–2575.

Belcher, A. M.; Whaley, S. R.; English, D. S.; Hu, E. L.; Barbara, P. F. Selection of

Sano, K.-I.; Shiba, K. A Hexapeptide Motif that Electrostatically Binds to the Surface of

Umetsu, M.; Mizuta, M.; Tsumoto, K.; Ohara, S.; Takami, S.; Watanabe, H.; Kumagai,

ACS Paragon Plus Environment

35

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 43

1

(29)

Niide, T.; Ozawa, K.; Nakazawa, H.; Oliveira, D.; Kasai, H.; Onodera, M.; Asano, R.;

2

Kumagai, I.; Umetsu, M. Organic Crystal-Binding Peptides: Morphology Control and One-Pot

3

Formation of Protein-Displaying Organic Crystals. Nanoscale 2015, 7, 20155–20163.

4

(30)

5

Nanoparticle Self-Similar Chain Structure Organized by DNA Origami. J. Am. Chem. Soc. 2010,

6

132, 3248–3249.

7

(31)

8

Plasmonic Devices: High-Yield Arrangement of Gold Nanoparticles on DNA Origami

9

Templates. ACS Nano 2016, 10, 5374–5382.

Ding, B.; Deng, Z.; Yan, H.; Cabrini, S.; Zuckermann, R. N.; Bokor, J. Gold

Gür, F. N.; Schwarz, F. W.; Ye, J.; Diez, S.; Schmidt, T. L. Toward Self-Assembled

10

(32)

Schreiber, R.; Santiago, I.; Ardavan, A.; Turberfield, A. J. Ordering Gold Nanoparticles

11

with DNA Origami Nanoflowers. ACS Nano 2016, 10, 7303–7306.

12

(33)

13

Oxide Nanoparticles and their Patterning onto Surfaces. J. Mater. Chem. 2012, 22, 12423–

14

12434.

15

(34)

16

Allwood, D. A.; Leggett, G. J.; Miles, J. J.; Staniland, S. S.; Critchley, K. Nano- and Micro-

17

Patterning Biotemplated Magnetic CoPt Arrays. Nanoscale 2016, 8, 11738–11747.

18

(35)

19

1947, 51, 631–636.

Galloway, J. M.; Staniland, S. S. Protein and Peptide Biotemplated Metal and Metal

Galloway, J. M.; Bird, S. M.; Talbot, J. E.; Shepley, P. M.; Bradley, R. C.; El-Zubir, O.;

Verwey, E. J. W. Theory of the Stability of Lyophobic Colloids. J. Phys. Colloid Chem.

ACS Paragon Plus Environment

36

Page 37 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1

(36)

Daniel, M.-C.; Astruc, D. Gold Nanoparticles: Assembly, Supramolecular Chemistry,

2

Quantum-Size-Related

3

Nanotechnology. Chem. Rev. 2004, 104, 293–346.

4

(37)

5

Biofunctionalization of Gold Nanoparticles and Surfaces with Anti-Gold Antibodies. J. Biol.

6

Chem. 2008, 283, 36031–36038.

7

(38)

8

Adschiri, T.; Kumagai, I. High Affinity Anti-Inorganic Material Antibody Generation by

9

Integrating Graft and Evolution Technologies: Potential of Antibodies as Biointerface

Properties,

and

Applications

toward

Biology,

Catalysis,

and

Watanabe, H.; Nakanishi, T.; Umetsu, M.; Kumagai, I. Human Anti-Gold Antibodies:

Hattori, T.; Umetsu, M.; Nakanishi, T.; Togashi, T.; Yokoo, N.; Abe, H.; Ohara, S.;

10

Molecules. J. Biol. Chem. 2010, 285, 7784–7793.

11

(39)

12

High-Affinity

13

Functionalization of Gold Nanoparticles as Biointerface Molecules. Bioconjug. Chem. 2012, 23,

14

1934–1944.

15

(40)

16

Res. 2014, 47, 731–739.

17

(41)

18

the Origin of the Reactivity of a 21st Century Catalyst Made by Pre-Columbian Technology.

19

ACS Catal. 2015, 5, 6263–6270.

Hattori, T.; Umetsu, M.; Nakanishi, T.; Sawai, S.; Kikuchi, S.; Asano, R.; Kumagai, I. A Gold-Binding

Camel

Antibody:

Antibody

Engineering

for

One-Pot

Wittstock, A.; Bäumer, M. Catalysis by Unsupported Skeletal Gold Catalysts. Acc. Chem.

Biener, J.; Biener, M. M.; Madix, R. J.; Friend, C. M. Nanoporous Gold: Understanding

ACS Paragon Plus Environment

37

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 43

1

(42)

Personick, M. L.; Madix, R. J.; Friend, C. M. Selective Oxygen-Assisted Reactions of

2

Alcohols and Amines Catalyzed by Metallic Gold: Paradigms for the Design of Catalytic

3

Processes. ACS Catal. 2017, 7, 965–985.

4

(43)

5

M. Gold Catalysts: Nanoporous Gold Foams. Angew. Chemie Int. Ed. 2006, 45, 8241–8244.

6

(44)

7

Support-Free Nanoporous Gold. J. Phys. Chem. C 2008, 112, 9673–9678.

8

(45)

9

Oxidation. J. Phys. Chem. C 2007, 111, 10382–10388.

Zielasek, V.; Jürgens, B.; Schulz, C.; Biener, J.; Biener, M. M.; Hamza, A. V.; Bäumer,

Yin, H.; Zhou, C.; Xu, C.; Liu, P.; Xu, X.; Ding, Y. Aerobic Oxidation of d -Glucose on

Zhang, J.; Liu, P.; Ma, H.; Ding, Y. Nanostructured Porous Gold for Methanol Electro-

10

(46)

Zeis, R.; Lei, T.; Sieradzki, K.; Snyder, J.; Erlebacher, J. Catalytic Reduction of Oxygen

11

and Hydrogen Peroxide by Nanoporous Gold. J. Catal. 2008, 253, 132–138.

12

(47)

13

Chen,

14

Semihydrogenation of Alkynes: Remarkable Effect of Amine Additives. J. Am. Chem. Soc.

15

2012, 134, 17536–17542.

16

(48)

17

Nanoporosity in Dealloying. Nature 2001, 410, 450–453.

18

(49)

19

Nanoporous Plasmonic Metamaterials. Adv. Mater. 2008, 20, 1211–1217.

Yan, M.; Jin, T.; Ishikawa, Y.; Minato, T.; Fujita, T.; Chen, L.-Y.; Bao, M.; Asao, N.; M.-W.;

Yamamoto,

Y.

Nanoporous

Gold

Catalyst

for

Highly

Selective

Erlebacher, J.; Aziz, M. J.; Karma, A.; Dimitrov, N.; Sieradzki, K. Evolution of

Biener, J.; Nyce, G. W.; Hodge, A. M.; Biener, M. M.; Hamza, A. V; Maier, S. A.

ACS Paragon Plus Environment

38

Page 39 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1

(50)

Cedervall, T.; Lynch, I.; Lindman, S.; Berggård, T.; Thulin, E.; Nilsson, H.; Dawson, K.

2

A.; Linse, S. Understanding the Nanoparticle–Protein Corona Using Methods To Quantify

3

Exchange Rates and Affinities of Proteins for Nanoparticles. Proc. Natl. Acad. Sci. U. S. A.

4

2007, 104, 2050–2055.

5

(51)

6

ACS Nano 2017, 11, 11773–11776.

7

(52)

8

Photoluminescence of Zinc Oxide with Different Morphologies and Dimensions. J. Appl. Phys.

9

2005, 98, 083502.

Ke, P. C.; Lin, S.; Parak, W. J.; Davis, T. P.; Caruso, F. A Decade of the Protein Corona.

Shi, W. S.; Cheng, B.; Zhang, L.; Samulski, E. T. Influence of Excitation Density on

10

(53)

Chou, T. P.; Zhang, Q.; Fryxell, G. E.; Cao, G. Z. Hierarchically Structured ZnO Film for

11

Dye-Sensitized Solar Cells with Enhanced Energy Conversion Efficiency. Adv. Mater. 2007, 19,

12

2588–2592.

13

(54)

14

Kim, H.; Yun Kim, H.; Hyoung Ryu, J.; Gu Kim, H.; Hong, C.-H. Enhanced Light Output Power

15

of GaN-Based Light-Emitting Diodes by Nano-Rough Indium Tin Oxide Film Using ZnO

16

Nanoparticles. J. Appl. Phys. 2011, 109, 093116.

17

(55)

18

Rev. A 1976, 13, 2287–2298.

19

(56)

20

Temperature of Individual Nanometer-Sized Metallic Clusters. Phys. Rev. B 1990, 42, 8548–

21

8556.

Deul Ryu, B.; Uthirakumar, P.; Hye Kang, J.; Jun Kwon, B.; Chandramohan, S.; Kyu

Buffat, P.; Borel, J.-P. Size Effect on the Melting Temperature of Gold Particles. Phys.

Castro, T.; Reifenberger, R.; Choi, E.; Andres, R. P. Size-Dependent Melting

ACS Paragon Plus Environment

39

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 43

1

(57)

Hüpkes, J.; Owen, J. I.; Pust, S. E.; Bunte , E. Chemical Etching of Zinc Oxide for Thin-

2

Film Silicon Solar Cells. ChemPhysChem 2012, 13, 66–73.

3

(58)

4

Dimethylformamide-Stabilized Gold Nanoclusters as a Catalyst for the Reduction of 4-

5

Nitrophenol. Nanoscale 2012, 4, 4148–4154.

6

(59)

7

Active Au Nanoparticles onto Gibbsite–Polydopamine Core–Shell Nanoplates. Langmuir 2015,

8

31, 9483–9491.

9

(60)

Yamamoto, H.; Yano, H.; Kouchi, H.; Obora, Y.; Arakawa, R.; Kawasaki, H. N,N-

Cao, J.; Mei, S.; Jia, H.; Ott, A.; Ballauff, M.; Lu, Y. In Situ Synthesis of Catalytic

Jia, H.; Schmitz, D.; Ott, A.; Pich, A.; Lu, Y. Cyclodextrin Modified Microgels as

10

“Nanoreactor” for the Generation of Au Nanoparticles with Enhanced Catalytic Activity. J.

11

Mater. Chem. A 2015, 3, 6187–6195.

12

(61)

13

S.; Yamamoto, Y.; Tanaka, N.; Ishikawa, Y.; Asao, N.; Yamamoto, Y.; Erlebacher, J.; Chen, M.

14

Atomic Origins of the High Catalytic Activity of Nanoporous Gold. Nat. Mater. 2012, 11, 775–

15

780.

16

(62)

17

D. Free-Standing Nanoparticle Superlattice Sheets Controlled by DNA. Nat. Mater. 2009, 8,

18

519–525.

19

(63)

20

C. A. DNA-Nanoparticle Superlattices Formed from Anisotropic Building Blocks. Nat. Mater.

21

2010, 9, 913–917.

Fujita, T.; Guan, P.; McKenna, K.; Lang, X.; Hirata, A.; Zhang, L.; Tokunaga, T.; Arai,

Cheng, W.; Campolongo, M. J.; Cha, J. J.; Tan, S. J.; Umbach, C. C.; Muller, D. A.; Luo,

Jones, M. R.; Macfarlane, R. J.; Lee, B.; Zhang, J.; Young, K. L.; Senesi, A. J.; Mirkin,

ACS Paragon Plus Environment

40

Page 41 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1

(64)

Zhang, Y.; Lu, F.; Yager, K. G.; van der Lelie, D.; Gang, O. A General Strategy for the

2

DNA-Mediated Self-Assembly of Functional Nanoparticles into Heterogeneous Systems. Nat.

3

Nanotechnol. 2013, 8, 865–872.

4

(65)

Brown, S. Protein-Mediated Particle Assembly. Nano Lett. 2001, 1, 391–394.

5

(66)

Thanh, N. T. K.; Rosenzweig, Z. Development of an Aggregation-Based Immunoassay

6

for Anti-Protein A Using Gold Nanoparticles. Anal. Chem. 2002, 74, 1624–1628.

7

(67)

8

Assembled via Specific Biological Cross-Linking. Langmuir 2004, 20, 6788–6795.

9

(68)

Hiddessen, A. L.; Weitz, D. A.; Hammer, D. A. Rheology of Binary Colloidal Structures

Norde, W.; Lyklema, J. The Adsorption of Human Plasma Albumin and Bovine Pancreas

10

Ribonuclease at Negatively Charged Polystyrene Surfaces: I. Adsorption Isotherms. Effects of

11

Charge, Ionic Strength, and Temperature. J. Colloid Interface Sci. 1978, 66, 257–265.

12

(69)

13

Reference to the Adsorption of Human Plasma Albumin and Bovine Pancreas Ribonuclease at

14

Polystyrene Surfaces. J. Colloid Interface Sci. 1979, 71, 350–366.

15

(70)

16

Technology”/Advanced Material. Adv. Mater. 2004, 16, 1897–1900.

17

(71)

18

Nanostructures. Nature 1999, 401, 548–548.

19

(72)

20

From Inorganic Oxides to Metals. Adv. Mater. 2000, 12, 531–534.

Norde, W.; Lyklema, J. Thermodynamics of Protein Adsorption. Theory with Special

Ding,

Y.;

Kim,

Y.-J.;

Erlebacher,

J.

Nanoporous

Gold

Leaf:

“Ancient

Velev, O. D.; Tessier, P. M.; Lenhoff, A. M.; Kaler, E. W. A Class of Porous Metallic

Velev, O. D.; Kaler, E. W. Structured Porous Materials via Colloidal Crystal Templating:

ACS Paragon Plus Environment

41

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 43

1

(73)

Wen, D.; Liu, W.; Haubold, D.; Zhu, C.; Oschatz, M.; Holzschuh, M.; Wolf, A.; Simon,

2

F.; Kaskel, S.; Eychmüller, A. Gold Aerogels: Three-Dimensional Assembly of Nanoparticles

3

and Their Use as Electrocatalytic Interfaces. ACS Nano 2016, 10, 2559–2567.

ACS Paragon Plus Environment

42

Page 43 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

ACS Paragon Plus Environment