Nanoparticle Synthesis - American Chemical Society

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

Bacteriophages as factories for Eu2O3 nanoparticle synthesis Piotr Golec, Kamila #elechowska, Joanna Karczewska-Golec, Jakub Karczewski, Adam Lesniewski, Marcin #o#, Grzegorz Wegrzyn, and Andrzej M. Klonkowski Bioconjugate Chem., Just Accepted Manuscript • Publication Date (Web): 08 May 2017 Downloaded from http://pubs.acs.org on May 10, 2017

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 free 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 accessible to all readers and 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.

Bioconjugate Chemistry 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 26

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

Bioconjugate Chemistry

1

Bacteriophages as factories for Eu2O3 nanoparticle synthesis

2

Running title: Eu2O3 NPs synthesis by modified M13 phage

3

4

Piotr Goleca*, Kamila Żelechowskab, Joanna Karczewska-Golecc,d, Jakub Karczewskib, Adam

5

Leśniewskie, Marcin Łośd, Grzegorz Węgrzync, Andrzej M. Kłonkowskif

6

a

7

Biochemistry and Biophysics, Polish Academy of Sciences, Wita Stwosza 59, 80-308 Gdansk, Poland

8

b

9

Technology, Narutowicza 11/12, 80-233 Gdansk, Poland

Laboratory of Molecular Biology (affiliated with the University of Gdansk), Institute of

Faculty of Applied Physics and Mathematics, Solid State Physics Department, Gdansk University of

10

c

Department of Molecular Biology, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk, Poland

11

d

Department of Bacterial Molecular Genetics, University of Gdansk, Wita Stwosza 59, 80-308

12

Gdansk, Poland

13

e

14

Poland

15

f

Institute of Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw,

Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308 Gdansk, Poland

16

17

* Corresponding author: Dr. Piotr Golec, Laboratory of Molecular Biology (affiliated with the

18

University of Gdansk), Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Wita

19

Stwosza 59, 80-308 Gdansk, Poland, Tel.: +48 58 523 6041, Fax: +48 58 523 6025, e-mail:

20

[email protected]

21

KEYWORDS: europium oxide, europium oxide nanoparticles, luminescent materials, phage

22

display 1 ACS Paragon Plus Environment


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 26

23

ABSTRACT

24

The use of phage display to identify peptides with an ability to bind Eu2O3 is demonstrated in

25

this report. This is the first report of modified phages specifically binding a lanthanide. The

26

peptides exposed on virions revealed very strong binding to Eu2O3 nanoparticles and the

27

ability to catalyze Eu2O3 nanoparticles’ formation from Eu(OH)3 and Eu(NO3)3 solutions. The

28

luminescence emission spectrum of Eu3+ ions indicated that these ions existed mostly in sites

29

deviated from the inversion symmetry in crystalline Eu2O3 aggregates and gelatinous

30

Eu(OH)3 precipitate. The ability of phage-displayed peptides to catalyze formation of Eu2O3

31

nanoparticles provides a useful tool for a low-cost and effective synthesis of lanthanide

32

nanoparticles, which serve as attractive biomedical sensors or fluorescent labels, among their

33

other applications.

34

35

INTRODUCTION

36

There are many potential applications of Eu3+, ranging from color displays to

37

biomedical sensors1-4. Light emission from Eu3+ ions varies depending on the site symmetry

38

and is associated with 5D0 → 7FJ (J = 0 - 4) transitions5. Under basic conditions, Eu3+ ions

39

commonly form europium hydroxide, which can be converted to europium oxide by high-

40

temperature annealing. As a matter of fact, Eu2O3 is considered to be one of the best oxide

41

phosphors, thus the interest in this material is undoubtedly justified. Interestingly, Eu2O3

42

nanostructures have a higher packing density and a larger percentage of active sites than the

43

bulk material6.

44

Nanocrystalline Eu2O3 can be synthesized in a number of processes, including sol-gel

45

techniques7, laser evaporation8, gas-phase condensation9, colloidal chemical method10, and

46

sonochemical techniques11. Ultrathin nanodisks as well as one-dimensional (1D) Eu2O3 2 ACS Paragon Plus Environment

Page 3 of 26

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


47

nanostructures, including nanotubes, nanorods and nanowires, have particularly interesting

48

optical properties12. While Eu2O3 nanorods were synthesized by thermal conversion (700°C)

49

of amorphous Eu(OH)3 nanorods prepared by ultrasonication13, Eu2O3 nanotubes and

50

nanorods were prepared by a hydrothermal method14, 15. More recently, Eu2O3 nanorods have

51

also been synthesized by a precipitation method16 and by a sol-gel template approach17.

52

Similarly, Eu2O3 spindles of different aspect ratio were prepared via a polyethylene glycol-

53

assisted reflux method18 and later by a room temperature (RT) precipitation method19.

54

Aside from the rare metals 1D-type structures, the hollow sphere nanostructures have

55

also attracted much attention and their potential applications in therapy, energy storage and

56

catalysis were proposed. For example, Li et al.20 obtained mesoporous Eu2O3 microspheres by

57

calcining an europium precursor synthesized hydrothermally. A similar two-step methodology

58

leading to europium oxide spheres was later reported21. In the first step, sphere precursors

59

were prepared in the hydrothermal reaction of EuCl3·6H2O and 6-aminonicotinic acid. Then,

60

a heat treatment of precursors under inert atmosphere yielded the final material, which

61

exhibited a spherical morphology and a well-defined mesoporous structure. Similarly,

62

europium oxide hollow spheres were synthesized in the improved sol-gel method using

63

polymeric microspheres as templates.

64

Besides the physicochemical approaches to the production of europium oxide

65

nanoparticles (NP) or Eu2O3 NPs-based materials, biological techniques such as phage display

66

could potentially be useful, however, such a possibility has not been explored for lanthanide

67

ions so far. Phage display, developed in 1985 by George P. Smith22, uses bacteriophages

68

(viruses that infect bacteria) as carriers to display short peptides. The most common phage in

69

this technique is M13, which is easy to manipulate genetically. A DNA sequence encoding

70

the peptide of interest may be inserted into M13 genes coding for phage coat proteins, e.g.

71

pIII or pVIII23. As a result, the protein of interest, which has usually seven or 12 amino acids, 3 ACS Paragon Plus Environment


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

72

is presented outside of the phage virion and is able to bind biological molecules (DNA

73

sequences, peptides or proteins), nanoparticles, or other materials24-27. With the use of a huge

74

phage display peptide library (i.e. a great number of phages that present various peptide

75

sequences), it is possible to identify a specific peptide capable of binding the materials or

76

molecules of interest. Although peptides with high affinity for diverse inorganic materials,

77

including metals (e.g. gold, silver or chromium), metal and semi-metal oxides (e.g. silicon,

78

germanium, titanium or zinc oxides) and metal sulfides (e.g. ZnS, CdS), were reported28, the

79

sequence of europium oxide binding peptide was not - to the best of our knowledge -

80

previously identified.

81

Page 4 of 26

In this paper, the Eu2O3 binding peptide is reported for the first time and a new method

82

for formation of Eu2O3 nanoparticles is described. We proved that the isolated peptide is able

83

to mineralize europium oxide in the form of nanoparticles from the europium salt solution

84

(europium nitrate) as well as from the Eu(OH)3 solution. It should be underlined that only

85

phages presenting the peptide sequence and the Eu2O3 precursor, and no thermal treatment,

86

were required for the synthesis of Eu2O3 quantum dots that exhibited light emission

87

properties.

88

89

RESULTS AND DISCUSSION

90

Isolation of Eu2O3-binding peptides

91

For isolation of phages that expose Eu2O3 binding peptides, we employed the

92

commercially available Phage Display Peptide Library (NEB), in which a random peptide

93

(12-mer) is expressed at the N-terminus of minor coat protein (pIII) of M13 phage. The phage

94

display library used consists of approximately 1011 various amino acid sequences (each of

95

them in approximately 100 copies) in one ml of the library and had successfully been 4 ACS Paragon Plus Environment

Page 5 of 26

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


96

employed previously in numerous applications, including epitope mapping29, and the

97

identification of: anti-microbial/anti-viral peptides30, material-specific peptides31, small

98

molecule binders26 and novel enzyme substrates32. After three rounds of biopanning

99

procedure, which was carried out with the efficiency of around 10%, 20 phages were

100

randomly chosen for sequencing. Eighteen phage clones contained a foreign peptide insertion

101

within the pIII protein. The identified peptide sequences are presented in Tab. 1. The

102

sequencing analysis revealed the same peptide sequence (SRTGNWTRIDQS) in phages: M13

103

PG-Eu-1, M13 PG-Eu-3 and M13 PG-Eu-12. A very similar sequence (SRTGIWTRTDRS)

104

was observed in phage M13 PG-Eu-4.

105

106

Table 1. Amino acid sequences of the identified Eu2O3-binding peptides exposed on pIII

107

protein of M13 phage after three rounds of a biopanning procedure. a Isoelectric points (pI)

108

were calculated using pI/molecular weight tool available at http://ca.expasy.org Clone No. M13 PG-Eu-1 M13 PG-Eu-2 M13 PG-Eu-3 M13 PG-Eu-4 M13 PG-Eu-5 M13 PG-Eu-6 M13 PG-Eu-7 M13 PG-Eu-8 M13 PG-Eu-9 M13 PG-Eu-10 M13 PG-Eu-11 M13 PG-Eu-12 M13 PG-Eu-13 M13 PG-Eu-14 M13 PG-Eu-15 M13 PG-Eu-16 M13 PG-Eu-17 M13 PG-Eu-18

Sequence S R T A R Y S R T S R T D G Q N S V K A S I N P W D P Y A G N T L S R T R L I Y R A W N G G S S H T R F A T

G F G G S H F Y W E L G D G P S Q S

N A N I D R Y A S P L N K Q K K W R

W R W W L A N Q R V N W K V L G S L

T H T T S A V H V S M T P V V S G I

R P R R P L T P M V D R D M S A S D

I T I T R G S T G P P I A N N F G T

D A D D P P L G P G S D F V G V Q L

Q M Q R P G N S N T V Q I G D T M A

S G S S H T E I T S V S T A G A F S

pIa 9.31 10.84 9.31 11.70 5.21 9.76 6.00 6.74 5.84 4.00 3.80 9.31 8.59 8.75 5.84 8.75 9.76 5.84

109

110 5 ACS Paragon Plus Environment


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

111

112

Page 6 of 26

The binding efficiency of the selected peptide to Eu2O3 The first 10 phages from Tab. 1 exposing various peptide sequences with a wide range

113

of isoelectric points (4.0 to 11.70) were chosen for further analyses. To evaluate the Eu2O3

114

binding efficiency (presented in Fig. 1), we calculated the output/input (O/I) ratio, as

115

described in the Experimental Procedures section. The highest O/I ratio was achieved in the

116

case of M13 PG-Eu-3 and M13 PG-Eu-1 phages, which expose the same peptide sequence

117

(SRTGNWTRIDQS). Binding efficiency of this peptide to Eu2O3 was around 0.1, which is

118

two or more orders of magnitude higher than the O/I ratio reported previously for binding

119

other small molecules, like titanium33 or ZnO26. This result suggests that the sequence reveals

120

a strong ability to bind Eu2O3 NPs under the tested conditions. Additionally, it is easy to see

121

that all examined sequences have a significantly higher ability to bind Eu2O3 NPs in

122

comparison to the control M13Ke phage. The identified sequences have various pI, and may

123

potentially bind Eu2O3 NPs with higher efficiency under different conditions than those used

124

in this study.

125

an attractive labeling agent to be used in immunoassays. Nanoparticles of Eu2O3 and Eu3+

126

were previously employed as fluorescent labels of atrazine, a selective triazine herbicide and

127

one of the most commonly detected pesticides in the US waters34, and of human prostate-

128

specific antigen (PSA), respectively35.

With such a substantial ability to bind Eu2O3 NPs, the identified peptides are

129 130

131

132

133

6 ACS Paragon Plus Environment

Page 7 of 26

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


134 135

Figure 1. Efficiency of Eu2O3 binding by peptides (listed in rows 1 to 10 in Tab. 1) exposed

136

on pIII protein on phage virions. The efficiency was estimated as O/I (Output/Input) ratio.

137

The patterned bars present efficiency of Eu2O3 binding by the same peptide sequence

138

(SRTGNWTRIDQS). In control experiments, the unmodified M13KE phage was used. The

139

presented results are average values from three experiments with SD represented by error

140

bars.

141

142

The high efficiency and the specificity of binding Eu2O3 nanoparticles by M13 PG-Eu-

143

3 was further confirmed with the use of atomic force microscopic (AFM) imaging (Fig. 2).

144

Figure 2a reveals the large number of M13 PG-Eu-3 phages on Eu2O3 modified mica surface.

145

It is easy to see that M13 PG-Eu-3 phages are put in order such that they bind to Eu2O3 NPs

146

with one end of the virion, where the SRTGNWTRIDQS peptide is presented. In a control

147

experiment, when M13 PG-Eu-3 phages were replaced with M13KE, there were only few

148

cases of interactions between phages and the modified mica (Fig. 2b).

149



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

a

Page 8 of 26

b

150 151

Figure 2. AFM images (5 x 5 µm) of phage M13 PG-Eu-3 (a) and M13KE (CTRL) (b)

152

interacting with mica modified with Eu2O3 nanoparticles. The white spots represent Eu2O3

153

NPs, and the filamentous structures are M13 bacteriophages.

154

155

Eu2O3 nanoparticles synthesis

156

The ability of M13 PG-Eu-3 to synthesize nanostructures was explored using two

157

different Eu(III) precursors, i.e. europium nitrate solution and europium hydroxide solution.

158

First, europium nitrate was analyzed. Scanning electron microscope (SEM) images of dried

159

samples are shown in Fig. 3a-c. The nanoparticles sized below 10 nm were synthesized only

160

in the presence of M13 PG-Eu-3 and can clearly be seen in Fig. 3b, along with virions present

161

in the analyzed sample. In the case of control experiments with phage M13KE, no

162

nanoparticles were detected and phages were covered with an amorphous layer of salt (Fig

163

3c). Energy-dispersive X-ray (EDX) spectroscopic analysis indicated that the structures of

164

about 1 µm, that can be spotted in the Fig. 3a and 3c, were composed mainly of sodium and

165

chlorine, which were TBS buffer components.

166


Page 9 of 26

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


167 168

Figure 3. SEM images of Eu2O3 NPs synthesized by phages used as bio-templates. M13 PG-

169

Eu-3 phages and Eu2O3 nanoparticles are shown in panels (a) and (b), while no nanoparticles

170

could be observed in the control experiment with phage M13KE (c). Nanostructures

171

synthesized by M13 PG-Eu-3 phages and forming cauliflower-like structures are shown in

172

panel (d), and single nanoparticles located at the virion tips are visible in panel (e). The

173

structures marked as 1 and 2 in the picture (d) were analyzed by EDX spectroscopy. Panel (e)

174

shows SEM image of nanoparticles synthesized by M13 PG-Eu-3, after centrifugation. Scale

175

bars represent 1 µm.

176 9 ACS Paragon Plus Environment


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 26

177

In the next step, the ability of M13 PG-Eu-3 phages to mineralize europium oxide

178

from europium hydroxide solution was verified. Since in the control experiment described

179

above we had observed the formation of salt crystals, in this experiment phages suspended in

180

distilled water were used. Dried samples were analyzed by SEM with EDX, in order to

181

determine the NPs morphology and elemental composition. Results are shown in Fig. 3d and

182

3e and listed in Table 2. In the synthesis with M13 PG-Eu-3 phages as biotemplates,

183

spectacular cauliflower-like nanostructures were formed. Further magnification of the

184

structures (see inset in Fig. 3d) revealed, that they are composed of very small (less than 5

185

nm) nanoparticles. Phages can be seen as bundles of fibrous structures. Along with the

186

cauliflower-like structures, the formation of single, small nanoparticles, placed near virion

187

tips, was detected (Fig. 3e). This diversity may be explained by inhomogeneous phage

188

dispersion and by the differences in precursor diffusion in the reaction mixture, leading to the

189

different distribution of NPs in the analyzed sample. Not surprisingly, NPs were located near

190

phage tips, as the Eu2O3-binding peptide is exposed on pIII protein of M13 placed on one of

191

the virion ends. The elemental composition of the sample with atomic and weight percentage

192

is listed in Table 2. The point EDX analysis confirmed the presence of europium in the

193

cauliflower-like structure (spot 1 in Fig. 3d) and the absence of europium on the phage capsid

194

surface (spot 2 in Fig. 3d), giving an indirect evidence of Eu2O3 mineralizing ability of M13

195

PG-Eu-3.

196

197

198

199


Page 11 of 26

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


200

Table 2. Elemental composition of the Eu2O3 nanomaterial obtained. Analysis was carried out

201

for the spots 1 and 2 depicted in Fig. 3d.

202 Element

CK

Na K

Cl K

KK

Eu L

Weight [%]

203

204

OK

Spot 1

29.95

29.79

28.97

6.54

0.72

4.03

Spot 2

57.06

23.38

14.6

3.55

1.4

–

Atomic [%] Spot 1

42.65

31.86

21.56

3.15

0.32

0.45

Spot 2

68.03

20.93

9.1

1.43

0.51

–

205

206

207

Since in the SEM images (Fig. 3d and 3e) amorphous solid layers were visible, the

208

sample was centrifuged (12 600 g, 10 min.) and the collected precipitate was washed with

209

deionized water. The dried precipitate was again analyzed by SEM with EDX. As shown in

210

Fig. 3f, the sample was composed of nanometric particles. The weight and atomic percentages

211

of europium in the sample were 35.48 wt% and 5.72 at%, respectively (see Table 3). The

212

increased concentration of europium in the analyzed sample as compared to the previous

213

results (see Table 2), confirmed that the soluble impurities were removed during

214

centrifugation, and that the sample was composed mainly of europium oxide nanoparticles.

215

The sample consisted of: Eu(OH)3 as a substrate (taking no part in the conversion process),

216

Eu2O3 as a product of this process, and M13 phages. However, Eu oxide, which is insoluble in

217

water but is able to absorb water, could form an additional amount of Eu(OH)336.

218

219

220



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 26

221

Table 3. Elemental composition of centrifuged nanoparticles synthesized by M13 PG-Eu-3,

222

analyzed in Fig. 3f.

223

224

Element

OK

Na K

Cl K

Eu L

Weight [%]

23.51

5.84

16.10

54.55

Atomic [%]

57.16

10.37

18.8

14.39

225

226

Additionally, the synthesis of Eu2O3 nanoparticles in the presence of chemically

227

synthesized SRTGNWTRIDQS peptide only was performed. Scanning electron microscopy

228

and transmission electron microscopy were used to study the morphology of the samples. The

229

results are depicted in Fig. 4 and 5.

230

a

b

231 232

Figure 4. SEM images of control sample (a) and Eu2O3 nanoparticles obtained in peptide-

233

assisted synthesis (b). Scale bar represents 1 µm.

234

235

236


Page 13 of 26

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


a

b

237 238

Figure 5. TEM images of control sample (a) and Eu2O3 nanoparticles obtained in peptide-

239

assisted synthesis (b). Scale bars represent 100 nm. Inset shows a single nanoparticle, with a

240

peptide shell, obtained in peptide-assisted synthesis. Scale bar represents 25 nm.

241

242

Figures 4a and 5a, indicate that in the case of the control sample an amorphous material was

243

obtained. In contrast, the peptide-assisted synthesis led to the formation of 25-30 nm Eu2O3

244

nanocrystals. In inset in Fig. 5b a single nanoparticle is shown, with a shell probably

245

composed of peptides.

246

X-ray powder diffraction spectroscopy (XRD) was used to verify the crystallinity of

247

the samples. The XRD pattern for nanoparticles synthesized in the presence of the selected

248

peptide is shown in Fig. 6B. Reflexes can be seen at 28.3°, 32.8°, 47.1° and 55.8°, and they

249

were indexed to (2 2 2), (4 0 0), (4 4 0) and (6 2 2), respectively. It indicates that body-

250

centered cubic Eu2O3 crystals were produced (JCPDS No. 34-0392). Particle size estimated

251

from the diffraction spectrum by using half-maximum widths was in the range of 26−30 nm,

252

which is in agreement with estimations based on SEM and TEM images. The XRD spectrum



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 26

253

of the control sample revealed its amorphous character, with broad bumps instead of well-

254

defined reflexes. Similar results were published by others 19, 37, 38.

255

256 257

Figure 6. XRD pattern of control sample (A) and Eu2O3 nanoparticles obtained in peptide-

258

assisted synthesis (B).

259

260

Luminescence spectra

261

Excitation and emission spectra of the Eu2O3 nanoparticles synthesized by M13 PG-

262

Eu-3 are displayed in Fig. 7a and 7b, respectively. The excitation spectrum (a) consists of four

263

distinct, sharp bands at 375, 394, 452 and 561 nm, corresponding to electronic transitions of

264

Eu3+ from 7F0 to 5L1, 5L6, 5D3 and 5D0, respectively. There is also a weak band at 465 nm

265

ascribed to 7F0 → 5D2 transition39. The emission spectrum (b) contains emission bands peaked

266

at 432, 467 and 536 nm, which are attributed to 5D3 → 7F2, 5D2 → 7F0 and 5D1 → 7F2,

267

respectively. The following bands are present above 550 nm: a very weak band related to 5D0

268

→ 7F0 at 579 nm and then more distinct bands at 593 (5D0 → 7F1), 615 (5D0 → 7F2), 646 (5D0

269

→ 7F3) and 698 nm (5D0 → 7F4). 14 ACS Paragon Plus Environment

Page 15 of 26

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


270 271

Figure 7. Excitation and emission spectra. Excitation spectrum of Eu2O3 synthesized in the

272

presence of M13 PG-Eu-3 (λem = 595 nm) (panel a), and emission spectrum of M13 PG-Eu-

273

3/Eu2O3 nanoparticles (λexc = 395 nm) (panel b).

274

275

Since Eu3+ ion can serve as a symmetry indicator of its nearest environment, the point

276

of interest is the intensity ratio of the lines at 593 and 615 nm. If in the emission spectrum the

277

5

278

possesses inversion symmetry. In the opposite case (no inversion symmetry), the line

279

domination is vice versa39. As observed in Fig. 7b, the intensity of the 5D0 → 7F2 band

280

distinctly dominates over the 5D0 → 7F1 one. Thus, concentration of the Eu3+ sites deviated

281

from the inversion symmetry was much higher than concentration of the Eu3+ occupied sites

282

of this symmetry. This effect indicates that the majority of the Eu3+ ions existed in Eu2O3 and

283

- especially in Eu(OH)3 - in sites of lower symmetry.

284

D0 → 7F1 line dominates over the 5D0 → 7F2 one, it means that the site of the europium ions

In conclusion, this study has for the first time reported peptides specifically binding

285

Eu2O3 nanoparticles. Moreover, we have for the first time demonstrated that bacteriophages

286

exposing selected peptides can efficiently synthesize Eu2O3 nanoparticles. The synthesis was

287

dependent solely on the presence of 1) the phage and 2) Eu(III) precursors. It should be noted

288

that -in contrast to other known methods for Eu2O3 NPs synthesis - no thermal treatment of



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 26

289

samples was required. As a result of phage-assisted synthesis, uniform Eu2O3 quantum dots

290

were obtained. The light emission of the Eu2O3 NPs obtained was demonstrated. While we

291

observed Eu3+ emission, in our experimental procedure it was not very high, which could be

292

explained by two different phenomena. Firstly, in Eu(OH)3 and Eu2O3 formed in aqueous

293

surrounding, Eu3+ ions coordinated with H2O ligands possessing O-H oscillators that

294

effectively quench emission of the central ion, were present40. Secondly, since concentration

295

of Eu was as high as 54 wt%, the concentration quenching effect was observed.

296

297

EXPERIMENTAL PROCEDURES

298

Materials

299

Phage clones isolated from Ph.D.™-12 Phage Display Peptide Library (New England

300

Biolabs, NEB) and the control phage M13KE with the wild-type pIII protein (NEB) were

301

used. Escherichia coli ER2738 (NEB) was used in all experiments. Materials were from

302

Sigma-Aldrich unless otherwise noted.

303

304

Bacteriophage propagation, concentration and purification

305

Propagation of phages was carried out in E. coli ER2738 in Luria-Bertani (LB) medium.

306

Bacteriophages were added to the bacterial culture in an early exponential growth phase and

307

propagated for 5 h at 37°C with vigorous shaking. Next, bacteriophages were concentrated

308

and purified with the use of polyethylene glycol 8000 according to the M13 Amplification

309

protocol (NEB). Then, depending on the type of further analyses, phages were suspended in

310

distilled water or TBS buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl). Titration of phages

311

was carried out on double-agar plates containing Xgal/IPTG26. 16 ACS Paragon Plus Environment

Page 17 of 26

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


312

Biopanning procedures

313

For isolation and identification of peptide sequences that specifically bind Eu2O3, a triple

314

biopanning procedure was used. Ten milligrams of 99.999% Eu2O3 trace metals basis was

315

incubated in TBST buffer (TBS and 0.1 % Tween-20) for 1 h at RT and next washed six times

316

with TBST buffer. Ten microliters of Phage Display Peptide Library was added to Eu2O3 in 1

317

ml TBST, incubated for 30 min at RT and next washed ten times with TBST. After the

318

washing procedure, Eu2O3-bound phages were eluted with a buffer containing 0.2 M Glycine-

319

HCl pH 2.2 and 1 mg/ml BSA and incubated with shaking for 10 min at RT. The eluted

320

phages were then centrifuged (5 min, 4000 g, RT). The supernatant containing the eluted

321

phages was immediately used in propagation and/or titration procedures. After the third

322

biopanning procedure, eluted phages were titrated on double-agar plates containing

323

Xgal/IPTG. Phages from single plaques were used in propagation procedures, as described

324

above. SsDNA was isolated from the propagated phages (according to a method described by

325

Wilson41) and sequenced with the use of 96 gIII sequencing primer (NEB).

326

327

Analysis of efficiency of Eu2O3 binding

328

Efficiency assay was developed on the basis of a previous report33. Specific concentration of

329

phages (108 - 109 plaque forming unit (pfu)/ml) propagated after three rounds of biopanning

330

procedure, was added to 10 mg Eu2O3 (prepared as described in a biopanning procedure

331

above). These phages are designated in this work as input phages (I). Eluted phages (obtained

332

in the procedure described above) were titrated on double-agar plates containing Xgal/IPTG

333

and designated as output phages (O). Binding efficiency was expressed as the ratio of the

334

output/input (O/I) phages. As a control, we employed M13KE phage with the wild-type pIII

335

protein. 17 ACS Paragon Plus Environment


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 26

336

Atomic force microscopic (AFM) analysis

337

Atomic force microscopic (AFM) images were obtained with a MultiMode AFM instrument

338

using a Nanoscope V controller (Bruker) operating in the tapping mode using an uncoated

339

RFESP tip (Bruker). Cantilever’s spring constant was 3 N/m. The scanning area was 5×5 µm.

340

Raw AFM data were processed with Gwyddion 2.32 SPM data analysis and visualization tool.

341

First, mica was cleaved in order to obtain a clean surface. Next, 10 mg of Eu2O3 was

342

dispersed in 0.5 ml of water by sonication. Ten microliters of the resulting Eu2O3 dispersion

343

was deposited on mica. The sample was left under ambient conditions to let the water

344

evaporate. A five-microliter droplet of M13 PG-Eu-3 or M13KE suspensions (with a

345

concentration of 1010 - 1011 pfu/ml) in TBS buffer was deposited on Eu2O3-modified mica and

346

left under cover at RT for 30 minutes to let the phages interact with Eu2O3 particles. The

347

cover was essential to prevent water evaporation. After incubation, the sample was rinsed

348

with water to remove the non-specifically bound phages and buffer residues, and dried in a

349

stream of argon. After drying, the sample was ready for imaging.

350

351

Nanoparticle synthesis

352

For Eu2O3 NP synthesis, we used both the phages presenting peptides on pIII protein and the

353

selected peptides only. In the synthesis with the use of phages, 4 µl of 10-3 M europium nitrate

354

solution was added to 30 ml of M13 PG-Eu-3 and M13KE phage suspensions (1011 pfu/ml).

355

The reaction mixtures were left overnight; after that time, the samples were subjected to

356

analyses. Simultaneously, 40 µl of 5·10-4 M Eu(OH)3 solution was added to 30 ml of M13

357

PG-Eu-3 and 30 ml of M13KE phage suspensions, and left overnight. The 5·10-4 M Eu(OH)3

358

solution was prepared by mixing the same volumes of 10-3 M Eu(NO3)3 solution and 3·10-3 M

359

NaOH solution. In the synthesis with the use of the selected peptide only, 20 mg of peptide


Page 19 of 26

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


360

(synthesized chemically by a commercial service) was added to the mixture of Eu(NO3)3 (5

361

mL, 0.2 mM) and NaOH (5 mL, 1.6 mM) under magnetic stirring. In the control experiment,

362

the same amounts of Eu(NO3)3 and NaOH solutions were mixed, but with no peptide added.

363

The mixtures were stirred overnight at RT. After that time, the precipitates were collected by

364

centrifugation, and washed several times with deionized water. The precipitate was dried

365

overnight in a vacuum dryer (40°C, 0.01 bar).

366 367

Transmission electron microscopy analysis

368

For TEM analysis, Eu2O3 NPs synthesized by the selected SRTGNWTRIDQS peptide were

369

placed on grids coated with a 2% collodion solution and carbon. Eu2O3 NPs were negatively

370

stained with 2% uranyl acetate or were examined without staining using Philips CM100

371

electron microscope at 80kV.

372 373

Scanning electron microscope (SEM) analysis

374

For SEM analysis (FEI Quanta FEG 250), aqueous solutions of the samples were dropped

375

onto a carbon conducting support and left overnight to dry at RT. If necessary, samples were

376

covered with thin films of gold, using a high vacuum sputter coater (Leica EM SCD 500).

377 378

X-ray Diffraction analysis

379

X-ray diffraction patterns (XRD) were recorded on a X-ray diffractometer (Xpert PRO MPD,

380

Philips) with Cu target Kα-ray (λ=0.15404 nm) irradiation. Scans were taken in the 2θ range

381

of 10-80°.

382



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 26

383

Energy-dispersive X-ray (EDX) spectroscopy

384

For chemical characterization of the samples, EDX spectroscopy was performed using the

385

EDAX Genesis APEX 2i with Apollo X SDD spectrometer at 10 kV.

386

387

Luminescence spectroscopy

388

Front-face emission and excitation spectra were measured at RT using a HORIBA Jobin Yvon

389

Fluoromax-4 spectrofluorometer with a reflection spectra attachment. None of the excitation

390

spectra was corrected for the lamp and photomultiplier response.

391

392

ACKNOWLEDGEMENTS

393

We would like to thank Dr. Magdalena Narajczyk for her help with TEM. This work was

394

supported by National Science Center (Poland) within the project grant no. N N302 181439

395

(to G.W.).

396

397

CONFLICT OF INTEREST

398

There are no conflicts of interest to declare.

399

400

REFERENCES

401

(1) Sayyadi, N., Justiniano, I., Connally, R. E., Zhang, R., Shi, B. Y., Kautto, L., Everest-

402

Dass, A. V., Yuan, J. L., Walsh, B. J., Jin, D. Y., et al. (2016) Sensitive Time-Gated


Page 21 of 26

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


403

Immunoluminescence Detection of Prostate Cancer Cells Using a TEGylated Europium

404

Ligand. Anal Chem 88, 9564-9571.

405

(2) Skaudzius, R., Enseling, D., Skapas, M., Selskis, A., Pomjakushina, E., Justel, T., Kareiva,

406

A., and Ruegg, C. (2016) Europium-enabled luminescent single crystal and bulk YAG and

407

YGG for optical imaging. Opt Mater 60, 467-473.

408

(3) Wu, T., Kessler, J., and Bour, P. (2016) Chiral sensing of amino acids and proteins

409

chelating with Eu-III complexes by Raman optical activity spectroscopy. Phys Chem Chem

410

Phys 18, 23803-23811.

411

(4) Sy, M., Nonat, A., Hildebrandt, N., and Charbonniere, L. J. (2016) Lanthanide-based

412

luminescence biolabelling. Chem Commun 52, 5080-5095.

413

(5) Choi, Y. I., Yoon, Y., Kang, J. G., and Sohn, Y. (2015) Photoluminescence imaging of

414

Eu(III) and Tb(III)-embedded SiO2 nanostructures. J Lumin 158, 27-31.

415

(6) Bazzi, R., Flores, M. A., Louis, C., Lebbou, K., Zhang, W., Dujardin, C., Roux, S.,

416

Mercier, B., Ledoux, G., Bernstein, E., et al. (2004) Synthesis and properties of europium-

417

based phosphors on the manometer scale: Eu2O3, Gd2O3 : Eu, and Y2O3 : Eu. J Colloid

418

Interf Sci 273, 191-197.

419

(7) Wu, G. S., Zhang, L. D., Cheng, B. C., Xie, T., and Yuan, X. Y. (2004) Synthesis of

420

Eu2O3 nanotube arrays through a facile sol-gel template approach. J Am Chem Soc 126,

421

5976-5977.

422

(8) Eilers, H., and Tissue, B. M. (1996) Laser spectroscopy of nanocrystalline Eu2O3 and

423

Eu3+:Y2O3. Chem Phys Lett 251, 74-78.

424

(9) Bihari, B., Eilers, H., and Tissue, B. M. (1997) Spectra and dynamics of monoclinic

425

Eu2O3 and Eu3+:Y2O3 nanocrystals. J Lumin 75, 1-10.

426

(10) Wakefield, G., Keron, H. A., Dobson, P. J., and Hutchison, J. L. (1999) Synthesis and

427

properties of sub-50-nm europium oxide nanoparticles. J Colloid Interf Sci 215, 179-182.



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 26

428

(11) Pol, V. G., Reisfeld, R., and Gedanken, A. (2002) Sonochemical synthesis and optical

429

properties of europium oxide nanolayer coated on titania. Chem Mater 14, 3920-3924.

430

(12) Si, R., Zhang, Y. W., You, L. P., and Yan, C. H. (2005) Rare-earth oxide nanopolyhedra,

431

nanoplates, and nanodisks. Angew Chem Int Edit 44, 3256-3260.

432

(13) Pol, V. G., Palchik, O., Gedanken, A., and Felner, I. (2002) Synthesis of europium oxide

433

nanorods by ultrasound irradiation. J Phys Chem B 106, 9737-9743.

434

(14) Wang, X., and Li, Y. D. (2003) Rare-earth-compound nanowires, nanotubes, and

435

fullerene-like nanoparticles: Synthesis, characterization, and properties. Chem-Eur J 9, 5627-

436

5635.

437

(15) Wang, X., Sun, X. M., Yu, D. P., Zou, B. S., and Li, Y. D. (2003) Rare earth compound

438

nanotubes. Adv Mater 15, 1442-1445.

439

(16) Yan, T. T., Zhang, D. S., Shi, L. Y., and Li, H. R. (2009) Facile synthesis,

440

characterization, formation mechanism and photoluminescence property of Eu2O3 nanorods.

441

J Alloy Compd 487, 483-488.

442

(17) Zhang, L. X., Jiu, H. F., Luo, J., and Chen, Q. W. (2007) High-yield synthesis of single-

443

crystal short Eu2O3 nanorods through a facile sol-gel template approach. J Cryst Growth 309,

444

192-196.

445

(18) Wang, S. F., Gu, F., Li, C. Z., and Lu, M. K. (2007) Synthesis of mesoporous Eu2O3

446

microspindles. Cryst Growth Des 7, 2670-2674.

447

(19) Zhang, D. S., Yan, T. T., Shi, L. Y., Li, H. R., and Chiang, J. F. (2010) Template-free

448

synthesis, characterization, growth mechanism and photoluminescence property of Eu(OH)(3)

449

and Eu2O3 nanospindles. J Alloy Compd 506, 446-455.

450

(20) Li, Y., Ge, M., Li, J. W., Wang, J. F., and Zhang, H. J. (2011) Synthesis of mesoporous

451

Eu2O3 microspheres and Eu2O3 nanoparticle-wires as well as their optical properties.

452

Crystengcomm 13, 637-641.


Page 23 of 26

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


453

(21) Viana, R. D., Falcao, E. H. L., Dutra, J. D. L., da Costa, N. B., Freire, R. O., and Alves,

454

S. (2014) New experimental and theoretical approach in Eu2O3 microspheres: From synthesis

455

to a study of the energy transfer. J Photoch Photobio A 281, 1-7.

456

(22) Smith, G. P. (1985) Filamentous Fusion Phage - Novel Expression Vectors That Display

457

Cloned Antigens on the Virion Surface. Science 228, 1315-1317.

458

(23) Pande, J., Szewczyk, M. M., and Grover, A. K. (2010) Phage display: Concept,

459

innovations, applications and future. Biotechnol Adv 28, 849-858.

460

(24) Sidhu, S. S., Lowman, H. B., Cunningham, B. C., and Wells, J. A. (2000) Phage display

461

for selection of novel binding peptides. Applications of Chimeric Genes and Hybrid Proteins,

462

Pt C 328, 333-363.

463

(25) Wolcke, J., and Weinhold, E. (2001) A DNA-binding peptide from a phage display

464

library. Nucleos Nucleot Nucl 20, 1239-1241.

465

(26) Golec, P., Karczewska-Golec, J., Los, M., and Wegrzyn, G. (2012) Novel ZnO-binding

466

peptides obtained by the screening of a phage display peptide library. J Nanopart Res 14.

467

(27) Vetter, S. W. (2013) Phage display selection of peptides that target calcium-binding

468

proteins. Methods Mol. Biol. 963, 215-35.

469

(28) Dickerson, M. B., Sandhage, K. H., and Naik, R. R. (2008) Protein- and Peptide-Directed

470

Syntheses of Inorganic Materials. Chem Rev 108, 4935-4978.

471

(29) Pavlidou, M., Hanel, K., Mockel, L., and Willbold, D. (2013) Nanodiscs Allow Phage

472

Display Selection for Ligands to Non-Linear Epitopes on Membrane Proteins. Plos One 8.

473

(30) Dharmasena, M. N., Jewell, D. A., and Taylor, R. K. (2007) Development of peptide

474

mimics of a protective epitope of Vibrio cholerae ogawa o-antigen and investigation of the

475

structural basis of peptide mimicry. J Biol Chem 282, 33805-33816.

476

(31) Seker, U. O. S., and Demir, H. V. (2011) Material Binding Peptides for Nanotechnology.

477

Molecules 16, 1426-1451.



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 26

478

(32) Sugimura, Y., Hosono, M., Wada, F., Yoshimura, T., Maki, M., and Hitomi, K. (2006)

479

Screening for the preferred substrate sequence of transglutaminase using a phage-displayed

480

peptide library - Identification of peptide substrates for TGase 2 and Factor XIIIA. J Biol

481

Chem 281, 17699-17706.

482

(33) Sano, K. I., and Shiba, K. (2003) A hexapeptide motif that electrostatically binds to the

483

surface of titanium. J Am Chem Soc 125, 14234-14235.

484

(34) Feng, J., Shan, G. M., Maquieira, A., Koivunen, M. E., Guo, B., Hammock, B. D., and

485

Kennedy, I. M. (2003) Functionalized europium oxide nanoparticles used as a fluorescent

486

label in an immunoassay for atrazine. Anal Chem 75, 5282-5286.

487

(35) Huhtinen, P., Kivela, M., Kuronen, O., Hagren, V., Takalo, H., Tenhu, H., Lovgren, T.,

488

and Harma, H. (2005) Synthesis, characterization, and application of Eu(III), Tb(III), Sm(III),

489

and Dy(III) lanthanide chelate nanoparticle labels. Anal Chem 77, 2643-2648.

490

(36) Greenwood, N. N., and Earnshaw, A. (1997) in Chemistry of the Elements pp 1227-1249,

491

Elsevier, Oxford.

492

(37) Kang, J. G., Jung, Y., Min, B. K., and Sohn, Y. (2014) Full characterization of Eu(OH)3

493

and Eu2O3 nanorods. Appl Surf Sci 314, 158-165.

494

(38) Kattel, K., Park, J. Y., Xu, W. L., Kim, H. G., Lee, E. J., and Bony, B. A. (2012) Water-

495

soluble ultrasmall Eu2O3 nanoparticles as a fluorescent imaging agent: In vitro and in vivo

496

studies. Colloids Surf A Physicochem Eng Asp 394, 85-91.

497

(39) Blasse, G., and Grabmaier, B. C. (1994) in Luminescent Materials pp 112-130, Springer-

498

Verlag, Berlin.

499

(40) Horrocks, W. D., and Sudnick, D. R. (1979) Lanthanide Ion Probes of Structure in

500

Biology - Laser-Induced Luminescence Decay Constants Provide a Direct Measure of the

501

Number of Metal-Coordinated Water-Molecules. J Am Chem Soc 101, 334-340.


Page 25 of 26

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


502

(41) Wilson, R. K. (1993) High-Throughput Purification of M13 Templates for DNA-

503

Sequencing. Biotechniques 15, 414-&.

504 505 506 507 508 509

510

511

512

513

514

515

516

517

518

519

520

521

522



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 26

523

524

525

TOC

526

Binding to Eu2O3 NPs

Binding to Eu2O3 NPs

Synthesis of Eu2O3 NPs

Synthesis of Eu2O3 NPs


Nanoparticle Synthesis - American Chemical Society

Recommend Documents