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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

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Bioconjugate Chemistry

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Bacteriophages as factories for Eu2O3 nanoparticle synthesis

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Running title: Eu2O3 NPs synthesis by modified M13 phage

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

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

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a

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

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b

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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

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c

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

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d

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

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Gdansk, Poland

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e

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Poland

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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

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

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

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

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[email protected]

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

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ABSTRACT

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

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

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

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

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

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deviated from the inversion symmetry in crystalline Eu2O3 aggregates and gelatinous

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

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

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

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other applications.

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INTRODUCTION

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

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

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

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

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

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

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

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bulk material6.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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polymeric microspheres as templates.

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Besides the physicochemical approaches to the production of europium oxide

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

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

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

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

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

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

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


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

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

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

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

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

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

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

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

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previously identified.

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

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

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

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

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

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

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properties.

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RESULTS AND DISCUSSION

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Isolation of Eu2O3-binding peptides

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

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

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

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

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

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

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

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

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

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

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

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

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

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was observed in phage M13 PG-Eu-4.

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

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

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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

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

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

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

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

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

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

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

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

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

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

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

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

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in this study.

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

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

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

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specific antigen (PSA), respectively35.

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

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

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

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

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

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

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bars.

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

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

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

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

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

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

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

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a

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b

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

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

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NPs, and the filamentous structures are M13 bacteriophages.

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Eu2O3 nanoparticles synthesis

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

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

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

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

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

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

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

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

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

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chlorine, which were TBS buffer components.

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

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

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

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

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

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

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

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bars represent 1 µm.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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PG-Eu-3.

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

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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

–

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

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

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

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

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

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

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

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

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

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

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

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

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analyzed in Fig. 3f.

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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

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

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synthesized SRTGNWTRIDQS peptide only was performed. Scanning electron microscopy

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

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results are depicted in Fig. 4 and 5.

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a

b

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

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

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b

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

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

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

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

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

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

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composed of peptides.

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

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

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

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

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

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

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



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

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

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

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assisted synthesis (B).

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Luminescence spectra

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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5

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

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

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

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

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

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

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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

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

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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



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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

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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


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Page 18 of 26

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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


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(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



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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

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TOC

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Binding to Eu2O3 NPs

Binding to Eu2O3 NPs

Synthesis of Eu2O3 NPs

Synthesis of Eu2O3 NPs


Bacteriophages as Factories for Eu2O3 ... - ACS Publications

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