Biomedical Engineering Institute...their toxicological potential as reflected by ROS generation in...

L’Hocine YAHIA

Laboratoire d'innovation et d'analyse de bioperformances

1

New Developments

in Sterilization and Decontamination

using Nanotechnologies

Biomedical Engineering Institute

2014 World Sterilization Congress of the WFHSS

INFECTIONS RELATED TO IMPLANTS

Introduction

Different sources of the infectious agent, such as:

A contaminated implant/device surface

The hands of the surgical staff during implantation/application

The patient’s own skin or mucus membrane

Distant local infections in the patient

Contaminated disinfectants Contact with other patients in the hospital, or family members after

intervention

http://www.google.ca/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=WZ77S3RnJHDzwM&tbnid=pKHnRypaatoCZM:&ved=0CAUQjRw&url=http://www.tigran.se/en/professional/application-areas/implantology/peri-implantitis/debridement-at-peri-implantitis/&ei=c0wvUuL6JefX2QWikYGACg&bvm=bv.51773540,d.aWc&psig=AFQjCNFz_65nKzsRmDG1TSclk7tWFMsf5Q&ust=1378917807351674http://www.google.ca/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=Hl61oQjFkx4xJM&tbnid=22NOtIvCe3dkYM:&ved=0CAUQjRw&url=http://www.hpc.org.ar/v2/v_art_rev.asp?id=15&offset=21&ei=4k4vUuXmDabZ2wXdhYHICA&bvm=bv.51773540,d.aWc&psig=AFQjCNEX0M689wdQKbL2NuiWZWHSgwRcnw&ust=1378918350288263http://www.google.ca/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=VBgn44yF8_4NgM&tbnid=cO5QEoc_3Tl7-M:&ved=0CAUQjRw&url=http://www.boloncol.com/boletin-24/cuidados-de-la-piel-en-el-paciente-oncologico.html&ei=CVEvUqLcDcTr2QXn54GYBg&bvm=bv.51773540,d.aWc&psig=AFQjCNEX0M689wdQKbL2NuiWZWHSgwRcnw&ust=1378918350288263http://www.google.ca/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=vzotV1gbnvNVhM&tbnid=kRojBe1wMCVuSM:&ved=0CAUQjRw&url=http://sistemainterno.com/web/comvalsa/category/sklar-instrumental-quirurgico/&ei=w1IvUsSCG-bQ2wWa24GgDQ&bvm=bv.51773540,d.aWc&psig=AFQjCNE1d0f07bbMBBiesCdITfhffSTv_A&ust=1378919447681366http://www.google.ca/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=I_B2VPttWwv0wM&tbnid=7ZSY8qfXkA1DvM:&ved=0CAUQjRw&url=http://www.farmalta.com/blog/?paged=2&ei=PlMvUqjFAer12wWx-YGADg&bvm=bv.51773540,d.aWc&psig=AFQjCNE1d0f07bbMBBiesCdITfhffSTv_A&ust=1378919447681366http://www.google.ca/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=hLJIrQ878gUwIM&tbnid=847Qtuo5TCCEIM:&ved=0CAUQjRw&url=http://www.rpp.com.pe/2013-02-04-inician-visita-de-hospitales-para-verificar-buen-servicio-a-pacientes-noticia_564066.html&ei=MlUvUpiyO8m52wXPyoCQBA&bvm=bv.51773540,d.aWc&psig=AFQjCNF6OOR5y800dButqHl2DVkkFYWqCg&ust=1378919776783026

PRINCIPAL BACTERIA ON IMPLANTS Introduction

Infections caused by: • Staphylococcus epidermidis • Staphylococcus aureus

70–90% of the implant related infections

IMPLANT BACTERIA

Catheter-related infections Staphylococcus aureus and Staphylococcus epidermidis

Orthopedic implants Staphylococcu aureus

E. coli , Enterobacteriaciae, P. aeruginosa

Lenses S. epidermidis

Skin grafts S. aureus and S. epidermitis

Cardiac valve S. epidermidis , S. aereus, E. faecelis, P. aeruginosa, and Candida albicans

Table 1. Inscidence of bacteria present after different surgery implants

BIOFILM FORMATION

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Introduction

Fig. 2 Schematic representation of biofilm formation.

Lerberg


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Nanomaterials

Copper et Copper Oxide NPs

Antibacterial material

Iron Oxide NPs (SPIONs)

Biocompatible

Hyperthermia

Can be guided with a magnetic field to desired target

NO-coupled Iron Oxide NPs

NO: molecule used by immune system

Known to be antibacterial

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T. Theivasanthi and M. Alagar, 2010

Zone of Inhibition Test for Antimicrobial

Activity, also called a Kirby-Bauer Test.

It is used clinically to measure antibiotic

resistance and industrially to test the

ability of solids and textiles to inhibit

microbial growth.

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Suresh et al 2013

Copper as a novel biocide

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Synthesis of copper nanoparticles

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There are three main routes—chemical, physical, and biological—being used

for the synthesis of metallic nanoparticles.

These methods for the production of copper nanoparticles are appropriate

for laboratory-scale synthesis but are not economical for a large-scale or

commercial setup.

The induction plasma system (Tekna) has been successfully used in the

synthesis and preparation of advanced materials such as new ceramics,

nanometric metallic powders, biomaterials powders (by HF-75, Tekna).

Making nanopowders by induction plasma (Tekna)

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ICP stands for “inductively coupled plasma”. Also known as induction

plasma, H.F (high frequency) or R.F. (radio frequency) plasma.

Nanopowders synthesis by ICP: Pure metals

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Copper and Copper Oxide NPs

• Average size of CuO NPs is 36.5 ± 16.6 nm

• Average size of Cu NPs is 84.8 ± 24.6 nm for the

core and 17.1± 5.0 nm for the shell 2014 World Sterilization Congress of the WFHSS 19


Minimal Inhibitory Concentration (MIC) for

◦ Cu, S.aureus: 0.325mg/mL

◦ Cu, E.coli: 0.750mg/mL

◦ CuO, both bacterial strains: 0.325mg/mL

At each MIC

◦ suspension was plated and incubated for 24h to determine if NPs are bacteriostatic or bactericidal

◦ Bacteriostatic for both NPs

2014 World Sterilization Congress of

the WFHSS 20



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Copper Microparticles (MPs)

MPs average size is 21.31 ± 7.75 m

Smallest microparticle is 6.205 m

Biggest microparticle is 39.7 m.


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Copper Microparticles (MPs)

MIC for S.aureus of MPs is 3mg/mL

◦ Bacteriostatic

◦ At 3mg/mL, suspension was plated and incubated for 24h growth

MIC for E.coli of MPs is 6mg/mL

◦ Bacteriocidal

◦ At 6mg/mL, suspension was plated and incubated for 24h no growth


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Copper Microparticles

S.aureus E.coli


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Discussion

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Cell wall thicker for Gram-positive (many layers of peptidoglycan)

Size effects

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The decline in the biotoxicity level of the

copper compounds in a series:

ions → nanoparticles → microparticles

was observed in good agreement with the

results of animal studies.

Oxidation & Corona

Because Cu is less chemically stable than Cu(II) and

Cu(I) oxides; thus, a major problem is the usual

occurrence of surface oxidation during its synthesis. A

mixture of metallic Cu, cuprous oxide (Cu2O) or cupric

oxide (CuO) generally accompanies the production of

Cu nanopowders.

Uncontaminated and surfactant-free Metal NPs are

difficult to obtain. The bacteria will «see» corona

covering the copper NPs. In fact, the MEM/EBSS or

RPMI-1640 culture media contains inorganic salts,

amino acids, vitamins and other components wich could

«contaminate» these «ultra-pure» NPs.

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Effects of Surface Chemistry on the Generation of Reactive Oxygen

Species by Copper Nanoparticles (Miao Shi et al. 2012, ACS Nano, Vol. 6, No. 3, 2157–2164)

It is essential to develop approaches to control surface chemistry to

better understand the origins of nanoparticle-induced toxicity, as subtle

differences in surface properties can dramatically change biological

responses.

Shi et al studied the ROS generating capacity of uniform copper

nanoparticles with different capping ligands to better understand the

relationship between nanoparticle physicochemical properties and

their toxicological potential as reflected by ROS generation in an

acellular assay. Three mercaptocarboxylic acids with different carbon

chain lengths were used for surface modification as they have been

shown to provide good colloidal stability for various nanoparticles.


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As-synthesized

copper NPs

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TEM micrographs of these copper NPs after surface modification with 8-mercaptooctanoic acid (MOA), 12-

mercaptododec- anoic acid (MDA), and 16-mercaptohexadecanoic acid (MHA).

The ROS generating capacities of these three copper nanoparticle types were measured.

All particles demonstrated dose-dependent increases in ROS activity.

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The ROS generating capacities of the oxidized copper samples. The oxidized copper NPs had

lower equivalent [H2O2] than the corresponding as-made ones.

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IRON OXIDE NPs & MAGNETOSOMES Rational

* SPIONs 1) Positive amine groups 2) Negative carboxylic groups 3) Bare

* MAGNETOSOMES (Natural nanocrystals)

Contrast agents Attachment of NO

NANOSCALE CHARACTERIZATION

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Methodology

Time-of- Flight Secondary Ion Mass Spectrometry (TOF-SIMS)

Transmission Electron Microscope (TEM)

Fourier Transform Infrared Spectroscopy (FT-TIR)

Vibrating Sample Magnetometer (VSM)

X-ray Photoelectron Spectroscopy (XPS)

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TEM SPIONS &

Magnetosomes

TEM

Sample Shape Size

Bare Spherical 9.1 ± 1.4 nm

Negative Spherical 9.7 ± 1.7 nm

Positive Spherical 10.1 ± 1.3 nm

Magneto-

somes

Hexagonal,

square and

spherical

73.7 ± 14.6 nm

length: 0.4-0.8 µm

Figure 5. TEM images of SPIONs a) bare, b) positive, c) negative and d) magnetosomes

Table 2. Shape and Size of SPIONs and magnetosones found by TEM characterization

SIZE DISTRIBUTION

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Figure 6. Size distribution histograms of SPIONs NPs a) bare, b) positive, c) negative and d) magnetosomes

VMS

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Magnetization

VMS

Sample Mmax

(emu/g) Behavior

Histeresi

s

Positive 28 Superparamag-

netic 0

Negative 24 Superparamag-

netic 0

Bare 27 Superparamag-

netic 0

Magneto-

somes 14.7 Ferrimagnetic 472 Oe

Figure 7. Magnetization curves of SPIONs and magnetosomes

Table 3. Magnetic properties od SPIONs and magnetosones found by VMS characterization

SURVEY MAGNETOSOMES (XPS)

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XPS

Fe peak ~ 700 eV

Fig 8. Magnetosome survey spectrum. The inset shows the almost complete absense of Fe2p at 710eV

Cytotoxicity : Effect of nanoparticle time of exposition

and dose on cell morphology

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Effect of nanoparticle functionalization on cell viability

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Effect of culture medium on cell viability

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Iron Oxide Nanoparticles

S.aureus E.coli


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Iron Oxide Nanoparticles

E.coli:

◦ No change is notable between control and different concentrations of SPIONs at all time (T1, T3, T24). No effect on

E.coli growth

S.aureus

◦ After 1h, all concentration, decrease bacteria viability is insignificant

• After 3 h, for all concentration, bacteria have a decrement of their

growth arount one log

• After 24h, at smallest concentration (2.5, 1.25) not a real change

between T3 and T24

• 5mg/mL appromatively a decrease of 2log

• 10mg/mL, decrease of 3log


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Nitric oxide

Nitrogen oxide can refer to a binary compound of oxygen and

nitrogen, a mixture of such compounds:

Nitric oxide, also known as nitrogen monoxide, (NO),

nitrogen(II) oxide

Nitrogen dioxide (NO2), nitrogen(IV) oxide

Nitrous oxide (N2O), nitrogen(-I,III) oxide

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IMPORTANCE OF USE NITRIC OXIDE Rational

NITRIC OXIDE

Endogenous

molecule

Vascular tone

Wound healing

Immune

response

Host defense

against infection

NO-releasing

Biomaterials

Inhibiting in vivo

infection

Facilitating tissue

integration of the

implants

Reducing bacterial and

platelet adhesion in vitro

Antimicrobial

properties

G+ G-

Biofilms

P.

aeruginosa

S. aureus S.

epidermidis

E. coli

Klebsiella St.

pneumoniae

DESIGN OF MNPs COATING WITH NO

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Functionalization

Figure 3. Schematic representation of the formation of thiolated Fe3O4 NPs coated with MSA and DMSA

mercaptosuccinic acid (MSA)

dimercaptosuccinic acid (DMSA).

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DESIGN OF MNPs COATING WITH NO

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NO delivery

Positive SPIONs Negative SPIONs

Figure 4. Schematic representation of iron oxide NPs functionalized with silane layers containing amine groups (positive SPIONs) and carboxilic acid groups (negative SPIONs)

Schematic of two different methods to synthesize diazeniumdiolate-

modified silica nanoparticles. Tetraethoxysilane (TEOS) and N-(6-

aminohexyl)aminopropyltrimethoxysilane (AHAP3) are representative

examples of tetraalkoxy- and aminoalkoxysilanes, respectively. (A)

Postformation method. (B) Pre-formation method (reproduced from ref.

43 with permission from the American Chemical Society).

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Zhang & Yahia

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Time-kill curves for the Staphylococcus aureus arrows, application of a second dose at the same concentration

Plate counting of A. baumannii cells on the wounded skin of mice after 24 h application of SG-80A based NO releasing and control patches. NO releasing and control patches were applied to wounds 24 h after inoculation with A. baumannii. After 24 h, skin tissue was harvested, homogenized, serially diluted and grown on agar plates. The data are means ± SEM (n = 3). ⁄p <

0.05.

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Fredman et al

Schoenfisch et al. Acta Biomaterialia 10 (2014) 3442–3448

NO SH NO /SH

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Concluding Remarks

Various studies have revealed that copper NPs can be

synthesized by chemical, physical, and biological routes.

The chemical methods are time-consuming and tedious.

Moreover, some chemical methods include use of

hazardous chemicals, which may exert adverse effects to

the user.

The induction plasma used for nanopowder synthesis has

many advantages over alternative techniques such as high

purity, ease of scale-up and ease of operation and control.

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Moreover, it was shown that the toxicity of nanosized oxides

(nano CuO) was much high than their bulk counterparts.

Therefore, development of an efficient approach for

preventing the oxidation of Cu nanopowders during synthesis

in an aqueous solution is required urgently.

Inaccuracies of NO measurement methods in biological

media (amperometer NO sensor, chemiluminescence

analyzer --- Significant variations between techniques).

Further investigation on the stability of the stability of the

NO-donors became a significant problem in the particle-

based systems because of the possibility of the release of NO

before reaching the target site.

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Due to the growing tolerance of bacteria to antibiotics, metal and metal oxide nanoparticles with antibacterial properties

represent a promising alternative approach to antibiotics. But

Al2O3 NPs was found to induce bacterial resistance !!!)

Nitric oxide-releasing nanoparticles (NO NPs) and metal-

containing nanoparticles all use multiple mechanisms simul-

taneously to combat microbes, thereby making development of

resistance to these nanoparticles unlikely.

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62 Dr. Yahye Merhi

Dr. Karim Maghni Dr. Theodre Veres

http://www.icm-mhi.org/fr/index.html


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Questions

Félix d'Hérelle

• Mais, en 1917, on le retrouve à l’Institut Pasteur, à Paris, où il fait une

découverte majeure : le bactériophage, un ultravirus (impossible à observer

à l’époque, car précédant la mise au point des microscopes électroniques)

qui s’attaque aux bactéries. Il en conclut à la possibilité d’utiliser ce

« microbe invisible » pour combattre toutes les épidémies. Sur la base de

l'article publié dans The Lancet par Frederick Twort en 1915, d'Hérelle

publie, en 1917 son fameux article princeps : Sur un microbe invisible

antagoniste des bacilles dysentériques (Comptes rendus de l'Académie des

Sciences, Paris, 1917. 165 :p. 373-5.

64 2014 World Sterilization Congress of the WFHSS

http://fr.wikipedia.org/wiki/Institut_Pasteurhttp://fr.wikipedia.org/wiki/Institut_Pasteurhttp://fr.wikipedia.org/wiki/Institut_Pasteurhttp://fr.wikipedia.org/wiki/Parishttp://fr.wikipedia.org/wiki/Bact%C3%A9riophagehttp://fr.wikipedia.org/wiki/Microscope_%C3%A9lectroniquehttp://fr.wikipedia.org/wiki/Microscope_%C3%A9lectroniquehttp://fr.wikipedia.org/wiki/Microscope_%C3%A9lectroniquehttp://fr.wikipedia.org/wiki/The_Lancethttp://fr.wikipedia.org/wiki/The_Lancethttp://fr.wikipedia.org/wiki/The_Lancethttp://fr.wikipedia.org/wiki/Frederick_Tworthttp://fr.wikipedia.org/wiki/Frederick_Tworthttp://fr.wikipedia.org/wiki/Frederick_Twort

• La paternité de la découverte des

bactériophages est souvent disputée entre

Frederick Twort et Félix d'Hérelle. La

publication de Twort dans The Lancet en 1915

est toutefois très différente de celle de Félix

d'Hérelle en 1917.

• Une collection Félix-d’Hérelle comprenant

420 virus a été montée par Hans Wolfgang

Ackermann, professeur à l’université Laval.

En 2003, au moment de sa retraite, cette

collection a été transférée à Sylvain Moineau,


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http://fr.wikipedia.org/wiki/Bact%C3%A9riophagehttp://fr.wikipedia.org/wiki/Frederick_Tworthttp://fr.wikipedia.org/wiki/The_Lancethttp://fr.wikipedia.org/wiki/Universit%C3%A9_Laval

Tawil et al


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Tawil et al


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Tawil et al


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Schematic illustration of various possible orientations of phage on surface. The bacterial capture

proteins are marked in green (a) tailed phage can adsorb head-down,tail-down or side-ways, (b)

icosahedral asymmetric phage, (c) filamentous phage can adsorb via either pole or side-ways, (d)

filamentous phage are prone to bundling oraggregation (left), efforts to orient them typically focus

on arranging them parallel on the substrate (right).


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Various methods of orienting phage: (a) electrostatic, (b) assembly at liquid–liquid interface,

reproduced with permission from Ref. [163], (c) molecular imprinting Ref. [167] (d) molecular

imprinting from Ref. [166]

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(a) SEM pictures of sample 3 copper nanoparticles;

(b) TEM pictures of sample 3 copper nanoparticles.

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Effects of Surface Chemistry on the Generation of Reactive Oxygen

Species by Copper Nanoparticles (Miao Shi et al. 2012, ACS Nano, Vol. 6, No. 3, 2157–2164)

It is essential to develop approaches to control surface chemistry to better

understand the origins of nanoparticle-induced toxicity, as subtle differences in

surface properties can dramatically change biological responses.

Shi et al studied the ROS generating capacity of uniform copper nanoparticles

with different capping ligands to better understand the relationship between

nanoparticle physicochemical properties and their toxicological potential as

reflected by ROS generation in an acellular assay. Three mercaptocarboxylic

acids with different carbon chain lengths were used for surface modification as

they have been shown to provide good colloidal stability for various

nanoparticles.


88

As-synthesized

copper NPs

89


TEM micrographs of these copper NPs after surface modification with 8-mercaptooctanoic acid (MOA), 12-

mercaptododec- anoic acid (MDA), and 16-mercaptohexadecanoic acid (MHA).

90


91


92


The ROS generating capacities of these three copper nanoparticle types were

measured. All particles demonstrated dose-dependent increases in ROS

activity.

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The ROS generating capacities of the oxidized copper samples. The oxidized copper

NPs had lower equivalent [H2O2] than the corresponding as-made ones.

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Other applications

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Schematic of the atherosclerosis treatment with LDL and ox-LDL removal from the bloodstream

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Iron carbides. The carbon surface can be chemically functionalized using physisorbed poly(ethylene imine) and iminodiacetic acid (d) or thiol-moieties which can be crosslinked with antibody fragments using a poly(ethylene glycol)-based crosslinker (e).

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