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Real-time toxicity testing of silver nanoparticles to Salmonella Enteritidisusing surface plasmon resonance imaging: A proof of concept

Florian Mallevre, Vincent Templier, Raphael Mathey, Loic Leroy, YoannRoupioz, Teresa F. Fernandes, Thomas J. Aspray, Thierry Livache

PII: S2452-0748(15)30017-3DOI: doi: 10.1016/j.impact.2016.02.004Reference: IMPACT 7

To appear in: NanoImpact

Received date: 25 November 2015Revised date: 15 February 2016Accepted date: 17 February 2016

Please cite this article as: , Real-time toxicity testing of silver nanoparticles to SalmonellaEnteritidis using surface plasmon resonance imaging: A proof of concept, NanoImpact(2016), doi: 10.1016/j.impact.2016.02.004

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Real-time toxicity testing of silver nanoparticles to Salmonella Enteritidis using Surface

Plasmon Resonance imaging: a proof of concept.

Florian Mallevre1, Vincent Templier

2,3,4, Raphael Mathey

2,3,4, Loic Leroy

2,3,4, Yoann

Roupioz2,3,4

, Teresa F. Fernandes1, Thomas J. Aspray

1,* and Thierry Livache

2,3,4

1 School of Life Sciences, NanoSafety Research Group, Heriot-Watt University, Edinburgh

EH14 4AS, United-Kingdom

2 Univ. Grenoble Alpes, INAC-SPRAM, F-38000 Grenoble, France

3 CEA, INAC-SPRAM, F-38000 Grenoble, France

4 CNRS, INAC-SPRAM, F-38000 Grenoble, France

* Corresponding author. Tel.: +44 (0)131 451 3974; fax: +44 (0)131 451 3009; E-mail

address: T.J.Aspray@hw.ac.uk, thomasaspray@gmail.com (Dr T. J. Aspray).

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Abstract

In this paper we report for the first time on the suitability of surface plasmon resonance

imaging (SPRi) for performing ecotoxicity testing of nanoparticles (NPs). Specifically, the

impact of silver NPs (using Ag NM-300K) and ions (using AgNO3 salt) on Salmonella

Enteritidis growth was assessed in Luria Bertani medium using the culture-capture-measure

(CCM) based SPRi method. Clear effects were observed at 10 mg L-1

Ag NPs characterised

by shifted SPRi detection times (TD) by ca. 2.6 h compared to the control. Comparable results

were obtained using 1 mg L-1

Ag ions. No clear effects were observed at 1 mg L-1

Ag NPs

and 0.1 mg L-1

Ag ions. Overall results match the current trend in nanoecotoxicology using

bacteria (e.g. impact of Ag NPs between 1 - 10 mg L-1

and higher toxicity of Ag ions

compared to Ag NPs). The dose dependent patterns of toxicity were coherent with those

obtained using a standard plating method; however, the SPRi approach was faster (i.e. results

within a few hours) and generated kinetic data (i.e. real-time monitoring). In addition, SPRi

presents many valuable intrinsic advantages (e.g. label-free, multiplex, bespoke and robust)

over current approaches. Consequently, a plethora of opportunities for future developments

and applications of SPRi in NP testing is associated with the proof of concept reported herein.

Keywords

Surface plasmon resonance imaging, Salmonella, Nanoparticle, Silver, Ecotoxicology,

Biosensor.

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1. Introduction

The risk assessment of engineered nanoparticles (NPs) is essential in supporting the

development of nanotechnologies (Krug, 2014; Seal and Karn, 2014). Nanoecotoxicology is

quickly growing but the toxicity testing of NPs remains overall a challenging task (Garner

and Keller, 2014; Juganson et al., 2015). Although bacteria are widely used as model

organisms (Holden et al., 2014; Juganson et al., 2015), methods based on plating and

spectrophotometry have clear limitations. Plating is long to perform and lacks accuracy (Pan

et al., 2014) while spectrophotometry is rarely suitable for analyses in complex and/or

coloured matrices (Oostingh et al., 2011). Approaches exploiting the advantages of

luminescent genetically modified bioreporters (GMB) have emerged (Deryabin et al., 2012;

Li et al., 2013; Mallevre et al., 2014) offering additional perspectives, but regulations and

concerns on applicability of GMB limit their broad use and relevance (Weimer, 2010).

Surface plasmon resonance imaging (SPRi) is a biosensor technology suitable for

biomolecular interaction assessment (Kodoyianni, 2011) and analyte detection (Abadian et

al., 2014a) using a microarray format. SPRi has proven to be robust over the years via the

emergence of various instruments, dedicated studies and companies worldwide (Hill, 2015;

Nguyen et al., 2015; Rich and Myszka, 2011). Due to an appealing portfolio of advantages

(e.g. real-time, label-free, rapid, multiplex, GMB-free, using small volumes and generating

little waste) and on-going refinements (e.g. increasingly operator friendly and compatible

with various types of biological materials, matrices and configurations), applications of SPRi

are expanding. SPRi applications with bacteria have emerged recently (Abadian et al., 2014b;

Bouguelia et al., 2013; Bulard et al., 2015; Mondani et al., 2014; Mondani et al., 2016). The

notion of real-time monitoring of the bacterial growth by SPRi was first reported by

Bouguelia et al. (2013) proposing a culture-capture-measure (CCM) method using the

advantages of specific interactions between monoclonal antibodies microarrays and bacteria

(e.g. Salmonella enterica, Escherichia coli). Additional applications with various bacteria

followed using the same method (Bulard et al., 2015; Mondani et al., 2014; Mondani et al.,

2016). Concomitantly, Abadian et al. (2014b) reported the use of SPRi for the monitoring of

E. coli and Pseudomonas aeruginosa biofilm attachment, formation and removal.

From a technical viewpoint (Hill, 2015; Nguyen et al., 2015), a surface plasmon is an electro-

magnetic wave propagating along the surface of a thin di-electric metal film (e.g. gold). The

resonance of the surface plasmons can be achieved by a polarised light beam undergoing total

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internal reflection at the film and medium interface. The resonance state is highly sensitive to

changes (e.g. stresses, interactions) in the medium adjacent to the surface and modifications

at this level can be quantified in terms of variation of reflectivity via the monitoring of the

reflected light intensity. As such, SPRi is simply the characterisation of the SPR signals on

the whole surface of a biochip (e.g. a gold coated prism) using a video camera (Fig 1A). The

CCM based SPRi method per se uses biochips functionalised with a bespoke series of

specific and non-specific antibody bearing spots. In this particular case, the surface plasmon

alteration at the spot level is generated by occurring interactions bacteria-antibodies (Fig.

1B). As originally demonstrated (Bouguelia et al., 2013), the method is specific (i.e. a

selected bacterial model will interact first and mainly with its specific antibody) and

quantitative to some extent (i.e. the higher the number of cells, the larger the surface

interactions and therefore the corresponding output signal). Consequently, by monitoring the

variation in reflectivity overtime one can derive the growth curve of one or several selected

models in a microarray format. Using independent chambers (i.e. four herein, Fig. 1C) one

biochip can be exposed to different conditions of growth (or different exposure conditions) at

the same time.

The use of SPRi with bacteria has been initiated in ecotoxicology for the testing of antibiotics

(Abadian et al., 2014b) and for the impact assessment of thermal stresses (Mondani et al.,

2014). However, no evidence of workability in nanoecotoxicology has yet been reported. In

light of this, we aimed to extend the CCM based SPRi method to nanoecotoxicological

studies to assess its suitability and potentially to propose new options for performing toxicity

testing of NPs and related ions when using bacteria as model organisms.

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2. Experimental Procedure

2.1. Model bacterium

Salmonella enterica subspecies enterica serovar Enteritidis (hereafter referred to as S.

Enteritidis) from the Scientific Institute of Hygiene and Analysis (ISHA, Massy, France) was

used as a model bacterium in this study.

2.2. Antibodies

An S. Enteritidis monoclonal antibody (hereafter referred to as SE103, kindly provided by Dr

H. Volland, CEA-Saclay, France) was used as a specific probe. A keyhole limpet

hemocyanin (KLH) specific monoclonal antibody (kindly sourced by Dr L. Bellanger, CEA-

Marcoule, France) was used as a negative control probe (Bouguelia et al., 2013).

2.3. SPRi apparatus and biochips

Experiments were performed in a polyether-ether-ketone reactor containing four independent

chambers in combination with a SPRi PlexII instrument (Horiba Scientific). The SPRi PlexII

was housed in a temperature controlled incubator maintained at 37 °C. Gold coated glass

prisms were used as biochips (Horiba Scientific). All the material is commercially available.

The biochips were arrayed with pyrrolylated SE103 and KLH antibodies by electrochemical

directed pyrrole-based polymerisation (Cherif et al., 2006). The biochips were freshly made

for each experiment.

2.4. Nanoparticles

Representative Ag NPs (Ag NM-300K or JRCNM03000a) were obtained from the European

Commission’s Joint Research Centre (Ispra, Italy). Ag NM-300K NPs (negatively charged

and polydisperse nanoparticles with a primary size ca. 15 nm) were characterised previously

(Klein et al., 2011; Losasso et al., 2014). We reported further information on this material

(e.g. hydrodynamic size, zeta potential, spectrum of absorption, and dissolution rate) in

various media (including Luria Bertani, the medium used herein) using dynamic light

scattering (DLS), UV-visible spectrophotometry (UV-vis) and atomic absorption

spectroscopy (AAS) elsewhere (Mallevre et al., 2014). Silver nitrate (AgNO3, Fisher

Scientific) salt was used as a source of Ag ions. All concentrations are in Ag terms (i.e. mg

Ag L-1

).

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2.5. SPRi based assays

This application extends the principle of the CCM method (Bouguelia et al., 2013). Prior to

the assays, S. Enteritidis was pre-cultured in Luria Bertani (LB) medium (10 g L-1

tryptone, 5

g L-1

yeast extract, 5 g L-1

NaCl) at 37 °C under agitating conditions (150 rpm) then used

diluted at 102 CFU mL

-1. Ag NPs were freshly prepared in LB (bath sonicated twice for 8

min), as previously described (Mallevre et al., 2014), prior to addition for testing at 0.1, 1, 5,

10 and 100 mg L-1

. Ag ions were tested at 0.01, 0.1, 1 and 10 mg L

-1. A typical template for

an assay included at least three replicated spots per antibody across four different conditions

of exposure which involved a non-exposed control, a test with the Ag NPs, a test with the Ag

ions, and a control with the sole Ag NPs (i.e. in the absence of the bacteria). The bacterial

growth was characterised as the evolution of the local variation of the reflectivity (ΔR, in %

terms) over time for the selected spots. Experiments were performed at least three times.

Results were tested for significance (when applicable) using two-way ANOVA (Holm-Sidak

corrected).

2.6. Plating based assays

S. Enteritidis suspensions were prepared as aforementioned then cultured at 37 °C under

shaking conditions (150 rpm) for 24 h in the presence/absence of the Ag NPs or ions. Ag NPs

were tested at 0.1, 1, 10 and 100 mg L-1

; Ag ions at 0.01, 0.1, 1 and 10 mg L-1

. Samples taken

at 0, 2, 4, 6, 8 and 24 h were serial diluted (till 10-8

), plated (Tryptic Soy Agar) and incubated

each for 24 h at 37 °C. Finally, the colony forming units (CFU) were counted and the

bacterial growth was characterised as the evolution of the CFU mL-1

data over time.

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3. Results and Discussion

3.1. Control experiments

The results obtained by SPRi in the absence of the Ag NPs or ions are shown in Figure 2A.

The real-time growth monitoring generated a typical exponential plot on the specific antibody

(SE103) bearing spots. This was characterised by a detection time (TD) derived at 4.6 ± 0.2 h

with a starting bacterial inoculum of 97 ± 7 CFU mL-1

(mean ± SEM, n = 5). Details of data

processing are shown in the supplementary material (Fig. S1). The quality of spots, the

specificity of interaction of S. Enteritidis with SE103 and the consistency of the generated

data between replicated spots were confirmed (Fig. S1). Corroborative results were reported

elsewhere in buffered peptone water using the same bacterial model (Bouguelia et al., 2013).

As characterised by plating (Fig. 2B), S. Enteritidis exhibited a generation time (TG) of 18.9 ±

0.6 min in the linear part of the growth curve; TG between 18 and 22 min were previously

reported in LB for Salmonella spp. (Fehlhaber and Krueger, 1998). Besides, the SPRi

detection threshold has already been shown to be in the 105

- 106 CFU mL

-1 range with

bacteria (Bouguelia et al., 2013; Bulard et al., 2015; Mondani et al., 2014). Starting from 102

CFU mL-1

, the 10

5 - 10

6 CFU mL

-1 range was expected to be reached after 4 to 5 h of growth

therefore supporting the aforementioned SPRi results.

Overall results in the absence of the Ag NPs or ions showed that the S. Enteritidis growth

related information from the SPRi was consistent between replicated spots and experiments,

coherent with results obtained via plating, and in line with previously published data.

3.2. Toxicity testing of the Ag NPs and ions

A dose dependent impact of the Ag ions on S. Enteritidis TD was observed using SPRi

(Fig. 3A). More specifically, TD ca. 7.2 h were calculated at 1 mg L-1

(i.e. significantly

delayed by ca. 2.6 h compared to the control, p < 0.01). Interestingly, the shape of the SPRi

growth curves was not affected; attesting to an early and temporal antimicrobial effect on the

bacterial population. Similar observations (i.e. shifts) were reported with various bacteria

using OD monitoring (Lok et al., 2007; Santimano and Kowshik, 2013). No clear impacts

were detected at 0.01 and 0.1 mg L-1

. No growth was obtained at 10 mg L-1

.

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As shown by plating (Fig. 3B), the Ag ions were lethal at 10 mg L-1

(i.e. clear impact from 2

h of exposure without recovery up to 24 h). A temporary impact was shown at 1 mg L-1

characterised by a delayed growth with a detrimental effect during the first hours of exposure

(TG ~ 260 min at 1 mg L-1

versus 40 min at 0 mg L-1

for the 0 - 2 h time window), followed

by recovery over time. No difference from the control was visible at 24 h. A delay of two

logs (in CFU mL-1

terms) resulted at 4 and 6 h, compared to the control. No visible impact

was observed at 0.01 and 0.1 mg L-1

. Overall, the plate count supports the aforementioned

shift in TD (but unchanged curve shapes) obtained by SPRi in the same condition of exposure

(Fig. 3, arrows) as well as the hypothesis of a temporal antimicrobial effect of the Ag ions to

explain the results in the tested regime.

Comparatively, the Ag NPs were found to be ten times less toxic than the Ag ions; the

antimicrobial impact (e.g. cell death, lysis) of the NPs is therefore likely to be mainly driven

by the released ions as discussed elsewhere (Juganson et al., 2015; Lok et al., 2007;

Santimano and Kowshik, 2013). In accordance with the plating results (Fig. S2), exposure to

10 mg L-1

Ag NPs led to significantly shifted S. Enteritidis TD from 4.6 h to 7.5 h (p < 0.01)

in SPRi whereas no visible effects were obtained at 0.1 and 1 mg L-1

(Fig. 4). The shape of

the SPRi growth curves was visibly affected (i.e. in a dose dependent manner) by the

presence of the Ag NPs (Fig. 4). This was attributed to an amplified sedimentation of NP-

bacteria aggregates (Kinsner-Ovaskainen et al., 2014) occurring especially on the specific

spots and eventually flattening the output signal (data not shown). This effect was limited by

performing assays in inverted SPRi systems, even when using more concentrated bacterial

inoculum (Fig. S3). Inverted SPRi systems may also prevent the deposition of dead cells on

the biochip surface and thereby further improve the background noise level of the approach.

3.3. Relevance and perspectives

The toxicity of Ag ions and NPs was respectively reported from 2 and 20 mg L-1

for

Salmonella spp. (Losasso et al., 2014) and, more generally, in the 0.1 - 1 mg L-1

and 1 - 10

mg L-1

ranges for bacteria (Juganson et al., 2015; Chernousova and Epple, 2013). Toxicity of

the tested Ag NPs was previously shown to be ten times lower than the ions across different

bacterial models (Losasso et al., 2014; Mallevre et al., 2014). The general higher toxicity of

Ag ions compared to Ag NPs was also commonly reported regardless of the technique or

model organism (Notter et al., 2014). Consequently, the reported SPRi results appear most of

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all in line with the main trends in nanoecotoxicology and therefore attest to the relevance and

suitability of the SPRi technique for the testing of NPs and related ions.

With the suitability of SPRi demonstrated in nanoecotoxicology, the advantages of such

platforms (Hill, 2015; Nguyen et al., 2015; Rich and Myszka, 2011) should be exploited and

implemented. Further assays using different types of cells (e.g. prokaryotic and mammalian

cells), matrices (e.g. artificial and real), configurations (e.g. with and without flow) and

toxicants (e.g. antibiotics, potential drugs, emerging contaminants) are anticipated. In

addition, the response of multi-species population (i.e. based on the advantages of using

specific antibodies) to toxicants (i.e. tested separately or as mixtures) may be investigated.

The herein proof of concept is considered as a breakthrough in nanoecotoxicology by the

authors as supporting the development of real-time (i.e. kinetics) and multiplex (i.e. high

throughput) studies of wide applicability.

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Funding information

We acknowledge Heriot-Watt University (Edinburgh, UK) and the MODENA COST action

TD1204 for providing FM with, respectively, an Annual Fund grant and a Short-Term

Scientific Mission (STSM) grant supporting the visits at CEA-Grenoble France.

Conflict of interest

No conflict of interest declared.

Acknowledgements

We thank the European Union's Seventh Framework Programme [FP7 2007-2013] under EC-

GA No. 263215 ‘MARINA’, for the provision of the nanomaterials used in this study. We

thank Professor Vicki Stone (Heriot-Watt University, Edinburgh, UK) for the critical reading

of the manuscript.

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Figure 1: Introduction to bacterial growth monitoring using the CCM based SPRi

method. The general SPRi set-up used herein (based on functionalised prisms with bespoke

series of antibody bearing spots) is shown in A. The principle of the CCM method is

schematised in B. An example of two spots, bearing non-specific (in red) or specific (in blue)

antibodies to the bacterial model of interest, is presented. R is the reflectivity for a defined

spot as determined optically using a CCD camera and ∆R is the variation of this reflectivity

(due to specific interactions bacteria-antibodies occurring at the surface of the prism)

registered overtime for a selected (fixed) measurement angle. An example of a used prism,

silicon seal and PEEK reactor of four chambers (altogether constituting the biochip when

assembled), is shown in C; a 1.5 mL Eppendorf tube provides a perspective on the scale of

the material. Additional information on the approach can be found in Bouguelia et al. (2013)

and in the patent WO 2012073202 A1.

Figure 2: S. Enteritidis growth monitoring in the absence of the Ag NPs or ions. The

normalised variation of reflectivity over time from the SE103 antibody bearing specific spots

(i.e. the S. Enteritidis growth curve) obtained by SPRi is shown in A (▬). The detection

times (TD) were obtained as described by Bulard et al. (2014) from the peak of the derivative

curves (─). Examples of raw data and results processing are shown in the supplementary

material (Fig. S1). The output data (in CFU mL-1

terms) from the S. Enteritidis growth

monitoring obtained by plating are shown in B (●). The generation times (TG) were

calculated from the linear fit (─) on the exponential part of the curves (Santimano and

Kowshik, 2013). Data are mean ± SEM.

Figure 3: S. Enteritidis growth monitoring in the presence of the Ag ions. The output

data from the S. Enteritidis growth monitoring operated by SPRi (∆R, in % terms) and by

plating (in CFU mL-1

terms) in the presence of 0, 0.01, 0.1, 1 and 10 mg L-1

Ag ions (i.e. in

Ag final concentration, from AgNO3 salt) are shown in A and B, respectively. The

corresponding detection times (TD) are indicated in the SPRi case (i.e. N/A stands for non-

applicable). The calculated times of generation (TG) for the 1 mg L-1

exposure condition are

proposed for the plating case.

Figure 4: S. Enteritidis growth monitoring in the presence of the Ag NPs by SPRi. The

output data from the S. Enteritidis growth monitoring operated by SPRi (∆R, in % terms) in

the presence of 0, 0.1, 1, 5 and 10 mg L-1

Ag NPs (i.e. in Ag final concentration, from Ag

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NM-300K suspensions) are shown. The corresponding detection times (TD) are indicated.

Results obtained by plating (Fig. S2) and results from the assays piloted with an inverted

SPRi system (Fig. S3) are shown in the supplementary material.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Graphical abstract

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Highlights

Toxicity testing of nanoparticles (NPs) to bacteria using SPRi is reported for the first

time.

The impact of Ag NPs and Ag ions on Salmonella Enteritidis is discussed.

Real-time SPRi data supported current trends in nanoecotoxicology.

SPRi was concluded as a suitable technology for performing Ag NP testing with

bacteria.