real-time toxicity testing of silver nanoparticles to ......a keyhole limpet hemocyanin (klh)...
Post on 04-Apr-2020
2 Views
Preview:
TRANSCRIPT
�������� ����� ��
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
This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.
ACC
EPTE
D M
ANU
SCR
IPT
ACCEPTED MANUSCRIPT
1
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).
ACC
EPTE
D M
ANU
SCR
IPT
ACCEPTED MANUSCRIPT
2
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.
ACC
EPTE
D M
ANU
SCR
IPT
ACCEPTED MANUSCRIPT
3
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
ACC
EPTE
D M
ANU
SCR
IPT
ACCEPTED MANUSCRIPT
4
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.
ACC
EPTE
D M
ANU
SCR
IPT
ACCEPTED MANUSCRIPT
5
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
).
ACC
EPTE
D M
ANU
SCR
IPT
ACCEPTED MANUSCRIPT
6
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.
ACC
EPTE
D M
ANU
SCR
IPT
ACCEPTED MANUSCRIPT
7
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
.
ACC
EPTE
D M
ANU
SCR
IPT
ACCEPTED MANUSCRIPT
8
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
ACC
EPTE
D M
ANU
SCR
IPT
ACCEPTED MANUSCRIPT
9
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.
ACC
EPTE
D M
ANU
SCR
IPT
ACCEPTED MANUSCRIPT
10
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.
ACC
EPTE
D M
ANU
SCR
IPT
ACCEPTED MANUSCRIPT
11
References
Abadian, P.N., Kelley, C.P., Goluch, E.D., 2014a. Cellular analysis and detection using
Surface Plasmon Resonance techniques. Analytical Chemistry 86, 2799-2812. DOI:
10.1021/ac500135s.
Abadian, P.N., Tandogan, N., Jamieson, J.J., Goluch, E.D., 2014b. Using Surface Plasmon
Resonance imaging to study bacterial biofilms. Biomicrofluidics 8. DOI: 10.1063/1.4867739.
Bouguelia, S., Roupioz, Y., Slimani, S., Mondani, L., Casabona, M.G., Durmort, C., Vernet,
T., Calemczuk, R., Livache, T., 2013. On-chip microbial culture for the specific detection of
very low levels of bacteria. Lab on a Chip 13, 4024-4032. DOI: 10.1039/c3lc50473e.
Bulard, E., Bouchet-Spinelli, A., Chaud, P., Roget, A., Calemczuk, R., Fort, S., Livache, T.,
2015. Carbohydrates as new probes for the identification of closely related Escherichia coli
strains using Surface Plasmon Resonance imaging. Analytical Chemistry 87, 1804-1811.
DOI: 10.1021/ac5037704.
Cherif, B., Roget, A., Villiers, C.L., Calemczuk, R., Leroy, V., Marche, P.N., Livache, T.,
Villiers, M.B., 2006. Clinically related protein-peptide interactions monitored in real time on
novel peptide chips by surface plasmon resonance imaging. Clinical Chemistry 52, 255-262.
DOI: 10.1373/clinchem.2005.058727.
Chernousova, S., Epple, M., 2013. Silver as antibacterial agent: ion, nanoparticle, and metal.
Angewandte Chemie - International Edition 52, 1636-1653. DOI: 10.1002/anie.201205923.
Deryabin, D.G., Aleshina, E.S., Efremova, L.V., 2012. Application of the inhibition of
bacterial bioluminescence test for assessment of toxicity of carbon-based nanomaterials.
Microbiology 81 (4), 492e497. DOI: 10.1134/S0026261712040042.
Fehlhaber, K., Krueger, G., 1998. The study of Salmonella enteritidis growth kinetics using
rapid automated bacterial impedance technique. Journal of Applied Microbiology 84, 945-
949. DOI: 10.1046/j.1365-2672.1998.00410.x.
Garner, K.L., Keller, A.A., 2014. Emerging patterns for engineered nanomaterials in the
environment: a review of fate and toxicity studies. Journal of Nanoparticle Research 16.
DOI: 10.1007/s11051-014-2503-2.
ACC
EPTE
D M
ANU
SCR
IPT
ACCEPTED MANUSCRIPT
12
Hill, R.T., 2015. Plasmonic biosensors. Wiley Interdisciplinary Reviews - Nanomedicine and
Nanobiotechnology 7, 152-168. DOI: 10.1002/wnan.1314.
Holden, P.A., Schimel, J.P., Godwin, H.A., 2014. Five reasons to use bacteria when assessing
manufactured nanomaterial environmental hazards and fates. Current Opinion in
Biotechnology 27, 73-78. DOI: 10.1016/j.copbio.2013.11.008.
Juganson, K., Ivask, A., Blinova, I., Mortimer, M., Kahru, A., 2015. NanoE-Tox: new and in-
depth database concerning ecotoxicity of nanomaterials. Beilstein Journal of Nanotechnology
6, 1788-1804. DOI: 10.3762/bjnano.6.183.
Kinsner-Ovaskainen, A., Colpo, P., Ponti, J., Rossi, F., 2014. Nanotoxicology. In Vitro
Toxicology Systems, 481-499. DOI: 10.1007/978-1-4939-0521-8_21.
Klein, C., Comero, S., Stahlmecke, B., Romazanov, J., Kuhlbusch, T., van Doren, E., Wick,
P., Locoro, G., Koerdel, W., Gawlik, B., Mast, J., Krug, H. F., Hund-Rinke, K., Friedrichs,
S., Maier, G., Werner, J., Linsinger, T., 2011. NM-300 silver characterisation, stability,
homogeneity. EUR - Scientific and Toxicological Sciences, Technical Research Reports, JRC
Publication No. JRC60709, EUR 24693 EN, Publications Office of the European Union.
Kodoyianni, V., 2011. Label-free analysis of biomolecular interactions using SPR imaging.
Biotechniques 50, 32-40. DOI: 10.2144/000113569.
Krug, H.F., 2014. Nanosafety Research - Are We on the Right Track? Angewandte Chemie -
International Edition 53, 12304-12319. DOI: 10.1002/anie.201403367.
Li, F.F., Lei, C.Y., Shen, Q.P., Li, L.J., Wang, M., Guo, M.L., Huang, Y., Nie, Z., Yao, S.Z.,
2013. Analysis of copper nanoparticles toxicity based on a stress-responsive
bacterial biosensor array. Nanoscale 5 (2), 653-662. DOI: 10.1039/c2nr32156d.
Lok, C.-N., Ho, C.-M., Chen, R., He, Q.-Y., Yu, W.-Y., Sun, H., Tam, P.K.-H., Chiu, J.-F.,
Che, C.-M., 2007. Silver nanoparticles: partial oxidation and antibacterial activities. Journal
of Biological Inorganic Chemistry 12, 527-534. DOI: 10.1007/s00775-007-0208-z.
Losasso, C., Belluco, S., Cibin, V., Zavagnin, P., Micetic, I., Gallocchio, F., Zanella, M.,
Bregoli, L., Biancotto, G., Ricci, A., 2014. Antibacterial activity of silver nanoparticles:
sensitivity of different Salmonella serovars. Frontiers in Microbiology 5. DOI:
10.3389/fmicb.2014.00227.
ACC
EPTE
D M
ANU
SCR
IPT
ACCEPTED MANUSCRIPT
13
Mallevre, F., Fernandes, T.F., Aspray, T.J., 2014. Silver, zinc oxide and titanium dioxide
nanoparticle ecotoxicity to bioluminescent Pseudomonas putida in laboratory medium and
artificial wastewater. Environmental Pollution 195, 218-225. DOI:
10.1016/j.envpol.2014.09.002.
Mondani, L., Delannoy, S., Mathey, R., Piat, F., Mercey, T., Slimani, S., Fach, P., Livache,
T., Roupioz, Y., 2016. Fast detection of both O157 and non-O157 shiga-toxin producing
Escherichia coli by real-time optical immunoassay. Applied Microbiology 62, 39-46. DOI:
10.1111/lam.12503.
Mondani, L., Roupioz, Y., Delannoy, S., Fach, P., Livache, T., 2014. Simultaneous
enrichment and optical detection of low levels of stressed Escherichia coli O157:H7 in food
matrices. Journal of Applied Microbiology 117, 537-546. DOI: 10.1111/jam.12522.
Nguyen, H.H., Park, J., Kang, S., Kim, M., 2015. Surface Plasmon Resonance: a versatile
technique for biosensor applications. Sensors 15, 10481-10510. DOI: 10.3390/s150510481.
Notter, D.A., Mitrano, D.M., Nowack, B., 2014. Are nanosized or dissolved metals more
toxic in the environment? A meta-analysis. Environmental Toxicology and Chemistry 33,
2733-2739. DOI: 10.1002/etc.2732.
Oostingh, G.J., Casals, E., Italiani, P., Colognato, R., Stritzinger, R., Ponti, J., Pfaller, T.,
Kohl, Y., Ooms, D., Favilli, F., Leppens, H., Lucchesi, D., Rossi, F., Nelissen, I., Thielecke,
H., Puntes, V.F., Duschl, A., Boraschi, D., 2011. Problems and challenges in the development
and validation of human cell-based assays to determine nanoparticle-induced
immunomodulatory effects. Particle and Fibre Toxicology 8. DOI: 10.1186/1743-8977-8-8.
Pan, H.M., Zhang, Y.B., He, G.X., Katagori, N., Chen, H.Z., 2014. A comparison of
conventional methods for the quantification of bacterial cells after exposure to metal oxide
nanoparticles. BMC Microbiology 14. DOI: 10.1186/s12866-014-0222-6.
Rich, R.L., Myszka, D.G., 2011. Survey of the 2009 commercial optical biosensor literature.
Journal of Molecular Recognition 24, 892-914. DOI: 10.1002/jmr.1138.
Santimano, M.C., Kowshik, M., 2013. Altered growth and enzyme expression profile of ZnO
nanoparticles exposed non-target environmentally beneficial bacteria. Environmental
Monitoring and Assessment 185, 7205-7214. DOI: 10.1007/s10661-013-3094-6.
ACC
EPTE
D M
ANU
SCR
IPT
ACCEPTED MANUSCRIPT
14
Seal, S., Karn, B., 2014. Safety aspects of nanotechnology based activity. Safety Science 63,
217-225. DOI: 10.1016/j.ssci.2013.11.018.
Weimer, M., 2010. Applying precaution in EU authorisation of genetically modified products
- Challenges and suggestions for reform. European Law Journal 16, 624-657. DOI:
10.1111/j.1468-0386.2010.00526.x.
ACC
EPTE
D M
ANU
SCR
IPT
ACCEPTED MANUSCRIPT
15
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
ACC
EPTE
D M
ANU
SCR
IPT
ACCEPTED MANUSCRIPT
16
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.
ACC
EPTE
D M
ANU
SCR
IPT
ACCEPTED MANUSCRIPT
22
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.
top related