March 7th 2011 HYBRID MATERIALS 2011: Second International Conference on Multifunctional, Hybrid and Nanomaterials; 6-10 March 2011 Strasbourg (France). Sabine Crunaire (1), Thu-Hoa Tran-Thi (1), Khanh-Quyen Ngo (1), Pierre R. Marcoux (2), Jean-Pierre Moy (2) and Frédéric Mallard (3) 1 Laboratoire Francis Perrin, CEA/DSM/IRAMIS/SPAM – CNRS URA 2453, Gif-sur-Yvette, France. 2 Commissariat à l'Energie Atomique (CEA), LETI, MINATEC, Grenoble, France. 3 bioMérieux, Grenoble, France. There is a well-established and growing interest in the detection and identification of microorganisms by measuring their release of volatile organic compounds (VOCs). Indeed, the measurement of the VOCs emitted by in vitro or in vivo bacterial culture could be used as a characteristic fingerprint for detection and identification. To be of greatest diagnostic value, real-time non-invasive measurements of breath or headspaces above urine, feces, blood, or sputum would replace time-consuming culture techniques. Our study deals with the detection of microbial VOCs with functionalised nanoporous materials. These sol-gel materials include a probe molecule. This probe is chosen in order to react specifically with a target VOC, in liquid or gas phase, so as to produce an absorbent and/or fluorescent molecule within pores. This transduction pathway is called direct optical transduction. We will focus on the detection of indole. This volatile metabolite comes out of the degradation of the amino acid tryptophan and its presence is tested in numerous identification schemes, especially to presumptively identify Escherichia coli, the gram-negative bacillus most encountered in diagnostic bacteriology. We have prepared hybrid materials showing high-surface area (~600 m2/g), using the sol-gel chemistry. They are doped either with DMACA (dimethylaminocinnamaldehyde) or DMABA (dimethylaminobenzaldehyde), two probes reacting with indole in a complete and fast way. Therefore, we can measure indole concentration in a few minutes, by following the absorption kinetics at a chosen wavelength. Indole production of model strains of E. coli and H. alvei has also been monitored with our detectors, in liquid as well as in gas phase. We have proved the ability of these detectors to discriminate indole-producing bacteria from the others.
S. Crunaire et al., HYBRID MATERIALS 2011 – 07/03/11 1 Funtionalised nanoporous materials with Funtionalised nanoporous materials with direct optical transduction for direct optical transduction for microbiological monitoring. microbiological monitoring. bioMérieux – CEA joint team, Grenoble (France). bioMérieux – CEA joint team, Grenoble (France). Pierre R. Marcoux Pierre R. Marcoux , , Jean-Pierre Moy. Jean-Pierre Moy. Frédéric Mallard. Frédéric Mallard. Hybrid Materials 2011, Symposium B (Monday 7 March): Hybrid Materials 2011, Symposium B (Monday 7 March): Functional hybrid nanomaterials, nanocomposites and their applications Functional hybrid nanomaterials, nanocomposites and their applications Laboratoire Francis Perrin – CNRS URA 2453, Laboratoire Francis Perrin – CNRS URA 2453, Gif-sur-Yvette (France). Gif-sur-Yvette (France). Sabine Crunaire, Sabine Crunaire, Khanh-Quyen Ngo, Khanh-Quyen Ngo, Thu-Hoa Tran-Thi. Thu-Hoa Tran-Thi.
Transcript
S. Crunaire et al., HYBRID MATERIALS 2011 – 07/03/11 1
Funtionalised nanoporous materials with direct Funtionalised nanoporous materials with direct optical transduction for microbiological monitoring.optical transduction for microbiological monitoring.
bioMérieux – CEA joint team, Grenoble (France).bioMérieux – CEA joint team, Grenoble (France).
Pierre R. MarcouxPierre R. Marcoux,,Jean-Pierre Moy.Jean-Pierre Moy.
Frédéric Mallard.Frédéric Mallard.
Hybrid Materials 2011, Symposium B (Monday 7 March):Hybrid Materials 2011, Symposium B (Monday 7 March):Functional hybrid nanomaterials, nanocomposites and their applicationsFunctional hybrid nanomaterials, nanocomposites and their applications
Laboratoire Francis Perrin – CNRS URA 2453,Laboratoire Francis Perrin – CNRS URA 2453,Gif-sur-Yvette (France).Gif-sur-Yvette (France).
S. Crunaire et al., HYBRID MATERIALS 2011 – 07/03/11 2
Intro: Microbial Volatile Organic Compound (MVOC)• Volatile metabolite. Usually small molecules.
• Less susceptible to various forms of inactivation (e.g. alteration by wet chemical reactions) their sphere of influence (“diffusion radius”) is greater.
• Used by bacteria as signaling molecules.
Mass spectra of the examined bacteria. All measured mass concentrations (ppbv) of one
bacterium are plotted on top of each other and the additive columns of all the measured bacteria are
compared.
Diagnosis of Bacteria In Vitro by Mass Spectrometric Fingerprinting: A Pilot Study, M. Lechner et al., Current Microbiology, 2005, 51, 267-269.
« MVOC profiles »
S
S
O
H
N
O
OH
O
O
C NH
S. Crunaire et al., HYBRID MATERIALS 2011 – 07/03/11 3
Intro: Detecting MVOC, the “electronic nose” approachMultiparametric fingerprint: array of colorimetric sensors
http://www.chemsensing.com
each MVOC interacts with
several sensors
each sensor interacts
with several MVOC
S. Crunaire et al., HYBRID MATERIALS 2011 – 07/03/11 4
Intro: Optical transduction within sol-gel material
fluid
MVOC5
MVOC3MVOC1
MVOC4
MVOC2
productproduct
fluorescencefluorescence
absorbanceabsorbance
11 (nm) (nm)
fluorescencefluorescence22 (nm) (nm) 11 (nm) (nm)
probeprobe
Abs.
(nm)t0
t1
t2
Specific interactionSpecific interactionbetween a given sensor between a given sensor
and a given MVOC.and a given MVOC.
Within hybrid organic-inorganic nanoporous
sensor: reaction between a probeprobe and a targettarget.
An absorbent and/or fluorescent product
is formed.
MVOC detection in liquid-phase, as well
as in gas-phase.
S. Crunaire et al., HYBRID MATERIALS 2011 – 07/03/11 5
Intro: Indole as a microbial VOC
Indole comes from hydrolysis of tryptophan.NH2
O
NH
H
NH
tryptophan
tryptophanase
indole4.1.99.1
• Indole is a widespread bacterial metabolite: many pathogens, both Gram+ and Gram-, produce large quantities of indole. Signal molecule.
• For example: among Enterobacteriaceae (Salmonella, E. coli, etc), emission of indole is checked when performing a biochemical test for identification.
N
O
H
N
O
H
MeO
O
H
OHOH
O
O
O
OH
OMe
DMABADMABA
DMACADMACA
MOB croconic acid
MON
DMABADMABA : included in Ehrlich reagent, Kovacs reagent, James reagent
DMACADMACA : less soluble, more expensive but more sensitive
S. Crunaire et al., HYBRID MATERIALS 2011 – 07/03/11 6
Chemical reaction of transduction
• Formation, in acidic conditions, of a strongly absorbent salt. Fast enough to be used as a transduction reaction.
NH
N
O
NH N
NH N
+ + Cl +Cl
targettarget
probeprobe
productproduct
azafulvenium chloride
HCl
wavelength (nm)
abso
rban
ce
max 624 nm=97000 M-1.cm-1
• Reaction kinetics is proportional to [indole] between 10-7 and 10-4 M.
DMACA
time (s)
OD
(62
4 nm
)
DMACAin excess
S. Crunaire et al., HYBRID MATERIALS 2011 – 07/03/11 7
Hybrid nanoporous sensor• Sol-gel synthesis (TMOS and APTS) as precursors.
• Preparation of sol at -15°C.
• DMACA probe and HCl are directly added to sol(one-pot synthesis)
Si OMeMeO
OMe
OMe
Si OMe
OMe
OMe
H2N
Nanoporous detectors of monocyclic aromatic compounds and other pollutants, S. Crunaire et al., Int. Pat., WO 2010/004225 A2.
pore size (Å) pore size (Å)
Vol
ume
of p
ores
(cm
3 /g)
Sur
face
of
pore
s (c
m2 /
g)
with DMACAwithoutDMACA
with DMACAwithout DMACA
S. Crunaire et al., HYBRID MATERIALS 2011 – 07/03/11 8
Transduction reaction in porous sensorReaction 3× faster in porous sensor than in solution-phase
[indole] µmol.L-1
reac
tion
kine
tics
(s-1)
reac
tion
kine
tics
(s-1)
[indole] µmol.L-1
Side-reactions with tryptophan derivatives: interfering molecules.
In solution-phase: DMACA reacts also with skatole and tryptophan to yield colored products.
NH2
NH
O
OHtryptophan
NH
skatole
Within nanopores: only skatole reacts.
skatole
tryptophan
S. Crunaire et al., HYBRID MATERIALS 2011 – 07/03/11 9
Example of detection in solution-phaseA large volume is inoculated at t=0t=0.
This volume is split into 4 mL fractions. Incubation at 35°C (250 rpm).
One measurement = one flask = one sensor: bacterial concentration is measured (cfu/mL).
Indole measurement: 20 µL of solution are dropped onto a pellet, then an absorbance spectrum is done every 2 secondes for 11
minutes (azafulvenium kinetics).OD (624 nm)
time
S. Crunaire et al., HYBRID MATERIALS 2011 – 07/03/11 10
Example of detection in solution-phaseExample of an indole-positive strain in LB nutrient medium (Lysogeny Broth, Lennox type): the more indole there is, the faster azafulvenium compound is formed.
Azafulvenium kinetics is proportional to [indole].
080925_E. coli 5_LB
0
0,02
0,04
0,06
0,08
0,1
0,12
0,14
0,16
0,18
0,2
0 2 4 6 8 10 12
temps (mn)
DO
624n
m
t=6h25 (1,7E9 cfu/mL)
t=4h25 (9E8 cfu/mL)
t=1h30 (3E8 cfu/mL)
Escherichia coliATCC 11775
time (min)
OD
624
nm
Formation of azafulvenium chloride
with a culture incubated for 1h30 (3×108 cfu/mL)
culture incubated for
4h25 (9×108 cfu/mL)
culture incubated for
6h25 (1,7×109 cfu/mL)
S. Crunaire et al., HYBRID MATERIALS 2011 – 07/03/11 11
Control samples: Hafnia alvei ATCC13337 (indole-negative strain) and non inoculated LB.
20 µM
Maximum [indole] is reached at the end of log-phase.
population
[indole]
S. Crunaire et al., HYBRID MATERIALS 2011 – 07/03/11 12
Example of detection in gas-phaseDetection of gaseous indole emitted by bacteria growing on agar:
1 2
2
1
33 4
4
3
1 Petri dish
2 agar nutrient medium(LB or DEV Tryptophan)
3 desiccant powder(anhydrous CaCl2)
4nanoporous sensor doped with DMACA
S. Crunaire et al., HYBRID MATERIALS 2011 – 07/03/11 13
Example of detection in gas-phaseAt t=0 : sensor is colorless, streaking of agar plates (except for non-inoculated control plates): LB agar, DEV Tryptophan agar.
E. Coli non-inoculated H. alvei
After one night at 37°C on agar plate: sensors in control plates turn red-orange, sensors in positive plates turn dark green:
Escherichia coli ATCC 11775
Hafnia alvei ATCC 13337
témoin sans bactéries
milieu tryptophane agar milieu LB agar
non-inoculated plates
Growth on DEV Tryptophan agar
Growth onLB agar
Control plates:1) non-inoculated2) with indole-negative strain
S. Crunaire et al., HYBRID MATERIALS 2011 – 07/03/11 14
Example of detection in gas-phaseStreaking at t=0 (108-109 bacteria).
Color change becomes eye-visible at t = 7-8 h.
With an initial population of 90 bacteria, color change becomes eye-visible at t = 17 h.
Application: the presence of an indole-positive strain can be checked after 1 day of incubation without opening Petri
dishes, even with low initial population.
S. Crunaire et al., HYBRID MATERIALS 2011 – 07/03/11 15
Conclusions
Sensor for indole based on a hybrid nanoporous material: sol-gel chemistry, large specific surface area, control of pore-size
Tasks on the run:
• using indole sensors in food matrices(indole +: E. coli O157:H7; indole : Salmonella typhimurium)
• sensors for other microbial VOC: ethanol, H2S, biogenic amines (such as cadaverine, putrescine).
distribution of pore diameters
chemical reaction for optical transduction(DMACA probe)
specificity of sensor
Low-cost sensors can detect volatile metabolite in liquid-phase, as well as in gas-phase.
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