3rd International Conference on Bio-Sensing Technology 2013 Laure-Hélène Guillemot, Marjorie Vrignaud, Pierre R. Marcoux, Department of Technology for Biology and Healthcare, CEA-LETI MINATEC, 17 avenue des Martyrs, 38054 Grenoble, France. Thu-Hoa Tran-Thi. Laboratoire Francis Perrin, URA CEA-CNRS 2453, CEA Saclay/DSM/IRAMIS/SPAM, bât. 522, 91191 Gif-sur-Yvette, France. Enzymatic substrates are powerful tools for microbiology tests and they are commonly used to detect, enumerate and identify microorganisms. (1) Nowadays, a large range of synthetic enzymatic substrates are commercially available for colorimetric or fluorimetric assays. However, a major drawback is the application to either coloured or fluorescent or scattering samples: blood, exudate or food colorants for example may compromise the assay. To overcome this drawback, a new type of synthetic substrates has been proposed: their cleavage yields a volatile compound, usually detected by mass spectrometry. (2,3) By probing the volatile analyte in a non-invasive manner, the detection method becomes simple, can be easily automated, and can reduce risks of cross-contamination between different samples. In the present study, we show the possibility of replacing mass spectrometry by a simple system, composed of an analyte-trapping matrix coupled with optical detection. The proof of concept is shown here with a glucuronate moiety as an enzymatic substrate, which is cleaved by the action of beta-D-glucuronidase, a hydrolase specific to E. coli. This cleavage frees a volatile metabolite which is trapped and becomes coloured in the nanoporous matrix (Figure 1). The detection of the microbial metabolite is performed in situ, via a continuous spectroscopic monitoring of the nanoporous matrix. Some other results on a fluorescent volatile metabolite released by the action of a peptidase will also be shown. [1] S. Orenga et al., J. Microbiol. Methods 79 (2009) 139-155. [2] N.J.C. Strachan et al., Anal. Chim. Acta 313 (1995) 63-67. [3] A.P. Snyder et al., Anal. Chem. 63 (1991) 526-529.

1. 2007200720072007
NonNon--invasive detection of bacteria via the sensing of volatile metabinvasive detection of bacteria via the sensing of volatile metabolites released by enzymatic activityolites released by enzymatic activity
Laure-Hélène Guillemot,1 Marjorie Vrignaud,1 Pierre R. Marcoux,1 Thu-Hoa Tran-Thi.2
INTRODUCTION
1 Department of Technology for Biology and Healthcare, CEA1 Department of Technology for Biology and Healthcare, CEA--LETILETI
MINATEC, 17 avenue des Martyrs, 38054 Grenoble, France.MINATEC, 17 avenue des Martyrs, 38054 Grenoble, France.
22 LaboratoireLaboratoire Francis Perrin, URA CEAFrancis Perrin, URA CEA--CNRS 2453,CNRS 2453,
CEA Saclay/DSM/IRAMIS/SPAM, 1191 GifCEA Saclay/DSM/IRAMIS/SPAM, 1191 Gif--sursur--Yvette, France.Yvette, France.
OUR INNOVATIVE CONCEPT
REFERENCES
[1] Orenga, S.; James, A. L.; Manafi, M.; Perry, J. D. and Pincus, D. H.
J. Microbiol. Methods, 2009, 79, 139.
[2] Snyder, A. P.; Miller, M.; Shoff, D. B.; Eiceman, G. A.; Blyth, D. A. and
Parsons, J. A. J. Microbiol. Methods, 1991, 14, 21.
[3] Guillemot, L.-H.; Vrignaud, M.; Marcoux, P.R.; Rivron, C. and Tran-Thi, T.-H.
submitted to Phys. Chem. Chem. Phys.
[4] Dupoy, M; Guillemot, L.-H.; Marcoux, P. and Tran-Thi, T.-H. Patent
FR2971846 A1, filed December 28, 2012.
[5] Crunaire, S. and Tran-Thi T.-H., Int. Patent, WO 2010/004225 A2.
Synthetic enzymatic substratesSynthetic enzymatic substrates are powerful tools in diagnostic microbiology,1 they are widely used to
detect, enumerate and identify microorganisms. Substrates have been customised for various microbial
assays, to detect an expanding range of both new enzymatic activities and target microorganisms:
Pseudomonas aeruginosaβ-alanyl arylamidase
SalmonellaC8-esterase
Escherichia coliβ-glucuronidase
Staphylococcus aureusα-glucosidase
Microorganism to be detectedEnzyme
Different types of synthetic enzymatic substrates, depending on the mode of detectionmode of detection:
fluorogenic substrates
chromogenic substrates (soluble dye; precipitating dye)
luminogenic substrates
Table 1. Examples of
targeted enzymatic activities
and their applications.
O
OH
OH
OH
OH O
O O
CH3
O O
CH3
OOH
4-MU
Equation 1. Example of a
fluorogenic substrate of
β-glucuronidase, commonly used
in the detection and enumeration
of E. coli in food samples.dye (non-
fluorescent)
glycoside
There are major difficulties in using all these substrates in optically unfavorable media, i.e.:
media showing intrinsic fluorescence
diffusing media
strongly coloured media
such as blood, exudate, food samples (meat, etc.)
Figure 1. Examples of
samples in which
optical detection of
hydrolysed substrates
is hard or impossible.
1) Using synthetic enzymatic substrates that are designed to generate volatile metabolites. In this way,
the classical enzyme/substrate reaction is monitored by probing the product analyte in gas phase:
Equation 2. (a) Enzymatic hydrolysis of
enzymatic substrate releasing volatile
metabolite, so as to be detected in gas
phase. (b) Examples of the hydrolysis of
4-nitrophenyl-β-D-glucuronide (pNPG).
The released metabolite is volatile and
displays interesting optical properties.
This concept has been reported by Snyder et al.,2 with GC-IMS (gas chromatography coupled with ion
mobility spectrometry) as a way of detecting the released volatile metabolite. However, GC-IMS is a
costly technique, time-consuming and needs high technical assistance.
Water Solution
Xerogel
Enzymatic
substrate
Bottle Headspace
(a)
Xerogel
Bottle Headspace
enzyme
Bacterium
Dissolved metabolites
(b)
Gaseous metabolites
Metabolites
in xerogel
Metabolite vaporisation
(Henry's law)
(c)
Figure 2. (a) The specimen to be tested is mixed with an aqueous solution of the enzymatic substrate. (b) The mixture is
incubated at 37°C, viable targeted microorganisms can cleave the synthetic substrate and give rise to a volatile organic
compound (VOC) dissolved in the aqueous phase. This VOC has been chosen so as to be detected easily by optical
transduction. (c) Because of Henry's law, VOC molecules are released in gas phase where they are trapped and
accumulated into a xerogel.
2) In our innovative concept, the enzyme cleaves the substrate to yield a volatile metabolite designedvolatile metabolite designed
for a detection in gasfor a detection in gas--phasephase, via optical transductionoptical transduction: 3,4
The metabolite emitted by bacteria shows properties (Henry’s constant H, molar extinction
coefficient ε, acidic constant pKa, etc.) optimised for an efficient transfer into gas phase and for an
optical detection: Hε has to be as high as possible; [molecular form]/[ionic form] as high as possible
The volatile fraction of the emitted metabolite is trapped inside a functionalised nanoporous
xerogel showing a high specific surface (more than 500 m2/g). 5
The pore size is tailored to trap the volatile metabolite, and the pore cavities are engineered to
chemically change the metabolite and improve the detection.
The transparent xerogel allows a quantitative measurement of trapped metabolites
CONCLUSION
The concept:
a volatile metabolitevolatile metabolite with optimised
properties
a tailored xerogel as an analyteanalyte
concentratorconcentrator
an optical transductionoptical transduction within xerogel
Our concept is very versatile as many different synthetic enzymatic substrates are available today:
very specific enzymatic activitiesspecific enzymatic activities for identification or screening of a specific species or strain
(for example, screening of MRSA carriers in exudate samples)
non specific activitiesnon specific activities (i.e. commonly found in most of species) for detection (for example: blood
culture = detection of pathogen in blood samples)
A system is currently under investigation in order to propose a new type of CMBCS (ContinuousContinuous
Monitoring Blood Culture SystemMonitoring Blood Culture System).
The advantages:
nonnon--invasiveinvasive and continuous monitoring
lowlow--costcost instrumentation and sensor
versatileversatile technique (detection of phenols,
thiophenols, naphthylamine, etc.)
Hε = 2.11M-1.cm-1Hε = 0.743M-1.cm-1
εmax = 3600M-1.cm-1εmax = 18000M-1.cm-1
pKa = 7.20pKa = 7.15
H = 5.85×10-4H = 4.13×10-5
Table 2. Properties of two
volatile metabolites for
glycosidases and esterases
activities. Hε is an interesting
parameter (volatility + optical
properties).
OH NO2 OH
O2
N
p-nitrophenol
(pNP)
o-nitrophenol
(oNP)
Figure 3. Detection of p-nitrophenol (pNP) released by E. coli ATCC11775 (β-glucuronidase enzymatic activity), and
trapped in a xerogel based on TMOS functionalised with 3% APTES (3-aminopropyltriethoxysilane) (a) UV-visible
spectrum of pNP trapped (anionic form) in the xerogel exposed to E. coli culture (initial concentration: 105 cfu/mL).
(b) Kinetics of trapping of pNP, at 383 nm, in a xerogel exposed to E. coli MES buffered culture (pH=6.1). (c) Set-up.
RESULTS
The concept is explored on a simple model: E. coli (ATCC11775), with an activity specific to E. coli species.
(a) (b) (c)xerogel before adsorption
xerogel after pNP adsorption
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