2007200720072007

NonNon --invasive detection of bacteria via the sensing of v olatile metabinvasive detection of bacteria via the sensing of v olatile metab olites released by enzymatic activityolites released by enzymatic activityLaure-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--LETI LETI MINATEC, 17 avenue des Martyrs, 38054 Grenoble, France.MINATEC, 17 avenue des Martyrs, 38054 Grenoble, France.

2 2 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

Enzymaticsubstrate

Bottle Headspace

(a)Xerogel

Bottle Headspace

enzyme

Bacterium

Dissolved metabolites

(b)

Gaseous metabolites

Metabolitesin 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 designed volatile 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

� CONCLUSIONThe concept:

� a volatile metabolitevolatile metabolite with optimised properties� a tailored xerogel as an analyte analyte 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 (Continuous Continuous 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 = 4.13×10-5Table 2. Properties of two

volatile metabolites for

glycosidases and esterases

activities. Hε is an interesting

parameter (volatility + optical

properties).

OH NO2 OH

O2N

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 adsorptionxerogel after pNP adsorption

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