Marjorie Vrignaud, Pierre R. Marcoux, Zoé Buniazet, Pierre L. Joly, Jean Hue, Isabelle Texier-NoguesCEA-LETI, Département des Technologies pour la Biologie et la Santé, 17 avenue des Martyrs, 38054 Grenoble cedex 9, France

Noninvasive bacteria detection based on the trapping of volatile metabolitesNoninvasive bacteria detection based on the trapping of volatile metabolites

Objectives: • In vitro diagnosis ; specific or nonspecific detection of bacteria

• Rapid and noninvasive detection of bacterial growth in optically unfavourable media (blood, food, …)• Low-cost and disposable optical sensor

• Detection via volatile organic compound (VOC)• Trapping of microbial VOC into a transparent nanoporous glass change of colour

Sensor made of nanoporous functionalised glass:• High surface for fast trapping of gas molecules: 600 – 1000 m2/g

• Sensor can be moulded according to many shapes

Microbial Volatile Organic Compounds (microbial VOC):Endogenous VOC:1

H2S CH3-SHNH NH2

O

CH3

These VOC do not have intrinsic optical properties a reaction with a probe moleculereaction with a probe molecule, within nanopores, is necessary. It yields a colored or fluorescent compound.

Exogenous VOCs:2

+Enzymatic substrate carrying a volatile moiety:

choice of the targeted enzymatic activitychoice of the targeted enzymatic activity: -glucuronidase (E. coli), -glucosidase (S. aureus), C-8 esterase (Salmonella),

-alanyl arylamidase (P. aeruginosa), etc. choice of the optical properties of the MVOC (no probe is no probe is

neededneeded)

Sensor containing a probe molecule Optical transduction (absorption, fluorescence)

Microbial VOC volatile moiety=

OH

NO2

OHNH2

NH2

for glycosidases for peptidases

o-nitrophenol (oNP) λmax = 415nm, ε = 3500 L.mol-1.cm-1

Nanoporous glass as an analyte concentrator Low-cost optical sensor

Noninvasive and continuous monitoring

Outline: 1 S. Crunaire et al., Procedia Chemistry, 2012, 6, 125–131; S. Crunaire et al., Chemosensors, 2014, 2, 171-181;Z. Buniazet et al., demande de brevet français n°14 60534, 2014.

2 P. Marcoux et al., Patent WO 2013001465; L.H. Guillemot et al., Phys. Chem. Chem. Phys., 2013, 15, 15840–15844

Indole (endogenous VOC) o-nitrophenol (exogenous VOC)

Sulfide endogenous VOC

E. coli (indole +) control H. alvei (indole –)NH

When this sensor is exposed to gaseous indole, a compound

absorbing at 633nm is formed.

When the sulfide sensor is exposed to gaseous H2S or CH3SH: red shift of the absorption spectrum

before incubation

after incubation (8h)

2×106 cfu/mL are detected in 5h

(C. freundii, 37C anaer.)

abs. max. at =415nmNanoporous glass is moulded following a retroreflector shape.

Retroreflector is placed into the cap:

Noninvasive and Noninvasive and continuous monitoring of continuous monitoring of the VOC concentration in the VOC concentration in

gas phasegas phase.

2 4 6 8 10 12 14 16 18

0,00

0,02

0,04

0,06

0,08

0,10

0,12

DO

à 4

15nm

Temps (heures)

Blood culture (37°C; 15mL) of E. coli

(100 cfu/mL at t=0).-glucuronidase substrate.

Detection at t=8h45.

Blood with bact.

Blood without bact.

• Monitoring through eye-reading or with a photometerdrying

Strategy:

demoulding

mould

Copyright © 2015 Pierre R. MARCOUX, pierre.marcoux@cea.fr