2007 2007 2007 2007 Non Non - - invasive detection of bacteria via the sensing of volatile metab invasive detection of bacteria via the sensing of volatile metab olites released by enzymatic activity olites 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, CEA 1 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 Laboratoire Laboratoire Francis Perrin, URA CEA Francis Perrin, URA CEA - - CNRS 2453, CNRS 2453, CEA Saclay/DSM/IRAMIS/SPAM, 1191 Gif CEA Saclay/DSM/IRAMIS/SPAM, 1191 Gif - - sur sur - - 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 substrates Synthetic 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 aeruginosa β-alanyl arylamidase Salmonella C8-esterase Escherichia coli β-glucuronidase Staphylococcus aureus α-glucosidase Microorganism to be detected Enzyme Different types of synthetic enzymatic substrates, depending on the mode of detection mode 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 O H O O O CH 3 O O CH 3 O O H 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 designed volatile metabolite designed for a detection in gas for a detection in gas - - phase phase, via optical transduction optical 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 m 2 /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 metabolite volatile metabolite with optimised properties a tailored xerogel as an analyte analyte concentrator concentrator an optical transduction optical transduction within xerogel Our concept is very versatile as many different synthetic enzymatic substrates are available today: very specific enzymatic activities specific enzymatic activities for identification or screening of a specific species or strain (for example, screening of MRSA carriers in exudate samples) non specific activities non 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 System Monitoring Blood Culture System). The advantages: non non - - invasive invasive and continuous monitoring low low - - cost cost instrumentation and sensor versatile versatile technique (detection of phenols, thiophenols, naphthylamine, etc.) Hε = 2.11M -1 .cm -1 Hε = 0.743M -1 .cm -1 ε max = 3600M -1 .cm -1 ε max = 18000M -1 .cm -1 pKa = 7.20 pKa = 7.15 H = 5.85×10 -4 H = 4.13×10 -5 Table 2. Properties of two volatile metabolites for glycosidases and esterases activities. Hε is an interesting parameter (volatility + optical properties). O H NO 2 O H O 2 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: 10 5 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 www.leti.fr