1. P. R. Marcoux et al., BIOSENSORS 2010 – 27/05/101 Random micro-confinement of bacteria into picolitre emulsion droplets for rapid detection and enumeration by enzymatic activity determination. bioMérieux – CEA joint team, Grenoble (France). Pierre R. Marcoux, Mathieu Dupoy, Pierre L. Joly, Florence Rivera, Sophie Le Vot, Jean-Pierre Moy. Armelle Novelli-Rousseau, Raphaël Mathey, Frédéric Mallard. BIOSENSORS 2010, Parallel Session 5D: Enzyme-based biosensors.

2. P. R. Marcoux et al., BIOSENSORS 2010 – 27/05/102 Intro: The use of enzymes in microbiology The enzymes of bacteria can be used in order: to identify micro-organisms to detect and to count micro-organisms Identification: Identification: the presence of a panel of enzymes is checked, so as to give a biochemical profile of the unknow bacteria (+--+-+--++…) api 20E Exemple of api 20E: ADH: arginine dehydrolase, LDC: lysine decarboxylase, ODC: ornithine decarboxylase, TDA: tryptophane deaminase, IND: tryptophanase, etc.

3. P. R. Marcoux et al., BIOSENSORS 2010 – 27/05/103 Intro: The use of enzymes in microbiology Detecting and counting bacteria: Detecting and counting bacteria: inside bacteria cell, enzymes transform a fluorogenous substrate into a fluorescent molecule that diffuses outside cell Most Probable Number Most Probable Number method: each well corresponds to a dilution tube and the size of the well corresponds to 1 to 3 levels of dilution: 2.25 µL; 22.5 µL; 225 µL. Number and size of positive wells (fluorescent or non-fluorescent) yield the number of microorganisms present in the initial sample (cfu/mL). The enzymes of bacteria can be used in order: to identify micro-organisms to detect and to count micro-organisms

4. P. R. Marcoux et al., BIOSENSORS 2010 – 27/05/104 Two advantages of micro-confinement: 1.The fluorophore concentration is increased  earlier detection. 2.A single cell can be detected: no need for culture and growth of population. Shall we confine ? Shall we confine ? : example of a single bacterium producing 10 6 molecules of fluorophore per minute: If the detection threshold is 1 µM of fluorophore: 10 000 h are necessary to reach the threshold if the cell (~1 fL) is confined in a volume of 1 µL 1 h100 pL 1 h to reach the threshold for a volume of 100 pL Intro: Confining bacteria into pL volumes 1 mm 1 µL 46 µm 100 pL How could we confine ? reverse emulsion 1.Water droplets in oil (fluorinated reverse emulsion). nebulisation 2.Water droplets in air on a surface (nebulisation).

5. P. R. Marcoux et al., BIOSENSORS 2010 – 27/05/105 Intro: rapid detection and enumeration of bacteria excitation of the reference fluorophore (fluorescein) 480 nm 360 nm excitation of the fluorophore produced by bacteria (4-MU) The ratio yields the number of bacteria / mL. reverse emulsion (water in oil) aqueous sample with bacteria

6. P. R. Marcoux et al., BIOSENSORS 2010 – 27/05/106 4-MU Experimental: a glucuronidase-based assay non fluorescent Fluorogeneous substrate: bacteria metabolise a non-fluorescent molecule and turns it into a fluorescent molecule enzyme activity fluorescent Biological model: Escherichia coli BL21 pUC18 DsRed Enzyme activity:  -glucuronidase Cell labelling: fluorescent protein DsRed fluorescence of DsRed,t=4 h fluorescence of DsRed,t=22 h

7. P. R. Marcoux et al., BIOSENSORS 2010 – 27/05/107 encapsulation the emulsification process must be stable (homodisperse droplets). storagereading interpretation reference fluorophore 4-MU emulsificationincubationfluo. measurement 1. Impede compositional ripening and coalescence (efficiency of confinement). 2. Avoid any movement of droplets during incubation. Measure fluorescence (fluorescein, 4-MU, DsRed) as a function of time in a maximum number of stored droplets. Experimental: a three-step process

8. P. R. Marcoux et al., BIOSENSORS 2010 – 27/05/108 FC-70: perfluorinated oil. Chemically inert, both hydrophobic and lipophobic, good solvent for gases (O 2 twenty times as soluble as in water), enough viscous, no toxicity for bacteria was observed. compositional ripening Major problem: compositional ripening = trend to equalise the composition in every droplet (the full drops fill the empty ones) DMP-PFPE: perfluorinated surfactant. Soluble in the oil phase, but not in water; good stabilisation of the water-oil interface, good elasticity of the interface. It was synthesised from Krytox® (DuPont TM ) by Virginie Héran (iSm2, University of Marseille). Experimental: emulsion formulation oil emulsifying agent fluorophilic hydrophilic fluorophilic

9. P. R. Marcoux et al., BIOSENSORS 2010 – 27/05/109 Results: droplet size, explored volume kinetics of the fluorescence for 3902 droplets average volume: 208 pL explored volume = the volume of the sample that is effectively analysed = 0.811 µL Threshold, then binarisation.

10. P. R. Marcoux et al., BIOSENSORS 2010 – 27/05/1010 2 h 5 h 9 h 11 h 17 h 6 h 10 h 14 h

11. P. R. Marcoux et al., BIOSENSORS 2010 – 27/05/1011 Results: enumeration based on enzymatic activity E. coli at 37°C in droplets, fluorescence kinetics of 4-MU (t=0 is the time when bacteria are encapsulated into droplets): 155 positive droplets were counted among all the observed droplets 1.9×10 5 cfu/mL Enumeration result: positive droplets/explored volume = 155 cfu/0,811 µL = 1.9×10 5 cfu/mL 2 nd method: based on Poisson’s law, the number of empty droplets (negative drops) is compared with the number of filled droplets (positive drops), and we assume that all the filled drops include a single bacterium at t=0. 155 filled drops for a total amount of 3902 observed drops  filling ratio = 155/3902 = 4 % 2.0×10 5 cfu/mL We deduce cV = 41×10 -3. If we assume that all the drops have the same volume V = 200 pL, then c = 2.0×10 5 cfu/mL.

12. P. R. Marcoux et al., BIOSENSORS 2010 – 27/05/1012 Results: Two kinds of control regarding enumeration 2.6×10 5 cfu/mL 1. DsRed labelling: plasmid coding for a fluorescent protein DsRed, 214 filled drops in the explored volume (811 nL)  2.6×10 5 cfu/mL 2.8×10 5 cfu/mL Poisson’s law: filling ratio = 5.5 %, it yields cV = 0.058 and c = 2.8×10 5 cfu/mL 2. Streaking on agar plates with the liquid sample of bacteria (dilution 1/100, then 0.1 mL are spread on a Petri dish) : 1,9.10 5 cfu/mL 188 cfu per plate  1,9.10 5 cfu/mL LB (lysogeny broth) chromID CPS3 Streaking on CPS3 medium is a standard method for the enumeration of E. coli.

13. P. R. Marcoux et al., BIOSENSORS 2010 – 27/05/1013 Results: Enzymatic enumeration vs. DsRed labelling 72% of encapsulated bacteria are detected in 22h à 37°C In the explored volume V = 0.811 µL: DsRed labelling yields 214 cfu but only 155 of them provided a detectable signal of 4-MU fluorescence after 22 h of incubation at 37°C  only 72% of encapsulated bacteria are detected in 22h à 37°C (this ratio is coherent with the enumeration results we got from the nebulisation device). DsRed + reference fluorophore (fluorescein) 4-MU

14. P. R. Marcoux et al., BIOSENSORS 2010 – 27/05/1014 4-MU fluorescence (normalised with respect to fluorescein) Only droplets 1 and 3 show a detectable enzymatic activity. Results: Enzymatic enumeration vs. DsRed labelling 1: filled 2: filled 4: filled 3: filled 5: filled 6: empty

15. P. R. Marcoux et al., BIOSENSORS 2010 – 27/05/1015 Results: Growth vs. enzymatic activity Fast growth and high enzymatic activity. Slow growth and no detectable enzymatic activity. Fast growth, but without any detectable enzymatic activity. Slow growth, but high enzymatic activity. empty drops

16. P. R. Marcoux et al., BIOSENSORS 2010 – 27/05/1016 Conclusions 74% More than a fast detection and enumeration method, we have a tool for the study of single cells (metabolism, growth, etc). Our enumeration results are in good agreement with the standard agar plate method. Detection: in less than 2 h. Enumeration: A plateau is reached after 10 h.