P. R. Marcoux et al., BIOSENSORS 2010 – 27/05/10 1

Random micro-confinement of bacteria into Random micro-confinement of bacteria into picolitre emulsion droplets for rapid detection and picolitre emulsion droplets for rapid detection and enumeration by enzymatic activity determination.enumeration by enzymatic activity determination.

bioMérieux – CEA joint team, Grenoble (France).bioMérieux – CEA joint team, Grenoble (France).

Pierre R. Marcoux, Mathieu Dupoy,Pierre R. Marcoux, Mathieu Dupoy,Pierre L. Joly, Florence Rivera,Pierre L. Joly, Florence Rivera,Sophie Le Vot, Jean-Pierre Moy.Sophie Le Vot, Jean-Pierre Moy.

Armelle Novelli-Rousseau,Armelle Novelli-Rousseau,Raphaël Mathey,Raphaël Mathey,Frédéric Mallard.Frédéric Mallard.

BIOSENSORS 2010, Parallel Session 5D:BIOSENSORS 2010, Parallel Session 5D:Enzyme-based biosensors.Enzyme-based biosensors.

P. R. Marcoux et al., BIOSENSORS 2010 – 27/05/10 2

Intro: The use of enzymes in microbiologyThe 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 (+--+-+--++…)

Exemple of api 20Eapi 20E: ADH: arginine dehydrolase, LDC: lysine decarboxylase, ODC: ornithine decarboxylase, TDA: tryptophane deaminase, IND: tryptophanase, etc.

P. R. Marcoux et al., BIOSENSORS 2010 – 27/05/10 3

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 NumberMost 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

P. R. Marcoux et al., BIOSENSORS 2010 – 27/05/10 4

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 106 molecules of fluorophore per minute:

If the detection threshold is 1 µM of fluorophore:

• 10 000 h are necessary to reach the threshold ifthe cell (~1 fL) is confined in a volume of 1 µL

• 1 h1 h to reach the threshold for a volume of 100 pL100 pL

Intro: Confining bacteria into pL volumes

1 mm

1 µL

46 µm

100 pL

How could we confine ?

1. Water droplets in oil (fluorinated reverse emulsionreverse emulsion).

2. Water droplets in air on a surface (nebulisationnebulisation).

P. R. Marcoux et al., BIOSENSORS 2010 – 27/05/10 5

Intro: rapid detection and enumeration of bacteria

bacterium

wateroil

1. oil is added

2. emulsification

excitation of the reference fluorophore (fluorescein)

480 nm360 nm

excitation of the fluorophore produced by bacteria (4-MU)

The ratiodrops of number total

drops tfluorescen of number

yields the number of bacteria / mL.

reverse emulsion

(water in oil)aqueous sample with

bacteria

P. R. Marcoux et al., BIOSENSORS 2010 – 27/05/10 6

O

OH

OH

OH

OH O

O O

CH3

O O

CH3

OOH

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

P. R. Marcoux et al., BIOSENSORS 2010 – 27/05/10 7

encapsulation

the emulsification process must be

stable (homodisperse droplets).

storage readinginterpretation

reference fluorophore

4-MU

emulsification incubation fluo. 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

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• FC-70: perfluorinated oil. Chemically inert, both hydrophobic and lipophobic, good solvent for gases (O2 twenty times as soluble as in water), enough viscous, no toxicity for bacteria was observed.

FF

FF FF

F

FF

FF

N

FF

FF

F

F

FF

F

FF

F F

F FF F

F

FF

F F

Major problem: compositional ripeningcompositional ripening = trend to equalise the composition in every droplet (the full drops fill the empty ones)

F3C

CF2

CF2

O

CF

CF2

O

CF3

CF3

O

O

P

O

N

N

O

O

• 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® (DuPontTM) by Virginie Héran (iSm2, University of Marseille).

Experimental: emulsion formulation

oiloil

emulsifying emulsifying agentagent

fluorophilic

hydrophilic

fluorophilic

P. R. Marcoux et al., BIOSENSORS 2010 – 27/05/10 9

Results: droplet size, explored volume

• kinetics of the fluorescencefor 3902 droplets

• average volume: 208 pL

• explored volume = the volume of the sample that is effectively analysed= 0.811 µL

Threshold, then binarisation.

P. R. Marcoux et al., BIOSENSORS 2010 – 27/05/10 10

2 h 5 h

9 h

11 h

17 h

6 h

10 h

14 h

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Results: enumeration based on enzymatic activityE. 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

Enumeration result: positive droplets/explored volume= 155 cfu/0,811 µL = 1.91.9××101055 cfu/mL cfu/mL

2nd 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.

!

)(x

ecVxP

cVx

155 filled drops for a total amount of 3902 observed drops filling ratio = 155/3902 = 4 %

21

104!1

)1(

cVcV

cVeecV

P

We deduce cV = 41×10-3. If we assume that all the drops have the same volume V = 200 pL, then c = 2.02.0××101055 cfu/mL cfu/mL.

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Results: Two kinds of control regarding enumeration

1. DsRed labelling: plasmid coding for a fluorescent protein DsRed, 214 filled drops in the explored volume (811 nL) 2.62.6××101055 cfu/mL cfu/mL

Poisson’s law: filling ratio = 5.5 %, it yieldscV = 0.058 and c = 2.8 2.8××101055 cfu/mL 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) :

188 cfu per plate 1,9.101,9.1055 cfu/mL cfu/mL

LBLB(lysogeny broth)(lysogeny broth)

chromID CPS3chromID CPS3

Streaking on CPS3 medium is a standard method for the enumeration of E. coli.

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Results: Enzymatic enumeration vs. DsRed labellingIn 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 72% of encapsulated bacteria are detected in 22h à 37°Cencapsulated 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

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0

2

4

6

8

10

12

14

2 4 6 8 10 12 14 16 18 20 22

incubation time (h)

4-M

U f

luo

resc

ence

(n

orm

alis

ed s

ign

al)

droplet1(filled)droplet2(filled)droplet3(filled)droplet4(filled)droplet5(filled)droplet6(empty)

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: filled1: filled

2: filled2: filled

4: filled4: filled

3: filled3: filled

5: filled5: filled

6: empty6: empty

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DsRed fluorescence vs. 4-MU fluorescence(normalised signals)

0

1

2

3

4

5

6

7

8

9

10

11

12

13

0 1 2 3 4 5 6 7 8

max(DsRed)

max

(4-M

U)

filled droplets

empty droplets

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

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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.