Transcript Results

Random micro-confinement of bacteria into
picolitre emulsion droplets for rapid detection and
enumeration by enzymatic activity determination.
bioMérieux – CEA joint team, Grenoble (France).
Armelle Novelli-Rousseau,
Raphaël Mathey,
Frédéric Mallard.
Pierre R. Marcoux, Mathieu Dupoy,
Pierre L. Joly, Florence Rivera,
Sophie Le Vot, Jean-Pierre Moy.
BIOSENSORS 2010, Parallel Session 5D:
P. R. Marcoux et al.,
BIOSENSORS 2010
– 27/05/10
Enzyme-based
biosensors.
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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: the presence of a panel of enzymes is checked, so
as to give a biochemical profile of the unknow bacteria (+--+-+--++…)
Exemple of api 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
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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
Detecting and counting bacteria: inside bacteria cell, enzymes
transform a fluorogenous substrate into a fluorescent molecule that
diffuses outside cell
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).
P. R. Marcoux et al., BIOSENSORS 2010 – 27/05/10
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Intro: Confining bacteria into pL volumes
Shall we confine ? : example of a single bacterium producing 106
molecules of fluorophore per minute:
1 mm
If the detection threshold is 1 µM of fluorophore:
1 µL
• 10 000 h are necessary to reach the threshold if
the cell (~1 fL) is confined in a volume of 1 µL
46 µm
• 1 h to reach the threshold for a volume of 100 pL
100 pL
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.
How could we confine ?
1. Water droplets in oil (fluorinated reverse emulsion).
2. Water droplets in air on a surface (nebulisation).
P. R. Marcoux et al., BIOSENSORS 2010 – 27/05/10
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Intro: rapid detection and enumeration of bacteria
480 nm
360
aqueous sample with
bacteria
reverse
emulsion
(water in oil)
1. oil is added
2. emulsification
bacterium
water
oil
number of fluorescen t drops
total number of drops
yields the number of bacteria / mL.
The ratio
excitationof
ofthe
thefluorophore
reference
excitation
fluorophore
(fluorescein)
produced
by bacteria
(4-MU)
P. R. Marcoux et al., BIOSENSORS 2010 – 27/05/10
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Experimental: a glucuronidase-based assay
Fluorogeneous substrate: bacteria metabolise a non-fluorescent
molecule and turns it into a fluorescent molecule
CH3
fluorescent
enzyme activity
O
HO
O
O
OH
OH
CH3
O
HO
O
O
O
non fluorescent
4-MU
OH
• Biological model: Escherichia coli BL21 pUC18 DsRed
• Enzyme activity: b-glucuronidase
• Cell labelling: fluorescent protein DsRed
fluorescence of
DsRed,t=4 h
P. R. Marcoux et al., BIOSENSORS 2010 – 27/05/10
fluorescence of
DsRed,t=22 h
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Experimental: a three-step process
encapsulation
emulsification
the emulsification
process must be
stable (homodisperse
droplets).
storage
incubation
reading
interpretation
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.
4-MU
P. R. Marcoux et al., BIOSENSORS 2010 – 27/05/10
reference
fluorophore
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Experimental: emulsion formulation
Major problem: compositional ripening = trend to equalise the
composition in every droplet (the full drops fill the empty ones)
emulsifying
agent
O
hydrophilic
O
fluorophilic
CF3
P
O
CF2
F3C
CF2
N
CF2
O
CF
CF3
F
F
O
F F F
F
F
F
F
F
FF
F
F
F
F
F
O
F F
F
oil
fluorophilic
F
F
O
• 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.
• 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).
P. R. Marcoux et al., BIOSENSORS 2010 – 27/05/10
F
N
F
N
F F F F
F F F F
F
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Results: droplet size, explored volume
Threshold, then binarisation.
• 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
P. R. Marcoux et al., BIOSENSORS 2010 – 27/05/10
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2h
5h
6h
9h
10 h
11 h
14 h
17 h
P. R. Marcoux et al., BIOSENSORS 2010 – 27/05/10
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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
Enumeration result: positive droplets/explored volume
= 155 cfu/0,811 µL = 1.9×105 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

cV  e  cV
P( x) 
x!
155 filled drops for a total amount of 3902 observed
drops  filling ratio = 155/3902 = 4 %
1

cV  e  cV
P(1) 
1!
 cVe cV  4 10  2
We deduce cV = 41×10-3. If we assume that all the drops have
the same volume V = 200 pL, then c = 2.0×105 cfu/mL.
P. R. Marcoux et al., BIOSENSORS 2010 – 27/05/10
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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)  2.6×105 cfu/mL
Poisson’s law: filling ratio = 5.5 %, it yields
cV = 0.058 and c = 2.8×105 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
 1,9.105 cfu/mL
LB
(lysogeny broth)
chromID CPS3
Streaking on CPS3 medium
is a standard method for the
enumeration of E. coli.
P. R. Marcoux et al., BIOSENSORS 2010 – 27/05/10
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Results: Enzymatic enumeration vs. DsRed labelling
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).
4-MU
DsRed + reference fluorophore (fluorescein)
P. R. Marcoux et al., BIOSENSORS 2010 – 27/05/10
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Results: Enzymatic enumeration vs. DsRed labelling
4-MU fluorescence
(normalised with respect to fluorescein)
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droplet1
(filled)
droplet2
(filled)
droplet3
(filled)
droplet4
(filled)
droplet5
(filled)
droplet6
(empty)
4-MU fluorescence (normalised signal)
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1: filled
4: filled
3: filled
2: filled
6: empty
8
5: filled
Only droplets 1 and 3
show a detectable
enzymatic activity.
6
4
2
0
2
4
6
8
10
12
14
incubation time (h)
16
18
P. R. Marcoux et al., BIOSENSORS 2010 – 27/05/10
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22
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Results: Growth vs. enzymatic activity
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12
11
10
DsRed fluorescence vs. 4-MU fluorescence
(normalised signals)
Slow growth, but high
enzymatic activity.
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Fast growth and
high enzymatic
activity.
max(4-MU)
8
7
6
5
filled droplets
empty droplets
4
3
empty drops
Fast growth, but
without any detectable
enzymatic activity.
2
1
0
Slow
growth
and no 2
0
1
detectable enzymatic activity.
3
4
5
6
7
max(DsRed)
P. R. Marcoux et al., BIOSENSORS 2010 – 27/05/10
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8
Conclusions
74%
Detection: in less than 2 h.
Enumeration: A plateau is reached after 10 h.
Our enumeration results are in good agreement
with the standard agar plate method.
More than a fast detection and enumeration method, we have a
tool for the study of single cells (metabolism, growth, etc).
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