Note: Descriptions are shown in the official language in which they were submitted.
CA 02853985 2014-06-11
MICROFLUIDIC DETECTION OF COLIFORM BACTERIA
Field of the Invention
[0001] The present invention relates to a microfluidic method and apparatus
for detecting
coliform bacteria, such as E. coli, in a sample.
Background of the Invention
[0002] Es.cherichia (E.coh) is a bacterium that is commonly found in the
lower intestine
of warm-blooded organisms [1,2]. E. coil can be found in, food products and
contaminated
drinkable water. Most E. coil are harmless, but some can cause serious food
poisoning in
humans, and are occasionally responsible for product recalls due to food
contamination [3]. The
CDC (Center for Disease Control and Prevention) estimates about 73,000 cases
of E,coli
infection occur each year in United States [3]. About 61 people die from the
illness [2]. Greatest
threat to drinkable water resources is contamination by microbial pathogens,
so water quality is
routinely assessed by testing for the presence of E. coli using pathogenic
activity.
[0003] Three main groups of detections methods for E.coll: 1) Molecular whole-
cell and
surface recognition methods [4,5]; 2) Enzyme/substrate methods [6-8]; and 3)
Nucleic acid
detection methods [9]. Antibody-antigen binding [4,5] and receptor-ligand
binding based
techniques comes under molecular whole-cell and surface recognition methods.
Capture
antibodies needs to be coated on the surface of the chips for capturing the E.
coli present in water
samples. The production of specific antibodies is time consuming and expensive
process.
Enzyme/substrate based methods are proven to be one of the best methods for
improved
specificity of bacteria detection. Fluorophore or color dye-tagged growth
substrate in. specific
broth is used in such techniques [6-8]. Upon growth specific enzymes of
bacteria cleave the
'fluoroph.ore or color compound from the substrate, causing the fluorescence
or color increase.
This type of detection takes 7 h to 24 h for detection. Buehler et al. [10]
identified specific
enzymes for E.coli. .E.coh can produce either p-glu.euronidase [7, 10] or P-
galactosidase, [11]
based on the type of E. coil strains. 'Hence, these enzymes have therefore
been considered to be a
suitable indicator for E. coif especially for the detection of fecal.
contamination of food and.
water. Nucleic acid methods, such as PCR (polymerase chain reaction) [9, 12-
15], microarrays
[16], and NASBA (Nucleic acid sequence based amplification) [17], are highly
specific and
CA 02853985 2014-06-11
sensitive techniques for detecting bacteria. Based upon known sequence
complementarity,
different specific strains of bacteria can be identified. There are .several
other lab based
conventional techniques (for example: membrane filtration, multiple tube
fermentation, etc.) to
detect E. coil in the contaminated water. All the above discussed methods are
expensive, time
consuming and not portable. In these cases, water samples need to be delivered
to the labs for
testing contaminants.
[0004] Therefore, there is a need to develop a hand-held, inexpensive and easy
to use
diagnostic system for fast and accurate results, Several groups have
implemented microfluidic
based biosensors [18-21] for rapid detection of E. coll. However, they are not
able to detect low
concentration of E. coil samples. Additionally some instruments such as
impedance analyzer,
microscopesor the like may be required for quantification.
Summary of the Invention
[0005] In the present work, we developed a new microfluidic method for
detecting E. coil in
contaminated water using "microspot with integrated wells" (MSIW). Improved
and modified
enzyme/substrate detection method is employed on the MSIW to enhance the
sensitivity,
specificity and selectivity of the detection method, In addition, amount of
reagents and time
required for the test has been reduced. MSIWs can detect low concentration of
samples since
there are no fluid movement within the MSIW. Methodology starts with
fabrication of M,SfW
and then applying the specifically formulated enzymatic method on micro-wells.
Further
measurements are conducted to quantify the color or fluorescence emitted by
the wells(MSIW).
Initially the detection protocol was optimized in microcentrifuge tubes and it
was found that
approx. 70 min was required to detect E. co/i. The optimized protocol was then
employed on
MSIWs, which reduced the detection time to 10 to 15 min It was observed that
MSIW reduces
the reaction time compared to conventional centrifuge tube based method,
likely due to the
increase in reaction surface area and decrease in volume of the samples.
[0006] In one aspect, the invention comprises a microfluidic system for
detecting coliform
bacteria in a water sample, comprising a silicon or PDMS substrate defining a
microspot having
a plurality of microwells, the microspot and each microwell coated with at
least one enzymatic
substrate which results in a detectable change caused by the action of an
enzyme, which enzyme
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CA 02853985 2014-06-11
is indicative of the presence of a coliform bacteria.
[0007] In another aspect, the invention comprises a method of detecting a
coliform bacteria in
a water sample, comprising the steps of depositing a volume of the sample in
each of a plurality
of microwells arranged in a microspot, wherein the microspot and each
microwell is coated with
at least one enzymatic substrate which results in a detectable change caused
by the action of an
enzyme, which enzyme is indicative of the presence of a coliform bacteria, and
detecting the
change or absence of the change.
[0008] In one embodiment, the coliform bacteria is E. coil.
[0009] Additional aspects and advantages of the present invention will be
apparent in view of
the description, which follows. It should be understood, however, that the
detailed description
and the specific examples, while indicating preferred embodiments of the
invention, are given by
way of illustration only, since various changes and modifications within the
spirit and scope of
the invention will become apparent to those skilled in the art from this
detailed description.
Brief Description of the Drawings
[00010] The invention will now be described by way of an exemplary embodiment
with
reference to the accompanying simplified, diagrammatic, not-to-scale drawings:
[00011] Figure 1A shows a schematic of one embodiment of a MSIW; Figure 1B
shows a
scanning electron microscope (SEM) image of the microwells inside a silicon
based MSIW;
Figure 1C shows a SEM image of the microwells inside a PDMS based MSIW.
[00012] Figure 2 shows microcentrifuge tube tests with E. coil with Red-gal
after incubating
the samples for 12 hours (a) at room temperature and (b) at 37 C; Label 1 and
l' on the tubes
indicate the Ecoli concentration of 10 cfu/ml, Labels 2 and 2' belongs to E.
coil concentration of
100 cfu/m1 and label3 and 3' related to Eco/i concentration of 1000 cfu/ml.
[00013] Figure 3 shows an optical image of the coated PDMS based MSIWs after
reacting
with E. coll.
[00014] Figure 4 shows graphs of intensity of color/fluorescent development
over incubation
3
CA 02853985 2014-06-11
time for reaction. (a) Color intensity produced by Red-gal substrate after
enzymatically reacting
with E. coil (b) Fluorescence intensity produced by MUG substrate after
enzymatically reacting
with E. coll.
[00015] Figure 5 shows (a) SEM image of dried PDMS MSIWs after complete
reaction of E.
coil with Red-gal; (b) Exploded view of dried well of MSIW
Detailed Description of Preferred Embodiments
[00016] When describing the present invention, all terms not defined herein
have their
common art-recognized meanings. To the extent that the following description
is of a specific
embodiment or a particular use of the invention, it is intended to be
illustrative only, and not
limiting of the claimed invention. The following description is intended to
cover all alternatives,
modifications and equivalents that are included in the spirit and scope of the
invention, as
defined in the appended claims.
[00017] The present invention relates to a microfiuidic system and method for
detecting E.
coil in a sample. The microfiuidic system comprises a microspot with
integrated wells. As used
herein, a "microspot" comprises an array of microwells formed in a substrate,
such as a silicon or
PDMS substrate. The microspot may be a well or depression, or may be defined
by a raised
border, In one embodiment, the microspot is a circular well, approximately 2
mm in diameter,
and the microwells are about 40 to 120 um in diameter and spaced approximately
80 to 150 pm
apart (measured from the center of each microwell), in a regular or irregular
pattern. Thus, a
microspot may feature approximately 200 to 450 microwells, as shown
schematically in Figure
1.
[00018] The microwells may be formed in a silicon or PDMS substrate using
conventional
photolithography or soft lithography methods known in the art. In general
terms, an oxide layer
is added to the substrate and then the microwell pattern added and etched.
[00019] The microwells are coated with the desired enzyme substrate using an
enzyme
substrate solution. During a positive assay, the enzyme substrate is
hydrolyzed or cleaved by a
coliform or an E.coli specific enzyme, which results in a detectable change in
colour or
fluorescence. In one embodiment, the microwells are coated with a solution
comprising 4-
4
CA 02853985 2014-06-11
Methylumbellifery1-13-D-glucuronide (4-MUG) substrate, 6-Chloro-3-indoly1-0-D-
ga1actoside
(Red-Gal) substrate in N,N-dimethylformamide (DME), ferric chloride in
deionized water and
bacteria protein extraction reagent.
[00020] E. coil produces P-D-glucuronidase, which hydrolyzes 4-MUG to 4-MU,
which
fluoresces blue color under exposure to long wavelength ultraviolet light (350-
360 nm). E. coil
(along with other coliforms) may produce I3-galactosidase, which hydrolyzes
Red-gal to produce
the development of red color, visible to the naked eye.
[00021] In use, a small volume, for example 5 pl, of the aqueous sample in
question may be
added to each microwell. Optionally, the microwell may be incubated briefly at
a slightly
elevated temperature, for example, about 37 C. Color and/or fluorescence
development may be
monitored visually or using commercially available readers. The maximum
intensity of the color
or fluorescence may be indicative of the concentration of coliform or E. coil
in the sample.
[00022] Without restriction to a theory, it is believed that increased
density of the substrates
(MUG or Red-gal) in the microwells reduces the time and concentration of E.
coil required for
detectable reactions. Due to high density of wells in MSIW, the reaction
surface area is increased
compared to other microfluidic approaches using the same sample volume. As a
result, the
microspot with integrated microwells may enhance the signal intensity even
with low
concentration samples.
[00023] Exemplary embodiments of the present invention are described in the
following
Examples, which are set forth to aid in the understanding of the invention,
and should not be
construed to limit in any way the scope of the invention as defined in the
claims which follow
thereafter.
[00024] Example 1 ¨ Methods and Materials
[00025] E.coli samples of different strains (0157, Castellani and Chalmers
(ATCC 11229),
Dh5a) were obtained from University of Alberta, Edmonton, Canada. 4-MUG
substrate (4-
Methylumbellifery1-13-D-glucuronide, trihydrate) was purchased from Bioworld,
Dublin, OH,
USA. Red-gal (6-Chloro-3-indoly1-13-D-galactoside) was purchased from Research
Organics,
Cleveland, OH, USA. Lauryl Tryptose Broth (LTB) and Bacteria protein
extraction reagent (B-
5
CA 02853985 2014-06-11
PER) were obtained from Fisher Scientific, Canada. N,N-Dimethylformamide
(DMF),
Anhydrous Fenic Chloride (FeC13)were bought from Sigma-Aldrich, USA. 100-mm-
diameter
silicon (Si) substrate was purchased from Silicon Valley Microelectronics
Inc., Santa Clara, CA,
USA. Polydimethylsiloxane (PDMS) was obtained from Dow Corning Corporation,
Midland,
MI, USA.
[00026] Fabrication of MSIP: We used silicon and polydimethylsiloxane (PDMS)
based
MSIWs in this work. The silicon MSIW were fabricated using the standard
photolithography
process. MSIW fabrication is similar to the fabrication of micropillars
reported elsewhere [22,23]
but briefly described here. A 100-mm-diameter Si substrate was taken and
cleaned with Piranha
solution. Further, a ¨ 0.52-micron thick oxide layer was thermally grown on
top of it followed by
patterning of MSIWs on silicon/silicon-dioxide substrate with standard
photolithography using
HPR504 (Fuji-film Electronic Materials Inc., Mesa, Arizona) positive
photoresist (PPR).
Subsequently oxide and silicon layers are anisotropically etched in plasma-
reactive ion etchers
(dry etching technique, DRIE). After etching the silicon for about 70 um, the
PPR on the
substrate was stripped off using acetone and the substrate was thoroughly
cleaned in Branson
PPR stripper. Then oxide layer was removed using plasma-reactive ion etchers.
PDMS based
MSIWs were fabricated using standard soft lithography process as described
elsewhere [24]. A
micro-spot of 2 mm diameter with square/staggered configuration of wells is
considered in this
work. A schematic of MSIW and SEM image of the wells inside the silicon MSIW
and PDMS
MSIW is shown in Figs. lA and 1B/1C respectively.
[00027] Fabricated MSIW were coated with specifically formulated enzyme
substrate solution
(mixture of 100 [1,1 of 1 % (w/v) 4-MUG and/or Red-Gal substrate in LTB, 10
,1 of 1 % (w/v)
ferric chloride in DI water, 100 u.1 B-PER) Mixture of above prepared solution
is dispensed into
WSW, which is kept at room temperature or 1 h for coating of the mixture to be
effective. After
coating, the MSIW were cleaned with LTB.
[00028] Enzymatic Test in MicroCentrifuge Tubes. Before the protocol for
detection of E.
coli is conducted for MSIW, the chemistry is first tested on micro-centrifuge
tubes of 1.5 ml size
(purchased from Fisher scientific, Canada). The color or fluorescence
producing conditions with
different concentrations of chemical reagents, substrates and E. coli samples
are optimized. We
6
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tried with different E.coli samples in LTB medium (0157, Castellani and
Chalmers (ATCC
11229), Dh5a) with Red-gal and 4-MUG substrates. In all experiments, E,coii
ATCC 11229
(Castellani and Chalmers) has been used for convenience and testing. Enzyme
substrate solution
was prepared as above, with a mixture of 4-MUG substrate, Red-Gal substrate,
ferric chloride
solution and B-PER, Then E. coil sample of known concentration is added and
incubated at room
temperature (or at 37 C) for development of color or fluorescence. The
presence of E. coil as red
color with Red-gal substrate is observed within 70 min of incubation. This
shows that some
strains of E.coli has P-galactosidase enzyme which cleaves the Red-gal
compound to produce red
color. With time, the intensity of the red color increased up to 12 h and then
remained constant.
The intensity of the red color produced by different concentrations of E. coil
with Red-gal
substrate solution is shown in Fig. 2.
[000291 Detection of E.coli: MSIWs coated in accordance with the example
described above
were used for detecting E. coll. Different concentrations of E. coil samples
of 1-5 tl volume were
dispensed on the coated MSIW and kept at room temperature, Glucuronidase A
(gusA) gene in
E. coil encodes P-D-Glucuronidase (GUS) to hydrolyze the substrate 4-MUG (in
the coating)
enzymatically which leads to the generation of the fluorogenic compound 4-MU.
The presence
or absence of an active P-galactosidase in E. coil was detected by Red-Gal
(substrate), which
produces a characteristic red color when cleaved by P-galactosidase enzyme,
thereby providing
an easy means of distinguishing the presence or absence of E. coil in water.
Using portable
optical color/fluorescent readers (Lateral flow reader and ESElog, Qiagen,
Germany) [25], the
average color/fluorescent intensity emitted by the MSIWs was measured. Based
on intensity, the
level of contamination can be predicted for early warnings.
[00030] The developed protocols were applied on to the MSIWs and measured the
intensity of
the developed color/fluorescence after enzymatic reaction. Color (red)
identification in silicon
based MSIWs is difficult compared to PDMS based MSIWs, since silicon material
itself emits a
color which interferes with red color produced in the reaction. Hence we used
PDMS based
MSIWs for the most of the present work. Figure 3 shows the optical image of
the PDMS based
MSIWs after treating E. coil with RedGal substrate. A light red-color was
obtained at the time of
enzymatic reaction within 5 to 10 min. It was observed that the intensity of
the color increased
over time. It was observed that MSIW reduces the reaction time compared to
conventional
7
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centrifuge tube based method, probably due to an increase in the reaction
surface area to sample
volume ratio, The average color or fluorescent intensity emitted by MSIW after
enzymatically
reacting with respective substrates was measured using portable optical
readers. Figure 4 shows
the increase in color or fluorescent intensity emitted by MSIWs with increase
in incubation time.
It is observed that intensity values increased linearly within first few
minutes and then slowly
increased (non linearly) up to a maximum value, and then remained relatively
constant . This
means that in first few min, E. coli was reacting with enzymatic substrates
and the reaction was
completed after 10 to 15 min.
[00031] A scanning electron microscope was employed to observe the coating and
reaction
zones of MSIWs. Figure 5 shows a dried PDMS MSIWs (after complete reaction
with E. coli). It
was found that coating of microwells was not uniform. Although non-uniformly
coated
microwells may provide qualitative results and rough quantitative results, it
would be preferable
to have uniformly coated MSIWs to allow for accurate measurement of intensity
values, which
may be correlated to concentration levels of the bacteria.
References
The following references are incorporated herein by reference (where
permitted) as if reproduced
in their entirety. All references are indicative of the level of skill of
those skilled in the art to
which this invention pertains.
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