Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02520812 2012-02-07
DETECTION OF SIGNALING MOLECULES IN A BIOLOGICAL
ENVIRONMENT
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FIELD OF INVENTION
[0002] This invention relates to the detection of signaling molecules in a
biological environment.
BACKGROUND OF INVENTION
[0003] Certain living species release and detect natural chemicals which
act as
signaling methods to other like neighbors. Cell to cell communication (quorum-
sensing) within bacterial populations can direct certain internal processes,
such as cell
division, sporulation, genetic transformation and virulence. Similar signaling
molecules, such as plant hormones, also control the way plants grow and
develop.
Bacteria, plant species and other micro-organisms release small signaling
molecules
into their intercellular space to communicate both with their interspecific
and
intraspecific neighbors. Certain bacteria and micro-organisms grouped in
critical
populations exhibit more information via the signaling molecules than the
individual
bacterium or micro-organism.
[0004] Traditional testing methods for bacteria are relatively expensive
and
time consuming. Most common testing methods require an environmental or
product
sample which is incubated in a separate media until enough bacteria exist to
visually
confirm their presence via culture plates or more elaborate immunoassays.
These
known methods are not real-time bacteria detection schemes. Other known
detection
methods, such as polymerase chain reaction (PCR), are faster but require a
more
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complicated and expensive procedure. Many known bioassay sensors are not
robust
enough and therefore not suited for portable applications, because they
require
specific growing media to operate. Known biosensors also have difficulty
adequately
stating a limit of detection or dynamic range. The time to prediction of
bacteria also
depends on the response time of the bacteria cell growth.
[0005] Bacteria are single celled organisms typically 0.5
to 1 micron (gm) in
diameter to 3-15 gm long (C.A. Hart, "Microterrors" Firefly Books Ltd, 2004)
and are
less mobile in their intercellular space than their small signaling molecules
categorized as autoinducers. Acoustic wave devices have been developed for the
direct detection of large bacterium, as described by Sang-Hun Lee, Desmond D.
Stubbs, John Cairney, and William D. Hunt in "Rapid Detection of Bacterial
Spores
Using a Quartz Crystal Microbalance (QCM) Immunoassay" IEEE SENSORS
JOURNAL, VOL. 5, NO. 4, AUGUST 2005. Lee et al., describe a method of instant
identification of Bacillus subtilis (nonpathogenic simulant for Bacillus
anthracis)
bacterium by constructing a dual quartz crystal microbalance (QCM)
immunosensing
system. A set of 10-MHz AT-cut QCMs operating in thickness shear mode are
employed in an enclosed flowcell. However, this method only detects the
presence of
a micro-organism, not the purpose of the micro-organism, such as cell
division,
sporulation, genetic transformation, virulence and species development.
SUMMARY OF INVENTION
[0006] The present invention provides a method of
detecting and identifying
bacteria, micro-organisms or plants in a liquid or gaseous medium, said
bacteria,
micro-organisms or plants being of the kind which produce signaling molecules
in
intercellular space, said method including positioning a biosensor in the
liquid or
gaseous medium, said biosensor having a biolayer matched to specific signaling
molecules to be detected whereby the biolayer is reactive thereto in a manner
which
varies operation of the sensor, and detecting such variation of the operation
of the
biosensor and thereby determining the presence and purpose of the bacteria,
micro-
organisms or plants in the liquid or gaseous medium. The sensor may be an
acoustic
wave biosensor.
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[0007] Signaling molecules, characterized as autoinducers, diffuse more
readily within the surrounding environment compared to the actual bacterium.
The
present invention is well suited for SAW (surface acoustic wave) geometries
which
are typically in the sub-micron range and can also function as RFID sensors
which
can be interrogated by a wireless system. SAW detectors can be small, simple
in
nature and provide microbial differentiation detection results in typically 10
seconds
or less.
[0008] Thus, the present invention also provides acoustic wave based
sensors
coated with specific bioreceptor molecules which can detect small signaling
molecules from an originating species in real-time and quantify the acoustic
wave
sensor data due to the linear relationship between the mass of the signaling
molecule
and the velocity of the acoustic wave and therefore identify both the presence
and the
purpose of the originating species.
[0009] Such biosensors can provide a medium for detecting harmful
biological agents without coming into direct contact with the bacteria
themselves. In
addition, acoustic wave biosensor techniques permit quantification through the
direct
relationship between the concentrations of small signaling molecules in
intercellular
space to the relative amount of signaling source present. This technology can
be used
for the real time detection of Bacillus related species, such as anthracis,
subtilis,
cereus, globigii and other species, such as Streptococcus pneumoniae,
Staphylococcus
aureus and Enteroccoctis faecalis. This technology can also be further used to
detect
sources of signaling chemicals, such as ethylene which communicates and
promotes
the premature ripening of foodstuffs within the agricultural industry.
[0010] The present invention is well suited for monitoring certain
environments which require the detection of various species of bacteria,
including but
not limited to airborne microorganisms, such as Bacillus subtilis, B. cereus,
B.
anthracis and plant hormones such as ethylene. This invention can also be used
for
the detection of fungii microbes, including mold forming organisms, and for
the
detection of both vegetative and spore forms of microbes. Also, the
agricultural
industry can benefit from such monitoring of signaling molecules. The present
invention can detect the release of certain gaseous plant hormones, such as
ethylene,
which control the way plants grow and develop. Ethylene is a gaseous plant
hormone
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which plays an important role in plant growth and development. Both fruit and
flowers may ripen or blossom prematurely when exposed to ethylene. Ethylene is
perceived by the agricultural industry as detrimental to product quality, and
significant efforts are made to minimize its effects. The present invention
also
permits the detection and identification of signaling molecules of certain
pests and
weeds to enable the application of pesticides and herbicides to be controlled.
[0011] Advances in industrial microbiology are also major factors for
innovation and progress in the food industry. Products such as yogurt, cheese,
chocolate, butter, pickles, sauerkraut, soy sauce, food supplements (vitamins
and
amino acids), food thickeners (produced from microbial polysaccharides),
alcohol
(beer, whiskeys, wines) and silage for animals are all products of microbial
activity.
The maintenance of the fermentation process which is commonly used in industry
becomes a high priority as the necessity for monitoring species population,
growth
and contamination becomes more stringently controlled. The present invention
is
thus useful in this field also.
[0012] The present invention is also useful in the bioterrorism field.
Bioterrorism applications of the invention include detecting harmful
biological agents
on the battle ground, in public places, including individual packages or
enclosures, or
in heating ventilation and air conditioning (HVAC) systems of buildings.
[0013] The present invention also has usefulness in the real-time clinical
detection of bacteria in various media, including blood and exhaled breath.
For
example, Bacteremia is an infection caused by the presence of bacteria in the
blood.
Such infection can cause damage of the heart valves, the lining of the heart
and the
lining of blood vessels. Early detection of elevated bacteria population in
blood may
mediate these symptoms. Certain pathogenic bacteria contain /uxS proteins
which
produce the autoinducer-2 (AI-2) signaling chemicals. Such signaling chemicals
assist in the co-ordinated gene expression depending upon the population
density of
the bacteria.
[0014] Another clinical example is the Neisseria meningitidis bacteria,
which
colonizes within the human nasopharynx and is the cause of meningitis. K.
Winzer et
al, show evidence in "Role of Neisseria meningitidid luxS in Cell-to-Cell
Signaling
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and Bacteremic Infection," Infection and Immunity, Vol. 70, No. 4, pp.2245-
2248
April 2002, that N. meningitidis possesses a functional luxS protein which is
necessary for AI-2 production and full meningoccal virulence. Further
detection of
signaling molecules within the nasopharynx can lead to the detection of the
Neisseria
meningitides bacteria. Technology incorporating the present invention can also
be
utilized in hospitals, schools, office buildings, transportation centers and
any other
environments to aid in determining signaling chemical concentrations.
Signaling
molecule detection systems can be implemented with a small portable device or
with
fixed devices. In addition, the present invention can be used for the
determination of
signaling chemicals in restaurants and other food handling facilities to
monitor the
amount of bacterial organisms around work stations.
[0015] This invention could also be used as a feedback element within a
control system for the detection and remediation of certain biological
entities. Such a
control system would identify a biological entity and then apply appropriate
corrective measures, such as pesticides, herbicides or other autoinducer
degrading
enzymes which would minimize the biological entity. For example, in the
technique
described by Jones et al. "Inhibition of Bacillus anthracis Growth and
Virulence-Gene
Expression by Inhibitors of Quorum-Sensing," Journal of Infectious Diseases,
June
2005, the use of antibiotics could be replaced by the detection and use of
signaling
molecule inhibitors in accordance with the present invention.
[0016] An analogy of this invention would be a microphone (biosensor)
placed outside an open door of a room containing an unknown amount of people
(bacteria or plants) from various different countries (various inter- and
intraspecies).
The microphone does not come into contact with the people but rather listens
to the
chatter (autoinducers or biological hormones) generated by the people. Using
an
algorithm attached to the microphone, it is then possible to calculate the
estimated
number of different dialects from various countries that the people originated
from,
the number of people associated with each specific dialect and the purpose or
intent of
each conversation.
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DESCRIPTION OF THE DRAWINGS
[0017] Embodiments of the invention will now be described, by way of
example, with reference to the accompanying drawings, of which:
[0018] FIG.1 is a diagrammatic view showing a gram-negative bacteria cell,
species RhII (P.aeruginosa), producing AHL autoinducers, namely N-(2-0xo-
tetrahydro-furan-3-y1)-butyramide.
[0019] FIG.2 is a similar view showing a gram-negative bacteria call,
species
AinS (Vfischerii), producing AHL autoinducers, namely octanoic acid (2-oxo-
tetrahydro-furan-3-y1)-aimide.
[0020] FIG.3 is a similar view showing a gram-negative bacteria cell,
species
ExpI (E.carotorova), producing AHL autoinducers, namely 3-0xo-hexanoic acid.
[0021] FIG.4 is a similar view showing a gram-negative bacteria cell,
species
CviI (Chromobacterium Violaceum), producing AHL autoinducers, namely hexanoic
acid (2-oxo-tetrahydro-furan-3-y1)-amide.
[0022] FIG.5 is a diagrammatic view of a quorum-sensing system depicting
Bacillus anthracis and their AHL autoinducers diffusing a few metres away, and
[0023] FIG.6 is a similar view of a SAW device having proteins/antibodies
with certain autoinducers binding, a bacterium being placed beside the SAW
device to
enable dimensions to be compared.
DESCRIPTION OF PREFERRED EMBODIMENTS
[0024] Bacteria are for the most part free-living and, when provided with
habitable environments, they can reproduce by the process of binary fission
such that
one bacterial cell can produce up to 10 million descendants within 18 to 2
hours. The
physical shape of bacteria may be rod shaped (bacilli), spherical (cocci), or
spiral
(spirochaetes). Also, bacilli can be either straight or bent, and cocci can be
arranged
in pairs (diplococci), in clusters (staphylocobcci) or in long chains
(streptococci). The
design of a selective detection system dependent on the physical shape of
specific
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bacteria would be an enormous challenge. However, within the framework of
quorum
sensing, it has been reported by S.A. Voloshin & A.S. Kaprelyants, "REVIEW:
Cell-
Cell Interactions in Bacterial Populations," Biochemistry (Moscow), Vol 69,
No. 11,
2004, pp. 1268-1275, that it is possible to use certain detection proteins
which can
selectively distinguish between the chemical characteristics of the signal
molecules
(autoinducers) used for cell-cell interactions in bacterial populations.
Detecting such
chemical signal molecules is a precursor to the whereabouts of specific
bacteria.
[0025] Numerous pathogenic bacteria contain certain proteins, which are
required for the production of autoinducers as outlined by Michiko Taga and
Bonnie
Ba4er, "Proc. Nat. Acad. Sci.", vol. 100, pp.14549-14554, November 2003. The
nature of autoinducers generated by certain proteins within bacteria is
dependent on
whether the bacteria cell is gram-positive or gram-negative. Gram-negative
cells
generate low molecular weight signaling molecules such as N-acyl homoserine
lactones (AHLs). Gram-positive cells secrete more complex but still relatively
small
oligopeptides or proteins. FIG.1 depicts a typical Gram-negative LuxIR circuit
100
consisting of a bacteria cell, species RhII (P.aeruginosa) 110 producing
within the
LuxI proteins 120 AHL autoinducers 130 of composition N-(2-0xo-tetrahydro-
furan-
3-y1)-butyramide 135. The AHL autoinducers are received by the LuxR protein
140
and affect the virulence enzyme production and bio film formation of the
species RhII
(P.aeruginosa) 110.
[0026] Analogous AHL autoinducers share a common homoserine lactone
moiety and typically differ only in their acyl side chain moieties. FIG.2
depicts
another typical Gram-negative LuxIR circuit 200 consisting of a bacteria cell,
species
AinS (Vfischerii) 210, producing within the LuxI proteins 220 AHL autoinducers
230
of composition Octanoic acid (2-oxo-tetrahydro-furan-3-y1)-aiinide 235. The
AHL
autoinducers are received by the LuxR protein 240 and affect the
bioluminescence of
the species AinS (V.fischerii) 210. FIG.3 depicts another typical Gram-
negative
LuxIR circuit 300 consisting of a bacteria cell, species ExpI (E.carotorova)
310,
producing within the LuxI proteins 320 AHL autoinducers 330 of composition 3-
0xo-
hexanoic acid 335. The AHL autoinducers are received by the LuxR protein 340
and
affect the synthesis of carbapenom (an antibiotic) of the species ExpI
(E.carotorova)
310. FIG.4 depicts another typical Gram-negative LuxIR circuit 400 consisting
of a
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bacteria cell, species CviI (Chromobacterium Violaceum) 410, producing within
the
LuxI proteins 420 AHL autoinducers 430 of composition hexanoic acid (2-oxo-
tetrahydro-furan-3-y1)-amide 435. The AHL autoinducers are received by the
LuxR
protein 440 and affect the generation of a deep violet pigment named violacein
of the
species CviI (Chromobacterium Violaceum) 410.
[0027] For the gram-positive bacterial species Bacillus anthracis, a
signaling
molecule autoinducer-2 (AI-2) is synthesized via a LuxS-type protein. The LuxS
protein converts S-ribosylhomocysteine to 4,5-dihydroxy1-2,3-pentanedione,
catalysing the formation of the AI-2.
[0028] The species RhII (P.aeruginosa) 110, AinS (V.fischerii) 210, ExpI
(E.carotorova) 310, CviI (Chromobacterium Violaceum) 410 and similar other
species produce a variety of signaling molecules which perform diverse
functions
such as cell division, sporulation, genetic transformation, virulence and
species
development. Detection of a specific autoinducer/signalling molecule from the
same
micro-organism or plant depends on the specificity of the bioreceptor layer of
the
detection system.
[0029] The small signaling molecules produced by bacteria have a very low
vapor pressure and can only be detected by using very sensitive sensors. An
example
of how quorum sensing can be used for the detection and purpose of harmful
biological agents without coming into physical contact with the bacterium
themselves
in accordance with the invention is shown in FIG.5. Within a quorum-sensing
detection system 500, a subject species 510 which may for example contain
Bacillus
anthracis is positioned on a surface 520 within an environment 530 such as a
room,
office cubicle, warehouse or any military/bioterrorism scenario where harmful
biological threats may be present.
[0030] Signaling chemicals 540 such as autoinducers AI-2 specific to
Bacillus
anthracis would be present in the intercellular vapor space surrounding the
subject
species 510 within the environment 530. An acoustic wave biosensor 550 is
positioned at a safe distance from the subject species 510 to sample the
surrounding
vapor for the signaling chemicals 540 within the environment 530 and perform a
real-
time evaluation within the algorithm of the acoustic wave biosensor. The
acoustic
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wave biosensor 550 shown in FIG.5 may have the form factor of a self contained
wireless handheld type of unit or a remote fixed monitoring station with wired
or
wireless capability. The acoustic wave biosensor may be an RFID type sensor as
described by Edmonson et al. in "A surface acoustic wave sensor or
identification
device with biosensing capability", U.S. Patent Application No.11/139477 filed
May
31, 2005.
[0031] A further advantage of using an acoustic wave biosensor is to
capitalize on the promiscuous nature of the bioreceptors so that detection of
multiple
analogs of signaling chemicals is possible. An example of such detection using
a
multiple acoustic wave detector is described in Edmonson et al.
"Differentiation and
identification of analogous chemical or biological substances with
biosensors", U.S.
Patent Application 11/088809 filed March 25, 2005,
[0032] The scenario seen in FIG.5 can be repeated for numerous other
examples. In other words, other instances in which pathogenic bacteria with
Gram-
negative or Gram-positive cells generate autoinducers can be applied to the
scenario
seen in FIG.5. For agricultural examples, the plant hormone ethylene can be
detected
to control premature ripening of certain fruits within warehouses. For
background
information in this respect, see P.J. Davies, "Plant Hormones, Biosynthesis,
Signal
Transduction, Action." Kluwer Academic Publishers, Dordrecht, The Netherlands,
2004. The commonality is that the acoustic wave biosensor 550 must have its
biolayer matched to the quorum-sensing chemical signaling molecules, so that
the
specific bioreceptor molecules on the biosensor are receptive to the analogous
signaling chemical. Further examples for the detection of molds and fungi can
also be
applied to the scenario shown in FIG.5. It should also be noted that the
intercellular
space in which the autoinducers/signaling chemicals are present may be a
liquid or a
gaseous medium with the detection taking place within the liquid or gaseous
medium.
[0033] A major advantage of detecting autoinducers within an intercellular
space is that both the presence and purpose of bacteria rather than only the
actual
bacteria themselves can be identified. This is especially essential for
harmful
bacteria. Further, it permits the use of detectors based on thin-film
fabrication with
sub-micron geometries. Acoustic wave biosensors and other similar
nanotechnology
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devices can be made with very small-scale dimensions and with sub-circuit
structures
smaller than the actual bacteria. Such devices are 3-dimensional in nature
with a
typical biolayer of the acoustic wave biosensor being composed of a cross-link
layer
and antibody layer overlaid onto the acoustic wave structure. Similarly,
nanotechnology devices are also 3-dimensional in nature since beams deflect
depending upon the selective attachment of specific substances.
[0034] FIG.6 shows
a diagrammatic view illustrating the importance of scale
to such detection devices. For this embodiment, an acoustic wave biosensor 600
is
constructed on a piezoelectric material 605. The typical width of an
interdigital
transducer (IDT), finger electrode 610 and space 612 at a frequency of 2.44
GHz is
0.33 pm. Several of the finger electrode widths 610 and adjoining spaces 612
are
shown and may constitute a portion of the IDT or a part of a multi-fingered
reflector
array. A 3-dimensional biolayer 620 is located above certain selected fmger
electrodes 610 and spaces 612. The biolayer 620 has three main components,
namely
a bioreceptor molecule, Antibody A 623, a heterobifimctional molecule, Protein
A
625 and an organic agarose hydrogel 627. The actual binding of a selected
autoinducer with a specific bioreceptor occurs within a rigid thin layer of a
specific
bioreceptor molecule. Antibody A 623 may be, but is not limited to, an
antibody,
enzyme, lipid or protein. A thin layer of a specific bioreceptor molecule,
Antibody A
623, is attached to the piezoelectric material 605 or the acoustic wave finger
electrodes 610 via a heterobifunctional molecule, Protein A 625, such as but
not
limited to a protein A, alkanethiol. A thin sheath of an organic agarose
hydrogel 627
is applied to the device to provide a semi-aqueous environment important for
maintaining the three dimensional structure of the receptor molecule.
[0035] Above the
biolayer 620 are various bacteria autoinducers of species A
630 in vapor form which will bind to an equivalent matched bioreceptor
antibody A
623. A bacterium 640 secretes the autoinducer A 630 and is placed above this
portion
of acoustic wave device 600 to illustrate the scale and difficulty of using
this
technique to directly detect the bacteria. As the bacteria autoinducers of
species A
630 emitted by Bacterium species A 640 bind within the biolayer 620,
parameters
within the piezoelectric material 605 are altered and therefore change the RF
characteristics of the Interdigital Transducer. The IDT fmger electrodes 610
are
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electrically connected to a mechanism to detect this change in RF
characteristics. The
specificity of the bioreceptor molecule, antibody A 623 is chosen so that the
autoinducers of species B 650 are orthogonal or semi-orthogonal with the
bioreceptor
molecule, antibody A 623.
[0036] This example of an acoustic wave biosensor as shown in FIG.6
illustrates the expediency of detecting signaling molecules such as
autoinducers 630
rather than the large Bacterium species 640, such as a Bacillus Subtilis spore
which
measures lum in width at the cross section of the oval shaped organism. The
weight
of the Bacillus Subtilis spore has been estimated to be approximately 1 pico-
gram,
which would dampen out any acoustic wave when the organism made contact with
either the biolayer 620 or the piezoelectric material 605.
[0037] The advantages of the present invention will now be readily
apparent
to a person skilled in the art from the foregoing description of preferred
embodiments.
Other embodiments of the invention will also now be readily apparent, the
scope of
the invention being defined in the appended claims.
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