Pathogen Sensor
The present invention relates to a pathogen sensor.
Pathogens are agents that cause infection or disease, especially microorganisms such as
bacteria, protozoan, viruses and fungi.
Phytopathology or plant pathology relates to the diagnosis and management of plant
diseases caused by infection agents or diseases that attack plants and environmental
conditions. Organisms that cause diseases in plants include for example: fungi (including
molds and yeasts), viruses, oomycetes, bacteria, viroids, phytoplasmas, protozoa,
nematodes and parasitic plants.
In farming it is conventional to monitor the health of a crop through visual inspection of the
crop. Growth of a pathogen on a crop may be identified via this visual inspection,
whereupon a suitable agent such as a fungicide may be applied to the crop. In addition to
visual inspection of the crop, a farmer may take into account environmental conditions such
as the weather (including predicted future environmental conditions). Although this
approach may work in some instances it is desirable to provide an apparatus which is
capable of indicating that a pathogen is growing or is likely to be growing in a crop.
According to a first aspect of the invention there is provided a pathogen sensor comprising
a growth medium upon which and/or within which a pathogen may grow, the growth
medium being provided with nutrients which facilitate growth of the pathogen, wherein the
pathogen sensor further comprises an electronic detection apparatus configured to detect
an event mediated by the pathogen.
The event mediated by the pathogen may be the production of a chemical or biological
agent. The chemical or biological agent may be one of the following: an organic acid, a
nucleic acid, a protein, an enzyme, a toxin, a hormone, a metabolite, a peptide, a
carbohydrate or a lipid.
The chemical agent to be detected may be oxalic acid. Oxalic acid is an organic
compound with the formula H2C20 4. This colourless solid is a dicarboxylic acid and is about
3,000 times stronger than acetic acid. Oxalic acid is a reducing agent and its conjugatebase, known as oxalate (C20 2 ) , is a chelating agent for metal cations. Typically oxalic acid
occurs as the dihydrate with the formula C20 H2-2H20 .
Oxalic acid and derivatives thereof such as oxalates are present in many plants.
Consequently, oxalic acid, and salts or derivatives thereof is a suitable candidate for
detection in a pathogen sensor of the present invention.
The electronic detection apparatus may be configured to detect an electrochemical change
in the growth medium.
The electronic detection apparatus may comprise an enzyme that interacts with the
chemical or biological agent, the interaction leading to an electronically detectable signal.
The interaction may generate an electroactive species or lead to the generation of an
electroactive species. The electronic detection apparatus may further comprise an
electrode configured to detect the presence of the electroactive species.
The electrode may have been modified by a biochemical and/or chemical recognition
element. This may for example include incorporating an enzyme, antibody, DNA or
chemical species into the electrode which may enhance or change the electrochemical
response of the electrode.
The enzyme may be located in a biocompatible polymer. The biocompatible polymer may
be a hydrophilic polymer, or may be formed from hydrophilic monomers. The enzyme may
be immobilised on a surface of the electrode. The enzyme may be immobilised in a
biocompatible polymer. The enzyme may be oxalate oxidase. The pathogen sensor may
further comprise horseradish peroxidase.
Horseradish peroxidase is a 44,173.9-dalton glycoprotein with four lysine residues for
conjugation to for example a labeled molecule. It produces a coloured, fluorimetric, or
luminescent derivative of the labeled molecule when incubated with a proper substrate,
allowing it to be detected and quantified.
The pathogen sensor may further comprise a nutrient reservoir which is configured to
provide a supply of nutrients to the growth medium. The nutrient reservoir may be
configured to supply nutrients to the growth medium for a period which is longer than 10
hours.
The growth medium may be a nutrient liquid.The pathogen sensor may further comprise a fluid reservoir which is configured to provide a
supply of fluid to the growth medium to prevent dehydration of the growth medium. The
fluid reservoir may be configured to supply fluid to the growth medium for a period which is
longer than 10 hours. The nutrient reservoir and the fluid reservoir may be the same
reservoir.
The growth medium may have one or more properties which mimic an entity upon which
and/or within which the pathogen will grow. The one or more properties may include at
least one of the following: lighting of the growth medium, humidity or moisture conditions at
the growth medium, pH conditions at the growth medium, the orientation of the growth
medium, and the temperature of the growth medium.
The entity may be a plant.
The growth medium may be provided with one or more fungicides, antibiotics or
antimicrobials which do not prevent growth of the pathogen.
The pathogen may be a fungal pathogen. The pathogen may be Sclerotinia sclerotiorum.
Sclerotinia sclerotiorum is a plant pathogenic fungus that can cause a disease called white
mold if conditions are correct. S. sclerotiorum can also be known as cottony rot, watery soft
rot, stem rot, drop, crown rot and blossom blight. A key characteristic of this pathogen is its
ability to produce black resting structures known as sclerotia and white fuzzy growths of
mycelium on the plant it infects. These sclerotia give rise to a fruiting body in the spring that
produces spores in a sac, which is why fungi in this class are called sac fungi
(Ascomycetes). This pathogen can occur on many continents and has a wide host range of
plants. When S. sclerotiorum is onset in the field by favorable environmental conditions,
losses can be great.
Sclerotinia sclerotiorum proliferates in moist environments. Under moist field conditions, S.
sclerotiorum is capable of completely invading a plant host, colonizing nearly all of the
plant's tissues with mycelium. Optimal temperatures for growth range from 15 to 2 1
degrees Celsius. Under wet conditions, S. sclerotiorum will produce an abundance of
mycelium and sclerotia.
The pathogen may be a bacterial pathogen. The pathogen may be from the Burkholderia
genus.According to a second aspect of the invention there is provided a sensor apparatus which
comprises the pathogen sensor according to the first aspect of the invention and which
further comprises measurement electronics configured to receive a signal from the
electronic detection apparatus and to generate an output if the signal indicates that an event
mediated by the pathogen has occurred. The sensor apparatus may include any of the
above features of the pathogen sensor.
The pathogen sensor may be releasably engageable with the sensor apparatus such that
the pathogen sensor may be replaced with another pathogen sensor. The pathogen sensor
may be one of a plurality of pathogen sensors provided in a cartridge which is releasably
engageable with the sensor apparatus.
According to a third aspect of the invention there is provided a method of detecting a
pathogen comprising providing nutrients which facilitate growth of the pathogen on and/or in
a growth medium for a period which is sufficiently long to allow an event mediated by the
pathogen to occur, then using an electronic detection apparatus to detect the mediated
event. The growth environment may be a favourable growth environment. The favourable
growth environment may be an environment which facilitates growth of the pathogen at a
rate which is faster than the rate at which the pathogen will grow on a plant or other entity
adjacent to which the pathogen sensor is provided.
The event mediated by the pathogen may be the production of a chemical or biological
agent. The chemical or biological agent may be one of the following: an organic acid, a
nucleic acid, a protein, an enzyme, a toxin, a hormone, a metabolite, a peptide, a
carbohydrate or a lipid. The chemical agent may be oxalic acid.
The electronic detection apparatus may detect an electrochemical change in the growth
medium.
The electronic detection apparatus may comprise an enzyme which interacts with the
chemical or biological agent, the interaction leading to an electronically detectable signal.
The interaction may lead to the generation of an electroactive species. The method may
further comprise detecting the presence of the electroactive species using an electrode.
The enzyme may be oxalate oxidase which catalyses the production of hydrogen peroxide
from the oxalic acid. The pathogen sensor may further comprise horseradish peroxidase
which reduces the hydrogen peroxide.Detecting the presence of the electroactive species using the electrode may comprise
applying a first potential and a second different potential to the electrode and measuring the
resulting current.
The method may further comprise supplying nutrients to the growth medium for a period
which is longer than 10 hours. The method may further comprise supplying fluid to the
growth medium for a period which is longer than 10 hours.
The growth medium may have one or more properties which mimic an entity upon which
and/or within which the pathogen will grow. The one or more properties may include at
least one of the following: lighting of the growth medium, humidity or moisture conditions at
the growth medium, pH conditions at the growth medium, the orientation of the growth
medium, and the temperature of the growth medium.
The pathogen may be a fungal pathogen. The pathogen may be Sclerotinia Sclerotiorum.
The pathogen may be a bacterial pathogen. The pathogen may be from the Burkholderia
genus.
The method may comprise exposing the growth medium to the air and monitoring for the
mediated event and then subsequently exposing a second growth medium to the air and
monitoring for the mediated event.
A method of detecting the presence of a pathogen in the environment comprising exposing
to air the pathogen sensor of any preceding paragraph and monitoring for the mediated
event.
The pathogen sensor may be provided in a crop or adjacent to a crop, such that the method
provides an indication of whether a pathogen is growing in the crop or is likely to be growing
in the crop. The pathogen sensor may be provided in a storage area in which a crop is
stored after the crop has been harvested (e.g. a warehouse or barn).
The pathogen sensor may be one of a plurality of pathogen sensors distributed over an
area. The method may comprise analysing outputs from the pathogen sensors to obtain
information regarding the progress of the pathogen through the area.Analysis of information provided from the pathogen sensor may be combined with analysis
of information provided from one or more sensors which sense one or more of: temperature,
humidity, wind direction, wind speed, pressure sensor and ambient light.
According to a fourth aspect of the invention there is provided a pathogen sensor
comprising a growth medium upon which and/or within which a pathogen may grow, the
growth medium comprising nutrients which facilitate growth of the pathogen, wherein the
pathogen sensor further comprises an electronic detection apparatus configured to detect
an electrochemical change mediated by the pathogen.
The electrochemical change may be caused by a chemical or biological agent produced by
the pathogen.
The growth medium may be a liquid media which contains potato dextrose broth. The
growth medium may be potato dextrose agar.
The pathogen may be from the Sclerotinia species. The pathogen may be Sclerotinia
Sclerotiorum.
According to a fifth aspect of the invention there is provided a sensor apparatus which
comprises the pathogen sensor of any preceding aspect of the invention, and further
comprises measurement electronics configured to receive a signal from the electronic
detection apparatus and to generate an output if the signal is indicative of an
electrochemical change mediated by the pathogen.
The sensor apparatus may further comprise a control apparatus which is configured to
expose the pathogen sensor to the air, incubate the pathogen sensor for a predetermined
period of time, and then use the electronic detection apparatus to monitor for the
electrochemical change.
The sensor apparatus may further comprise a puncturing apparatus configured to puncture
a barrier which separates the growth medium from the electrode.
According to a sixth aspect of the invention method of detecting a pathogen comprising
providing nutrients which facilitate growth of the pathogen on and/or in a growth medium for
a period which is sufficiently long to allow a pathogen to mediate an electrochemical
change, then using an electronic detection apparatus to detect the electrochemical change.The electrochemical change may be caused by a chemical or biological agent produced by
the pathogen.
According to a seventh aspect of the invention there is provided a sensor apparatus which
comprises the pathogen sensor of any preceding claim and further comprises measurement
electronics configured to receive a signal from the electronic detection apparatus and to
generate an output if the signal is indicative of an electrochemical change mediated by the
pathogen.
The sensor apparatus may further comprise a control apparatus which is configured to
expose the pathogen sensor to the air, incubate the pathogen sensor for a predetermined
period of time, and then use the electronic detection apparatus to monitor for the
electrochemical change.
The sensor apparatus may further comprise a puncturing apparatus configured to puncture
a barrier which separates the growth medium from the electrode.
According to an eighth aspect of the invention there is provided use of a pathogen sensor
according to any preceding aspect or a sensor apparatus according to any preceding
aspect for detecting an electrochemical change in crops arising from the presence of one or
more of: fungi (including molds and yeasts), viruses, oomycetes, bacteria, viroids,
phytoplasmas, protozoa, nematodes and parasitic plants on the crop.
According to a ninth aspect of the invention there is provided use of a pathogen sensor as
described in relation to any of preceding aspect or a sensor apparatus according to any
preceding aspect in the treatment of wheat and barley.
Features of different aspects of the invention may be combined with one another.
Specific embodiments of the invention will now be described by way of example only, with
reference to the accompanying figures in which:
Figure 1 shows schematically in cross-section a pathogen sensor according to an
embodiment of the invention;
Figure 2 shows schematically in cross-section a pathogen sensor according to an
alternative embodiment of the invention;Figure 3 is a graph which demonstrates that oxalic acid may be detected using a pathogen
sensor according to an embodiment of the invention;
Figure 4 is a graph which demonstrates that oxalic acid may be detected using a pathogen
sensor according to an embodiment of the invention, including particular growth media;
Figure 5 shows schematically in cross-section a pathogen sensor according to a further
alternative embodiment of the invention;
Figure 6 shows schematically in cross-section a pathogen sensor according to a further
alternative embodiment of the invention;
Figure 7 shows schematically in cross-section a pathogen sensor according to a further
alternative embodiment of the invention;
Figure 8 shows schematically in cross-section a pathogen sensor according to a further
alternative embodiment of the invention;
Figure 9 shows schematically in cross-section a pathogen sensor according to a further
alternative embodiment of the invention;
Figure 10 shows schematically a sensor apparatus according to an embodiment of the
invention; and
Figure 11 shows schematically an alternative sensor apparatus according to an
embodiment of the invention.
Figure 1 shows schematically in cross-section a pathogen sensor 1 according to an
embodiment of the invention. The pathogen sensor 1 comprises a support structure 2 , a
nutrient reservoir 4 , an electrode 6 and a gel 8 . The nutrient reservoir 4 is annular, and
extends around a central portion of the support structure 2 . The support structure may for
example be formed from plastic or some other suitable material. The gel 8 is provided on
top of the electrode 6 and has an upper surface which is exposed to the atmosphere. The
electrode 6 is supported on a substrate (not shown). A cylindrical channel 10 extends
downwardly from the electrode 6 and may accommodate a wire or wires (not shown) which
are connected to the electrode. Additional electrodes such as a reference electrode and a
counter electrode (not shown) may be provided. A one-way membrane 12 is provided
around an outer wall of the cylindrical channel 10, thereby forming an inner wall of the
nutrient reservoir 4 . The one-way membrane 12 is configured such that water based
nutrients may pass through it from the nutrient reservoir 4 and may then travel to the gel 8 .
The one-way membrane 12 does not allow the water based nutrients to flow from the gel 8
into the liquid nutrient reservoir 4 . An upper surface of liquid nutrient reservoir 4 is covered
by an annular gas permeable sealing layer 13. The gas permeable sealing layer 13 allows
gas (e.g. air) to pass into the nutrient reservoir 4 and thereby prevents a pressure dropoccurring when water based nutrients leave the nutrient reservoir. In addition, the gas
permeable sealing layer 13 allows oxygen to be absorbed into the water based nutrients.
This is desirable because oxygen is one of the components of an electrochemical reaction
which will take place in the pathogen sensor when a pathogen is present (as is described
further below).
The gel 8 may be a non-water based gel which is configured to adhere to the surface of the
electrode 6 . The gel 8 may be considered to be an example of a growth medium upon
which and/or within which a pathogen may grow. The gel 8 may for example be potato
dextrose agar (PDA). The gel 8 absorbs water based nutrients through the one-way
membrane 12 via osmotic pressure. The osmotic pressure is generated by evaporation of
liquid from the gel 8 . The membrane 12 may deliver the water based nutrients to the gel 8
via a wicking action. The membrane 12 may for example be a polyethylene material which
is sulphonated on one side to make it hydrophilic and which is naturally hydrophobic on the
other side (similar to a membrane used in a diaper). Alternatively, functional groups other
than sulphonates may be applied to one side of the polyethylene material to ensure one
side of the material is hydrophilic. The functional groups may be for example, but are not
limited to, hydroxyl, carboxyl, amino, phosphate and sulfhydryl groups. The water based
nutrients may for example comprise potato dextrose broth (PDB), a sunflower derived
nutrient or some other nutrient.
The one-way membrane 12 provides a supply of water based nutrients to the gel 8 until the
nutrient reservoir 4 is empty. Providing a supply of nutrients to the gel 8 is advantageous
because it replaces nutrients as they are used by a pathogen growing on the pathogen
sensor. A further advantage of providing the supply of water based-nutrients is that this
ensures that the gel 8 remains hydrated. If the gel 8 were to dry out then growth of a
pathogen on the gel could be inhibited. In addition, the ability of the pathogen sensor 1 to
detect the presence of a pathogen could be compromised if the gel 8 were to dry out.
The pathogen sensor 1 may be provided with a seal (not shown) on its upper surface which
acts to prevent the gel 8 (and optionally the nutrient reservoir 4) being exposed to air until
operation of the pathogen sensor is desired, the seal being removed in order to initiate
operation of the pathogen sensor. This prevents evaporation of water from the gel 8
occurring before operation of the pathogen sensor is desired and hence the drying out of
the gel.The gel 8 may for example be 500-1000 microns thick and may for example have a
diameter of 3mm. The electrode 6 may for example have a thickness of 100 microns and
may for example have a diameter of 2mm. The nutrient reservoir 4 may for example be 1-
2mm deep and may for example have a diameter of 10mm. These dimensions are given
merely as examples, and the gel, electrode and nutrient reservoir may have other
dimensions.
An oxalate oxidase enzyme may be provided on the electrode 6 or in the vicinity of the
electrode.
In enzymology, an oxalate oxidase is an enzyme that catalyzes the chemical reaction of
oxalate to carbon dioxide and hydrogen peroxide as illustrated below.
oxalate + 0 2 + 2 H+ ¾ C0 2 + H20 2
The substrates of this enzyme are therefore oxalate (derived from oxalic acid), oxygen (0 2) ,
and hydrogen ions (H+) , whereas the two products are C0 2 and H20 2.
Oxalate oxidases belong to the family of oxidoreductases, specifically those enzymes acting
on an aldehyde or oxo group of a donor with oxygen as an acceptor. The systematic name
of this enzyme class is oxalate:oxygen oxidoreductase. However, other common names
include for example aero-oxalo dehydrogenase, and oxalic acid oxidase. This enzyme
participates in glyoxylate and dicarboxylate metabolism.
The oxalate oxidase is provided in such a manner that it retains its activity and stability. As
explained below, oxalate oxidase enzymes will catalyse the generation of hydrogen
peroxide when oxalic acid/oxalate and oxygen are present at the oxalate oxidase. The
presence of the hydrogen peroxide may be detected via the electrode 6 . The detected
hydrogen peroxide may indicate that a pathogen has grown on the gel 8 and has released
oxalic acid (some plant pathogens release oxalic acid when they grow). Thus, the oxalate
oxidase may be considered to form part of an electronic detection apparatus which detects
the oxalic acid. The electrode may also be considered to form part of the electronic
detection apparatus.
The pathogen sensor 1 may be provided at a location where it is desired to monitor for the
presence of a pathogen. The seal may be removed from the pathogen sensor, thereby
exposing the gel 8 to the atmosphere. Removing the seal also exposes the water based
nutrients in the nutrient reservoir 4 to the atmosphere via the gas permeable sealing layer
13. Water based nutrients are drawn by the gel 8 through the one-way membrane 12,thereby ensuring that the gel remains supplied with water based nutrients and remains
hydrated. This facilitates growth of a pathogen which may arrive at the sensor and then
germinate and grow. The pathogen may grow for a period of time on or in the gel using the
water based nutrients provided from the nutrient reservoir 4 . The pathogen may then
release oxalic acid, the catalytic breakdown of the oxalic acid being detected by the
electrode 6 as is explained further below. The release of oxalic acid and the subsequent
catalytic breakdown of the oxalic acid may be considered to be an event which is mediated
by the pathogen.
It may take a considerable period of time (e.g. 10 hours to 2 days, 4 days or more) for the
pathogen to grow sufficiently that it may mediate the event (e.g. the release and catalytic
breakdown of oxalic acid). It is desirable that the pathogen sensor 1 is capable of operating
for a period of time which is longer than the period required for the pathogen to grow and
mediate the event. The pathogen sensor may for example be capable of operating for 10
hours, 24 hours, 2 days, 3 days, 4 days or more. The pathogen sensor may thus for
example be capable of providing a supply of nutrients to the gel 8 for 10 hours, 24 hours, 2
days, 3 days, 4 days or more, and may be capable of keeping the gel 8 hydrated for 10
hours, 24 hours, 2 days, 3 days, 4 days or more.
When the mediated event takes place it is detected by the electrode 6 as is explained
further below. This indicates that the pathogen is present and is growing. When the
presence of the pathogen has been detected, measurement electronics connected to the
pathogen sensor may provide an output indicating the presence of the pathogen. This for
example allows a farmer to take appropriate measures to protect from the pathogen crops
which are located in the vicinity of the pathogen sensor.
The pathogen sensor 1 may for example be configured to detect Sclerotinia Sclerotiorum.
Where this is the case the pathogen sensor provides a growth medium (the gel 8) upon
and/or within which S. sclerotiorum may grow, and provides nutrients which nourish the S.
sclerotiorum over a period of time which is sufficient to allow the S. sclerotiorum to grow to
an extent that it will produce oxalic acid. In addition, the nutrients may facilitate the
production of oxalic acid by the S. sclerotiorum. The nutrients may facilitate growth of S.
sclerotiorum via metabolic pathways which provide more oxalic acid production than
alternative metabolic pathways (the alternative metabolic pathways producing less oxalic
acid). Selective fungicides, antibiotics or antimicrobials may be incorporated in the
pathogen sensor to inhibit the growth of other microorganisms which may inhibit S.sclerotiorum growth and/or produce oxalic acid or some other interferent electroactive
species.
The pathogen sensor may detect S. sclerotiorum by detecting oxalic acid released by the S.
sclerotiorum. Detection of oxalic acid may be used in the pathogen sensor to detect the
presence of other fungal pathogens which produce oxalic acid. Examples of such fungal
pathogens include: Ascomycetes, and may include Aspergillus fonsecaeus, Aspergillus
niger, Botrytis cinerea, Cryphonectria parasitica, Saccharomyces cerevisiae,
Saccharomyces hansenii, Penicillium bilaii, Penicillium oxalicum, Sclerotium cepivorum,
Sclerotium delphinii, Sclerotium glucanicum, Sclerotium rolfsii, Sclerotinia sclerotiorum,
Sclerotinia trifoliorum. Examples also include Deuteromycetes, and may include
Cristulariella pyramidalis, Leucostoma cincta and Leucostoma persoonii. Examples also
include Basidiomycetes, and may include Rhizoctonia solani, Postia placenta, Fomitopsis
palustris and Wolfiporia cocos. Examples also include other wood rotting fungal species
that secrete oxalic acid.
Measurement electronics (not shown) are configured to apply a potential at the electrode 6
which is stepped between a first value at which no electroactive reactions occur and a
second value at which an electroactive reaction occurs when hydrogen peroxide is present
at the electrode. The change of potential from the first value to the second value and back
again may for example be applied intermittently. The detection methodology used by the
electronic detection apparatus may be referred to as chronoamperometry, and may be
considered to be an example of electrochemical detection. The hydrogen peroxide is
generated as a result of the breakdown of oxalic acid released by the pathogen (e.g. S.
sclerotiorum ) , the generation of the hydrogen peroxide taking place in the presence of
oxygen and the oxalate oxidase provided at the electrode 6 . The potential change at the
electrode 6 caused by the hydrogen peroxide results in a characteristic charging and decay
current which is proportional (e.g. directly proportional) to the concentration of the hydrogen
peroxide at the electrode.
The second value of the potential applied to the electrode 6 (i.e. the value at which the
electroactive reaction occurs) may be chosen for optimal electron transfer to the hydrogen
peroxide, thereby maximising the current caused by the hydrogen peroxide. Similarly, the
time period during which the second potential value is applied to the electrode may be
chosen to facilitate detection of the hydrogen peroxide. An explanation of this detection
methodology may be found in Electroanalysis by C.M.A. Brett and A.M. Oliveira Brett, 1998,
which is herein incorporated by reference.An alternative embodiment of the invention is shown schematically in cross-section in figure
2 . In the embodiment shown in figure 2 , a working electrode 6 and a reference electrode 16
are provided, the reference electrode being separated from the working electrode. The
working electrode 6 may for example have a surface area of 3mm2 and the reference
electrode 16 may for example have a surface area of 0.5mm2. The working electrode 6 and
reference electrode 16 are provided on a substrate 14. The substrate 14 may for example
be 50mm long and 10mm wide. Wires 18 extend from the working electrode 6 and the
reference electrode 16, the wires passing through openings in the substrate 14 to
measurement electronics (not shown). A nutrient liquid 8 is provided over the electrodes 6 ,
16. The nutrient liquid 8 is held in place by walls (not shown), with an upper surface of the
nutrient liquid being exposed to the atmosphere. The nutrient liquid 8 is an example of a
growth medium.
An oxalate oxidase 20 is attached to the working electrode 6 . The oxalate oxidase was
generated in a purified form by taking the oxalate oxidase gene from barley (Hordeum
vulgare) and expressing it in a Pichia (a type of yeast) expression system. In more detail,
the method used to obtain the purified oxalate oxidase is as follows: the mature Hordeum
vulgare (Barley) oxalate oxidase open reading frame (GenBank reference no. 289356) was
codon-optimised for expression in Pichia pastoris and synthesised as an Xhol/NotI fragment
designed to create an in-frame fusion with the yeast a-mating factor when cloned into the
vector pPICZaA (Invitrogen). The assembled oxalate oxidase extracellular expression
vector was used to transform competent P. pastoris according to published protocols by
Whittaker MM and Whittaker JW, Journal of Biological Inorganic Chemistry, 2002 Jan;7(1-
2): 136-45 (herein incorporated by reference). A large scale (5 litres) high density X33 (a
strain of Pichia pastoris) fermentation was carried out as described in the same paper.
120mg of protein was purified from the supernantant broth using cation exchange
chromatography and size exclusion chromatography, which exhibited enzymatic activity in a
colorimetric assay. Oxalate oxidase protein identification was confirmed by peptide mass
fingerprinting (MALDI-TOF) and whole mass spectroscopy using Q-ToF.
The oxalate oxidase was stored as a lyophilised powder, and was prepared as a 1mg/ml
aqueous solution in a 2X buffer and a 2X stabiliser solution. The buffer was 100mM
succinic acid, 200 mM KCI, pH 3.8. Q20901 1D10, which is available from Applied Enzyme
Technology of Pontypool, United Kingdom, may be used as the stabiliser solution. Other
suitable buffers and stabilisers (e.g. sugars and polyelectrolytes) may be used.The oxalate oxidase solution was pipetted onto the working electrode 6 (e.g. 10µ Ι of oxalate
oxidase solution; other quantities of solution may be used). The solution was then allowed
to dry completely (e.g. drying for several hours). This dried version of the oxalate oxidase is
stable at room temperature for many weeks. The nutrient liquid 8 was subsequently
provided on top of the working electrode 6 . When this was done the oxalate oxidase
rehydrated and became active again but stayed on the surface of the working electrode 6
(the oxalate oxidase was adsorbed to the working electrode). Rehydration of the oxalate
oxidase was necessary in order to allow the oxalate oxidase to catalyse the generation of
hydrogen peroxide when oxalic acid/oxalate and oxygen are present.
An alternative oxalate oxidase which comprises a partially purified form of oxalate oxidase
derived from barley seedlings may be used. However, this form of oxalate oxidase has
been found to provide a less strong response to the presence of oxalic acid than the purified
oxalate oxidase. The partially purified oxalate oxidase is available as product 04127 from
Sigma-Aldrich of St Louis, USA.
Instead of using simple adsorption to attach the oxalate oxidase to the working electrode,
coupling chemistry may be used. The coupling chemistry may for example use
glutaraldehyde. Experiments have shown that the glutaraldehyde allows the oxalate
oxidase to remain active. However, adsorption may provide better retention of oxalate
oxidase on the electrode than glutaraldehyde.
In general, a number of different methods may be used to attach an enzyme (e.g. oxlate
oxidase) to an electrode or to keep the enzyme adjacent to the electrode. For example,
surface adsorption, with or without stabilisers, may be used. Physical entrapment, wherein
the enzyme is kept in the vicinity of the electrode surface by attaching a permeable
membrane over the top of the electrode, may be used. The membrane may be cellulose
acetate, collagen, polycarbonate or general purpose dialysis tubing. Polymer entrapment,
wherein a polymer is deposited electrochemically on the surface, may be used, the enzyme
being entrapped in the polymer or subsequently covalently or electrostatically attached to
the polymer. Covalent binding, for example gold-thiol bonds formed between enzyme
cystein residues and a gold electrode, may be used. Immobilisation via lysine residues, for
example using carbodiimide or N-hydroxysuccinimide mediated coupling, may be used.
The working electrode 6 may be formed from carbon paste and the reference electrode 16
may be formed from a 60:40 combination of silver and silver chloride paste. The reference
electrode 16 provides a stable reference equilibrium potential which may be used as astable reference point against which the potential at the working electrode 6 may be
measured. The reference electrode may partially encircle the working electrode. The
pathogen sensor 1 may have an electrode configuration which includes a counter electrode
(e.g. formed from carbon paste) in addition to the reference electrode. The sensor may for
example comprise sensor BE2050824D1 which is available from Gwent Electronic Materials
Ltd of Pontypool, United Kingdom.
The carbon paste of the working electrode 6 includes Prussian blue (ferric
hexacyanoferrate) which acts as a mediator (the oxidised form of Prussian blue being used
to pre-oxidise the working electrode 6). The oxidised form of Prussian blue catalyses the
reduction of hydrogen peroxide at the working electrode 6 (it acts as an artificial peroxidise)
and allows detection of hydrogen peroxide at significantly lower potentials than would be the
case in the absence of a mediator (e.g. it allows detection at less than 0.6 volts). Applying a
lower potential to the working electrode in this manner is advantageous because it reduces
the detection of other electroactive species, thereby increasing the accuracy with which
hydrogen peroxide is detected.
The nutrient liquid 8 may for example contain potato dextrose broth. The nutrient liquid may
for example be obtained by mixing 1% of potato dextrose broth with a minimal salt solution
(i.e. a solution containing inorganic salts). Other concentrations of potato dextrose broth
may be used. The minimal salt solution, which may also be referred to as minimal media,
may for example be a recipe in the literature and made up as: 1000mg/L
(NH4)2S04; 500mg/L K2HP04; 500mg/L KH2P04; 450mg/L NaCI; 250mg/L
MgS04.7H20; 5mg/L Na-NTA; 0.5mg/L FeCI3.6H20; 0.5mg/L CuS04.5H20; 0.5mg/L
ZnCI2; 0.5mg/L MnS04.H20; 0.5mg/L Na2Mo04.2H20 and pH adjusted to pH 5 using 1M
HCI). The minimal salt solution may alternatively be M9 minimal salts, available from BD of
New Jersey, USA. Other minimal salt solution may be used.
It is known from the published literature that potato dextrose based nutrients promote the
growth of S. sclerotiorum and the production of oxalic acid by S. sclerotiorum. Published
papers which mention growth of S. sclerotiorum and the production of oxalic acid in potato
dextrose based nutrients include:
"Mycelial growth and production of oxalic acid by virulent and hypovirulent isolates of
Sclerotinia sclerotiorum"; T Zhou and G J Boland; Can. J . Plant. Pathol. 21: 93-99 (1999);
Oxalic acid production and its role in pathogenesis of Sclerotinia sclerotiorum"; P
Magro, P Marciano and P Di Lenna; FEMS Microbiology Letters 24 (1984) 9-12;Oxalic Acid, a Pathogenicity Factor for Sclerotinia sclerotiorum, Suppresses the
Oxidative Burst of the Host Plant"; S G Cessna, V E Sears, M B Dickman and P S Low; The
Plant Cell, Vol. 12, 2191-2199, November 2000;
5 Nutrient liquid containing potato dextrose broth has been found to be effective in promoting
growth of S. sclerotiorum and promoting production of oxalic acid by S. sclerotiorum. For
example, growth of S. sclerotiorum and production of oxalic acid by S. sclerotiorum has
been seen in a nutrient liquid containing 2.4 % potato dextrose broth.
10 When the pathogen sensor is in use, the nutrient liquid 8 provides nutrients which allow S.
sclerotiorum to grow in the nutrient liquid. Nutrients used by the S. sclerotiorum over time
may be replaced from a nutrient reservoir (not shown), for example in the manner described
further above in connection with figure 1. After growing in the nutrient liquid 8 for a period of
time, the S. sclerotiorum produces oxalic acid. The catalytic activity of the oxalate oxidase
15 20 with the oxalic acid generated by the S. sclerotiorum (and with oxygen) causes the
generation of hydrogen peroxide at the working electrode 6 along with carbon dioxide. As
described above, the presence of the hydrogen peroxide at the working electrode 6 is
detected by applying a potential to the working electrode and then measuring a current
generated by reduction of the hydrogen peroxide at the working electrode. The reduction of
20 hydrogen peroxide at the working electrode is catalysed by the Prussian blue in the
electrode.
The potential applied to the working electrode 6 is stepped between a first value at which no
electroactive reduction of the hydrogen peroxide occurs and a second value at which
25 electroactive reduction of the hydrogen peroxide occurs. The potential step may for
example be applied intermittently. The potential may for example be stepped between 0
volts and around 0.6 volts (or lower). The value of the potential applied to the working
electrode 6 may be measured relative to the reference electrode 16. The change of
potential at the working electrode 6 causes a characteristic charging and decay current
30 which is proportional (e.g. directly proportional) to the concentration of the hydrogen
peroxide at the electrode surface. The resulting current is monitored by measurement
electronics (not shown) which identify the presence of oxalic acid based on the monitored
current, and which thereby identify the presence of S. sclerotiorum in the nutrient liquid 8 .
35 An experiment has been performed using the sensor described above (without potato
dextrose broth) to confirm that the sensor electrochemistry is capable of detecting the
presence of oxalic acid. The working electrode 6 and the reference electrode 16 werecovered with 100µ Ι of electrolyte (e.g. 50 mM succinic acid 100 mM KCI pH 3.8 buffer).
Oxalic acid was then added to the electrolyte such that the concentration of the oxalic acid
increased gradually. The electrochemical measurement was carried out by applying a
potential of -0.1 V to the working electrode (measured relative to the reference electrode)
for 50 seconds and measuring the resulting current. The current after 40 seconds was
recorded and plotted in a graph as a function of oxalic acid concentration. The results are
shown in Figure 3 , both for the purified form of oxalate oxidase and the partially purified
form of oxalate oxidase. In Figure 3 squares indicate data obtained using the purified form
of oxalate oxidase, and diamonds indicate data obtained using the partially purified form of
oxalate oxidase. As may be seen from Figure 3 , for both types of oxalate oxidase the size
of the measured current increases significantly as the concentration of oxalic acid is
increased. The slope of the graph is downwards because the current is a negative current
(the magnitude of the current increases). As may be seen from Figure 3 , purified oxalate
oxidase provided a stronger response than partially purified oxalate oxidase. These results
confirm that the pathogen sensor described above may be used to detect oxalic acid.
Experiments have also been performed using the sensor described above, with various
different liquid nutrient media being provided over the electrodes 6 , 16 (the nutrient media
are listed below). The liquid nutrient media were prepared as a 1% w/v solution in minimal
media pH 5 (the minimal media is from a recipe in the literature and made up as: 1000mg/L
(NH4)2S04; 500mg/L K2HP04; 500mg/L KH2P04; 450mg/L NaCI; 250mg/L
MgS04.7H20; 5mg/L Na-NTA; 0.5mg/L FeCI3.6H20; 0.5mg/L CuS04.5H20; 0.5mg/L
ZnCI2; 0.5mg/L MnS04.H20; 0.5mg/L Na2Mo04.2H20 and pH adjusted to pH 5 using 1M
HCI). 25mM glucose was also added to promote Sclerotinia growth. The pH was further
adjusted to 3.8 before the experiment was performed. This was done because it is
expected that the pH of the nutrient medium will drop after fungal growth and oxalic acid
production by S. sclerotiorum. Furthermore, 3.8 may be the optimum pH for activity of the
oxalate oxidase. In addition, the electrochemistry used by the pathogen sensor is more
effective at more acidic pH than at less acidic pH.
For each liquid nutrient, increasing amounts of oxalic acid were added to the liquid nutrient
such that the concentration of the oxalic acid increased gradually. The electrochemical
measurement was carried out by applying a potential of -0.1 V to the working electrode
(measured relative to the reference electrode) for 50 seconds and measuring the resulting
current. The current after 40 seconds was recorded and plotted in a graph as a function of
oxalic acid concentration. Results from the experiment are shown in Figure 4 , which is agraph which shows the detected current as a function of oxalic acid concentration for a
variety of different liquid media. The media are labelled in Figure 4 as follows:
E45 - 50mM succinic acid 100mM KCI pH 3.8
E57 - 1% potato dextrose broth minimal media pH 3.8
E58 - 1% Yeast nitrogen base without amino acid minimal media pH 3.8
E59 - 1% YPD broth in minimal media pH 3.8
E60 - 1% sabouraud dextrose liquid medium in minimal media pH 3.8
E43- 1% soytone in minimal media pH 3.8
E61 - 1% czapek dox liquid medium in minimal media pH 3.8
E62 - 1% yeast tryptone broth in minimal media pH 3.8
E63 - 1% LB Lennox broth in minimal media pH 3.8
E64 - 1% yeast extract in minimal media pH 3.8
E65 - 1% mycological peptone in minimal media pH 3.8
E66 - 1% tryptone soya broth in minimal media pH 3.8
E67 -1% beef extract in minimal media pH 3.8
E68 1% granulated tryptone in minimal media pH 3.8
As may be seen from Figure 4 , some nutrient media provide a significantly increased
current as the concentration of oxalic acid increases. These are: 1% potato dextrose broth
minimal media pH 3.8, 1% sabouraud dextrose liquid medium in minimal media pH 3.8, 1%
Yeast nitrogen base without amino acid minimal media pH 3.8, and 1% czapek dox liquid
medium in minimal media pH 3.8. 50mM succinic acid 100mM KCI pH 3.8 and 1% YPD
broth in minimal media pH 3.8 also provide an increased current as the concentration of
oxalic acid increases, but the increase is significantly less.
As noted further above, it is known from the published literature that potato dextrose based
nutrients promote the growth of S. sclerotiorum and the production of oxalic acid by S.
sclerotiorum. Since potato dextrose broth provides growth of S. sclerotiorum and oxalic
acid production, and provides a strong current increase as oxalic acid concentration
increases, potato dextrose broth may be used in the pathogen sensor to detect S.
sclerotiorum. Potato dextrose broth is preferred over potato dextrose agar because the
detection of oxalic acid in a liquid medium is significantly easier than detection of oxalic acid
in a solid medium such as a gel.It has been found via experimentation that Czapek dox does not promote growth of S.
sclerotiorum and oxalic acid production by S. sclerotiorum. Czapek dox should therefore
not be used in the pathogen sensor when monitoring for S. sclerotiorum.
Sabouraud dextrose liquid medium is expected to promote growth of S. sclerotiorum and
oxalic acid production by S. sclerotiorum.
Other media provide little or no increased current as the concentration of oxalic acid
increases, because they interfere with the electrochemistry of oxalic acid detection.
Carbohydrate based media (such as potato dextrose based media) may give rise to little or
no interference with the electrochemistry of oxalic acid detection. However, soytone based
media inhibit oxalate oxidase on the electrode, therefore interfering with the enzyme
mediated electrochemical detection.
Alternative embodiments of the invention are shown in Figures 5-9. In figure 5 the working
electrode 26 comprises carbon paste without a mediator. Some features of the
embodiment shown in figure 5 correspond with those of the embodiment shown in figure 2
and are provided with the same reference numerals. This embodiment of the invention
may require a higher voltage to be applied in order to detect the presence of hydrogen
peroxide (compared with the case when a mediator such as Prussian blue is present in the
electrode). A potential drawback of the embodiment shown in figure 5 is that in addition to
hydrogen peroxide, reduction reactions may also generate other electroactive species in the
liquid 8 . These other electroactive species may modify the current measured from the
working electrode 6 and this may give rise to erroneous results.
Some fouling of the electrode may occur. In this context fouling may refer to proteins and
other chemical species being non-specifically adsorbed at the working electrode 26.
Adsorbed proteins or other chemical species may form a layer on the working electrode 26
which inhibits diffusion of electrons or ions at the electrode, thereby limiting the reduction of
the hydrogen peroxide (and thereby limiting the current generated as a result of the oxalic
acid produced by the S. sclerotiorum). One way in which fouling may be minimised or
avoided is by keeping the liquid away from the electrode until a measurement is to be
performed (as described further below in relation to Figure 10).
It may be possible to prevent interfering species from reaching the working electrode 6
using pre-oxidation (e.g. with metal oxides), thereby improving the accuracy with which the
hydrogen peroxide concentration is measured. An oxidant may for example be provided asnanoparticles which are interspersed on the electrode surface with the oxalate oxidase 20,
or may for example be provided as a layer which lies over the oxalate oxidase, or may for
example be provided in a multilayer stack which alternates between the oxidant and the
oxalate oxidase. The oxidant catalyses the oxidation of interfering electroactive species into
chemically inert forms before they reach the electrode 6 . This prevents or reduces the
detection of interfering species at the electrode 6 .
In an alternative embodiment, an ion selective membrane may be provided above the
oxalate oxidase, the ion selective membrane active to prevent or restrict interfering species
from reaching and reacting with the oxalate oxidase. Figure 6 shows this schematically in
cross-section. Some features of the embodiment shown in figure 6 correspond with those
of the embodiment shown in figure 5 and are provided with the same reference numerals.
A membrane or gel layer 11 is provided over the liquid growth media 9 . The membrane or
gel layer 11 (and optionally the liquid growth media 9) may be considered to be a growth
medium upon which and/or within which a pathogen may grow. An ion selective membrane
22 is provided in the liquid growth media 9 . The ion selective membrane 22 prevents or
restricts interfering species from reaching and reacting with the oxalate oxidase 20 but
allows oxalic acid to reach and react with the oxalate oxidase.
Although some illustrated embodiments of the invention do not include a membrane or gel
layer over the liquid growth media, a membrane or gel layer may be provided in connection
with any embodiment. The membrane or gel layer may for example provide a surface upon
which and/or within which the S. sclerotiorum (or other pathogen) may grow. However, a
membrane or gel layer is not needed; the S. sclerotiorum (or other pathogen) may grow in a
liquid nutrient without a membrane or gel layer.
Although illustrated embodiments of the invention comprise a liquid growth media, a gel
growth media may be used instead of the liquid. The gel may be kept hydrated using a
reservoir of fluid. For example, the gel may be kept hydrated using a reservoir of water
based nutrients as described further above in relation to figure 1. Keeping the gel hydrated
avoids the possibility that the growth of S. sclerotiorum on the gel is inhibited by the gel
being dry. In addition, it facilitates detection of oxalic acid produced by the S. sclerotiorum.
If the gel is not hydrated then oxalic acid produced by the S. sclerotiorum may not diffuse
freely to the oxalate oxidase. In addition, dehydration of the gel could destabilise or
denature the oxalate oxidase. Dehydration could also prevent the flow of electrons and ions
between the working electrode and the reference electrode, thereby restricting
electrochemical detection of the hydrogen peroxide.Figure 7 shows a further alternative embodiment of the invention in cross-section. In this
embodiment the oxalate oxidase 20 is immobilised in a biocompatible polymer 28. Other
features of this embodiment correspond with those shown in figure 5 and are provided with
the same reference numerals. The biocompatible nature of the polymer allows the oxalate
oxidase 20 to be retained in the vicinity of the working electrode 26 in its active form. The
biocompatible polymer 28 may for example be a conducting polymer such as polyaniline,
mucin/chitosan (mucin - a high molecular weight, heavily glycosylated protein
(glycoconjugate)/chitosan- a linear polysaccharide composed of randomly distributed β-(1-
4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit)),
mucin/carbapol®, (Carbopol
® is polymers commonly used as thickeners, suspending
agents and stabilizers available from Lubrizol limited) or any other suitable polymer. The
polymer may also be a hydrogel such as polymethylmethacrylate. The biocompatible
polymer 28 and immobilised oxalate oxidase 20 may be provided as a polymer film (e.g. a
thick polymer film) on the working electrode 26.
The biocompatible polymer 28 may help to confer stability to the oxalate oxidase 20. In
addition, it may block the electrode 6 from fouling by unwanted electroactive species. This
is because the biocompatible polymer 28 provides a steric barrier which prevents proteins
and oxidising species from being able to approach the surface of the working electrode 6 .
Prevention of fouling using the biocompatible polymer may be particularly beneficial
because the pathogen sensor 1 may be operated over a considerable period of time (e.g.
10 hours or more, 24 hours or more, 2 days or more, or 4 days or more), during which time
an accumulation of proteins and oxidising species at the working electrode 6 could lead to a
significant loss of sensitivity at the working electrode (and could also lead to interfering
background signals).
As mentioned above, the biocompatible polymer 28 may be a hydrogel such as a
methyacrylate based polymer. The methacrylate containing biocompatible polymer may be
formed by providing a thick film of polyglycerol monomethacrylate (PGMMA) on the working
electrode 6 , then polymerising and reacting the PGMMA with the oxalate oxidase through
NHS-EDC coupling chemistry (e.g. as described in Bioconjugate Techniques by G.T.
Hermanson (1996)). This provides a thick biocompatible polymer. The thickness of the
PGMMA may be controlled by selecting an appropriate thickness for the pre-polymerised
film.A further alternative embodiment is shown in figure 8 . The embodiment shown in figure 8
corresponds with that shown in figure 7 , except that the working electrode 6 comprises a
mediated carbon electrode (mediation being provided for example by Prussian blue). The
mediated carbon working electrode 6 inhibits or restricts the detection of electroactive
species other than hydrogen peroxide, as explained above in relation to figure 2 . Other
features of this embodiment correspond with those shown in previously described figures
and are provided with the same reference numerals.
A further alternative embodiment of the invention is shown in figure 9 . The embodiment
shown in figure 9 corresponds with that shown in figure 7 , except that the biocompatible
polymer 28 is provided with horseradish peroxidase 30 in addition to oxalate oxidase 20 (it
is a bienzyme system). Other features of this embodiment correspond with those shown in
figure 5 and are provided with the same reference numerals. The horseradish peroxidase
30 is a secondary enzyme which catalyses the reduction of hydrogen peroxide and
therefore allows detection of the presence of S. sclerotiorum using a lower applied potential
at the working electrode 26 (compared with the potential used for direct detection). This
may provide improved selective detection of the hydrogen peroxide, since using a lower
potential reduces the detection of other electroactive species. The embodiment shown in
figure 9 may however be more expensive to produce than other embodiments due to its
increased complexity.
The biocompatible polymer 28 may be used to immobilise an enzyme other than oxalate
oxidase or horseradish peroxidase.
Although the embodiment shown in figure 9 provides the oxalate oxidase 20 and
horseradish peroxidase 30 in a biocompatible polymer 28, the oxalate oxidase and
horseradish peroxidase may be provided in other ways. For example the oxalate oxidase
and horseradish peroxidase may be provided on the surface of the working electrode 6 .
Components of different embodiments of the invention may be combined with one another.
For example, a mediated working electrode may be used in any of the illustrated
embodiments of the invention.
The above described embodiments provide immobilisation of an enzyme (e.g. oxalate
oxidase) or enzymes (e.g. oxalate oxidase and horseradish peroxidase) in the vicinity of an
electrode 6 , 26. In this context the term 'in the vicinity' may be interpreted as meaning
sufficiently close that electroactive species (e.g. hydrogen peroxide) generated due to thepresence of oxalic acid and the enzyme may be efficiently detected using the electrode. I f
the nutrient were to be a gel, and the enzyme were to be located too far from the electrode
6 then the electroactive species generated due to the presence of the oxalic acid would
have little or no reaction with the electrode (the reaction rate will be limited by diffusion
kinetics in the gel 8). As a result the presence of the electroactive species might not be
detected. In these circumstances, moving the enzyme closer to the electrode 6 will
increase the strength of the reaction of the electroactive species with the electrode, and
increase the strength of an output provided from the electrode. Thus, it may be
advantageous to provide the enzyme on the electrode surface or adjacent to the electrode
surface (the term 'in the vicinity of the electrode' is intended to encompass both of these
possibilities). Since diffusion kinetics also apply in a liquid, it is also advantageous to
provide the enzyme on the electrode surface or adjacent to the electrode surface in a
nutrient liquid.
The immobilisation of the oxalate oxidase (and/or other enzymes) may be done in a manner
which allows the oxalate oxidase to retain activity and stability, and which may prevent or
inhibit the oxalate oxidase from leaching out from its initial position, and may prevent or
inhibit the oxalate oxidase from denaturing. For example, the oxalate oxidase may be
provided on the electrode in the manner described further above. In embodiments in which
the oxalate oxidase is provided on the electrode, modification of the surface of the electrode
by the oxalate oxidase should not adversely affect diffusion of hydrogen peroxide and
electrons between the oxalate oxidase and the electrode.
When providing the oxalate oxidase (and/or other enzymes) on the electrode, the electrode
may be treated in order to facilitate a more homogeneous deposition of the oxalate oxidase.
Binder chemicals which may be used when printing the electrode may make the electrode
surface quite hydrophobic. This may make it difficult to achieve regular homogeneous
oxalate oxidase (and/or other enzyme) deposition on the electrode surface. This may lead
to loss of activity or sensitivity. To overcome this the electrode surface may be modified by
detergents such as Triton X-100 or Brijj-30, thereby facilitating an even distribution and
adsorption of the oxalate oxidase (and/or enzymes). Other treatments may be applied to
the electrode surface such as plasma treatment (plasma is a partially ionized gas which has
enough energy to ionize other atoms e.g. the atoms on the electrode surface thus changing
the surface chemistry), or electrochemical pre-treatment of the working electrode.
The working electrode 6 , 26 shown in figures 2 to 7 is formed from carbon paste (which may
be mixed with a mediator such as Prussian blue). Electrodes formed from carbon pastemay be produced at low cost (compared with electrodes formed using some other
materials) and may be relatively easy to form using mass production techniques. The
carbon electrodes may for example include Prussian blue or cobalt phthalocyanine, which
may allow the electrode to selectively sense hydrogen peroxide (i.e. excluding other
electroactive species).
In an alternative embodiment, the electrode may be formed from indium tin oxide (ITO), for
example on a glass slide which acts as a substrate. A disadvantage of using an ITO
electrode is that it may not be compatible with the detection of hydrogen peroxide unless it
is pre-treated. This is because differences in the surface chemistry and properties of ITO
(compared with for example carbon paste) may cause reduction of atmospheric oxygen to
occur at the working electrode. This reduction of atmospheric oxygen may for example
occur when the working electrode is held a potential which is used to detect the presence of
hydrogen peroxide (e.g. -0.6 volts), and will add to a noise signal at the electrode.
A pre-treatment may be applied to an ITO electrode in order to allow it to detect hydrogen
peroxide reduction without generating a large noise signal due to atmospheric oxygen
reduction. The pre-treatment may comprise modifying the surface of the ITO electrode by
applying high voltages to it (e.g. as described in X. Cai, B. Ogorevc, G. Tavcar and J .
Wang, Indium-tin oxide film electrode as catalytic amperometric sensor for hydrogen
peroxide. Analyst 120 (1995), pp. 2579-2583). A disadvantage of pre-treating the ITO
electrode is that it may add considerable complexity to the manufacture of the pathogen
sensor.
In an alternative approach, instead of pre-treating an ITO electrode, horseradish peroxide
may be provided at the ITO electrode in combination with an oxalate oxidase. This may be
done for example using the arrangement shown in figure 9 or may be done for example by
providing the horseradish peroxidase and the oxalate oxidase on the electrode. The
horseradish peroxidase acts as a secondary enzyme which catalyses the reduction of
hydrogen peroxide at the electrode. This may allow electrochemical detection of hydrogen
peroxide to be performed using an ITO electrode at a more neutral applied potential (e.g.
less negative than -0.6 volts).
Additionally or alternatively, Prussian blue may by provided at the ITO electrode. As
explained above, the Prussian blue acts as an artificial peroxidise which catalyses the
reduction of hydrogen peroxide. Again, this may allow electrochemical detection ofhydrogen peroxide to be performed using an ITO electrode at a more neutral applied
potential (e.g. less negative than -0.6 volts).
In general, Prussian blue may be combined with a variety of different electrode materials,
including carbon paste, glassy carbon, graphite, carbon nanotubes, platinum, silver, silver
chloride, gold and ITO. When Prussian blue is used the detection limit for hydrogen
peroxide may be in the micromolar range. Prussian blue may be deposited onto electrodes
using a variety of techniques including electrochemical and chemical methods, and may
also be deposited as nanoparticles. Carbon electrodes which include Prussian blue or
cobalt phthalocyanine are commercially available and may for example be purchased from
Gwent Electronic Materials of Pontypool, United Kingdom. Although Prussian blue is less
stable at alkaline pH values compared with acidic pH values, this may not be a
disadvantage for the pathogen sensor because the gel 8 may be optimised at acidic pH
values.
Other biochemical and/or chemical elements which decrease the electrochemical sensing
potential of the electrode needed for an electroactive species to be detected (e.g. hydrogen
peroxide) may be used instead of Prussian blue as a mediator which mediates the
electrode. For example cobalt phthalocyanine may be used. Cobalt phthalocyanine
electrodes detect hydrogen peroxide at around +0.5 V; less that the potential required to
detect hydrogen peroxide on bare carbon electrodes. The detection of hydrogen peroxide
using cobalt phthalocyanine electrodes is described in: Crouch, E., Cowell, D. C , Hoskins,
S., Pittson, R. and Hart, J . P. (2005). Amperometric, screen-printed, glucose biosensor for
analysis of human plasma oxidase using a biocomposite water-based carbon ink
incorporating glucose oxidase. Analytical Biochemistry, 14, 17-23
At higher applied potentials (e.g. around +0.7 V), cobalt phthalocyanine will react directly
with oxalic acid to produce a current. This is described in Li and Guarr (1991)
Electrocatalytic oxidation of oxalic acid at electrodes coated with polymeric
metallophthalocyanines. Journal of Electroanalytical and Interfacial Electrochemisrry, 317,
189-202). Consequently, oxalic acid may be measured directly without the need for an
enzyme. However, an advantage of using an enzyme is that when an enzyme is used the
electrochemical reaction occurs at a lower overpotential, thereby reducing the risk of
unwanted currents being generated from other electroactive species present in the assay.
A more sensitive measurement was obtained using oxalate oxidase on a Prussian blue
mediated carbon electrode than was obtained using direct detection via a cobalt
phthalocyanine electrode.Any suitable mediator may be used to mediate an electrode of the pathogen sensor.
Mediators which could be used instead of Prussian blue (potassium hexacyanoferrate) or
cobalt phthalocyanine include Quinones, Ferrocene, Ferrocyanide, Methylene green,
Osmium complexes e.g. osmium polypyridyl, Polypyrrol, Ruthenium complexes, and
Pthalocyanines (i.e. pthalocyanines other than cobalt phthalocyanine).
The mediator may be freely diffusible to shuttle electrons between the enzyme and
electrode surface. The mediator may be tethered to the enzyme and electrode. Tethered
mediators are sometimes described as 'wired' enzymes. A conducting polymer such as
polypyrrole and glucose oxidase is an example of a wired enzyme system.
The mediator may be used with redox enzymes (such as horseradish peroxidase) which
depend on the activity of co-substrates which require high overpotentials for regeneration of
the redox active co-substrate species.
The electrode may for example be modified by a biochemical and/or chemical recognition
element. This may for example include incorporating an enzyme, antibody, DNA or
chemical species into the electrode which may enhance or change the electrochemical
response of the electrode.
The electrode may be formed from carbon, including screen printed carbon, glassy carbon,
carbon nanotubes, graphene, carbon fibre, pyrolytic graphite carbon, metallised carbons e.g.
platinised carbon. The electrode may be formed from composite materials composed of a
powdered electronic conductor e.g. carbon powder or carbon nanotubes, and a binding
agent such as polymeric material or paste. The electrode may be formed from indium tin
oxide, platinum, silver, silver chloride, nickel, iron, copper, mercury (including mercury
amalgams), palladium, iridium, or gold. Forming the electrode from gold may be relatively
costly and in addition may not be compatible with a biocompatible polymer in which the
oxalate oxidase may be provided. In general, the electrode may be formed from any
suitable material which conducts electrons.
As explained above, horseradish peroxidase catalyses the reduction of hydrogen peroxide
and allows hydrogen peroxide produced from the oxalic acid to be detected at lower
electrochemical potentials (compared with direct electrochemical sensing of hydrogen
peroxide). Since horseradish peroxidase is a redox enzyme, it may be beneficial to connect
it to the surface of the working electrode 6 either directly (to allow direct electron transfer) orindirectly using mediators such as ferrocene (to allow the catalytic cycle to proceed and
reduce hydrogen peroxide). In general, direct electron transfer methods using horseradish
peroxidase may not be ideal for biosensing applications, because the horseradish
peroxidase may denature at the electrode surface with the consequence that electron
transfer rates between the electrode and the active sites of the horseradish peroxidase
become slow. Mediators may be used to overcome slow heterogeneous electron transfer
rates between the electrode and horseradish peroxidase. The mediator should be freely
diffusible between the horseradish peroxidase and the electrode surface. It may be
desirable that the mediator has high heterogeneous electron transfer rates and high
reactivity with horseradish peroxidase. The mediator may be selected to not cross-react or
inhibit the oxalate oxidase. In general, materials included in the pathogen sensor may be
selected to not co-react with horseradish peroxidase.
The horseradish peroxidase may be applied such that it overlaps beyond edges of the
oxalate oxidise. This reduces the likelihood that the horseradish peroxidase has a spatially
limited activity which does not truly reflect the activity of the oxalate oxidase.
Oxalate oxidase and horseradish peroxidase have different optimal pH values. The optimal
pH for oxalate oxidase is 4 and the optimal pH for horseradish peroxidase is 7 . I f oxalate
oxidase and horseradish peroxidase are used, the pH in the pathogen sensor may for
example be selected to be a value which lies between these two values, that is, between pH
4 and pH 7 . More preferably the pH range is selected to be between pH 4.5 and 6.5. The
pH in the pathogen sensor may be neutral, as this may encourage S. sclerotiorum growth.
The pH of the pathogen sensor may change during the lifetime of the pathogen sensor, for
example becoming more acidic due to accumulation of oxalic acid produced by the S.
sclerotiorum. The pH dependence of the enzyme activity (e.g. oxalate oxidase and
horseradish peroxidase) may be modified by using enzymes from different sources, or by
using genetic engineering techniques to produce enzymes which have a wider pH tolerance
or optimal activity at a desired pH value.
Although above described embodiments of the invention monitor for the presence of
hydrogen peroxide at an electrode, alternative embodiments of the invention may monitor
for the presence of other electroactive species at an electrode.
The above described embodiments of the pathogen sensor use electrochemical
transduction (i.e. the conversion of chemical energy into electrical energy) to detect the
presence of oxalic acid. An advantage of using electrochemical transduction to detectoxalic acid is that it allows the pathogen sensor to be made relatively small, and allows it to
be made using mass manufacturing techniques at relatively low cost (compared to making a
pathogen sensor which uses other transduction methods).
In the embodiments shown in figures 2 to 7 wires 18 extend downwardly from the working
electrode 6 and the reference electrode 16, and pass through openings in the substrate 14
to measurement electronics. Similarly, in the embodiment shown in figure 1 a cylindrical
channel 10 extends downwardly from the working electrode 6 to accommodate wires which
pass to measurement electronics. Wires may however travel along other routes to
measurement electronics. For example, wires may pass along the top of a substrate of the
pathogen sensor. Where this is done a layer of insulation (e.g. a plastic layer) may be
provided over the wires to insulate them from the electrode.
A sensor apparatus 140 which includes a plurality of pathogen sensors 100a-f according to
an embodiment of the invention is shown schematically in figure 10. The pathogen sensors
100a-f are provided on a flexible tape 0 1. The flexible tape 101 may be provided with, for
example, around one hundred pathogen sensors, and may be wrapped around a reel (not
shown). A lead end of the flexible tape 101 may be connected to a second reel (not
shown), which may be driven such that over time the flexible tape is unrolled from the reel
and is rolled onto the second reel. The second reel may be driven such that every 24 hours
the pathogen sensors are moved by a distance which corresponds to the separation
between pathogen sensors (this movement is indicated by the arrow 102 in Figure 10). The
position of the second reel, and hence the positions of the pathogen sensors 100a-f, may
be controlled by a control apparatus (not shown). The control apparatus may also control
operation of puncturing arms and measurement electronics (described below).
Each pathogen sensor 100a-f may comprise a housing which is generally cylindrical (or has
some other shape), and which is open at an upper end. The housing may for example have
a depth of around 15 mm, and may for example have a diameter of around 6 mm. An
impermeable barrier 104 may be located above the bottom of the housing, for example
around 2 mm above the bottom of the housing, thereby defining a volume which is referred
to hereafter as sampling volume 105. An electrode 106 is located at the bottom of the
sampling volume 105. The electrode 106 may for example be provided with an oxalate
oxidase, for example as described further above. Nutrient liquid 108 may be provided in the
housing above the impermeable barrier 104. A film 106 (or other barrier) may be located
above the nutrient liquid 108.The pathogen sensor 100a may initially be located in a pre-sampling housing 109. A
puncturing arm 110 in the pre-sampling housing 109 may be used to puncture the film 106.
Following this, the pathogen sensor may be moved to a sampling location which is located
outside of the pre-sampling housing. The sampling location is a location which receives air
and airborne pathogens. In Figure 10 pathogen sensor 100b is located at the sampling
location.
The pathogen sensor 100b may remain at the sampling location for 24 hours, during which
time pathogen spores may pass into the pathogen sensor. The pathogen spores may for
example land in the nutrient liquid.
The pathogen sensor is then moved into an incubator 111. The incubator 111 has a
temperature of 25°C, the temperature being selected to promote growth of the pathogen.
Pathogen sensor 100c is shown in the incubator 109.
The pathogen sensor may be moved through the incubator 111 such that it is incubated for
three days, as shown by pathogen sensors 100c-e in Figure 10. The incubator may include
some form of covering (not shown) for the pathogen sensors 100c-e which acts to prevent
or inhibit evaporation of the nutrient liquid 108 from the pathogen sensors. Alternatively or
additionally, the apparatus may include a liquid nutrient replenishment apparatus which is
configured to periodically add liquid nutrient to the pathogen sensors 100c-e to replace
evaporated liquid nutrient. During incubation the pathogen will grow and will release oxalic
acid. The oxalic acid will mix with the nutrient liquid 108. During incubation the pathogen
and the nutrient liquid are isolated from the electrode 106 by the impermeable barrier 104.
After the pathogen sensor has been incubated for three days, a puncturing arm 112 is used
to puncture the impermeable barrier 104. This allows the nutrient liquid and oxalic acid to
pass into the sampling volume 105 at the bottom of the pathogen sensor (as shown for
pathogen sensor 100f). The nutrient liquid and oxalic acid thus come into contact with the
electrode 106. Measurement electronics 113 are configured to apply a potential at the
electrode 106, for example in the manner described further above. As described further
above, the oxalic acid reacts with the oxalate oxidase to generate hydrogen peroxide which
is detected by the electrode 106. This indicates that the pathogen has grown in the
pathogen sensor.
The housing 103 of the pathogen sensor may be formed from a polymer. The polymer may
include a coating which prevents or inhibits the release of volatile organics that could inhibitgrowth of the pathogen. The sampling volume 105 of the pathogen sensor may include a
hydrophilic element which is arranged to draw the liquid nutrient and oxalic acid into the
sampling volume. The sampling volume 105 may for example have a volume of 200µ Ι
An apparatus (not shown) which is arranged to draw air into the pathogen sensor may be
provided at the top of the pathogen sensor.
Although the above description refers to sampling for 24 hours and incubating for 3 days
before measurement, any suitable sampling and incubating periods may be used.
Incubation may for example be for between 3 and 7 days. The incubation may be at any
suitable temperature. The temperature may for example be chosen to provide optimal
growth of the pathogen. Any suitable number of pathogen sensors may be provided on the
flexible tape 101. For example, sufficient pathogen sensors may be provided to allow
pathogen sensing to be performed over an entire growing season.
Any suitable apparatus may be used to isolate a nutrient medium and a pathogen from an
electrode during incubation of the pathogen. Similarly, any suitable apparatus may be used
to end that isolation such that the nutrient medium and pathogen come into contact with the
electrode when a measurement is to be performed. Keeping the nutrient medium and
pathogen away from the electrode during incubation is advantageous because it avoids
deterioration of the electrode that could occur if the nutrient liquid and pathogen were in
contact with the electrode during incubation.
In an alternative arrangement, the pathogen sensors on the tape may not be pre-filled with
liquid nutrient. The liquid nutrient may be delivered into the pathogen sensor after sampling
takes place. The liquid nutrient may for example be delivered via a pump. The pump may
be sterile, and the apparatus may include a washing apparatus arranged to wash the pump
and keep it sterile.
The puncturing arms 110, 112 are examples of puncturing apparatus. The sensor
apparatus 140 may include any suitable puncturing apparatus.
Each pathogen sensor 100a-f may be provided with an air sampling apparatus which is
arranged to sample air and to direct spores from the air into the pathogen sensor. Such air
sampling apparatus are well known in the art and are therefore not described here.A sensor apparatus 40 which includes a pathogen sensor 1 according to an embodiment of
the invention is shown schematically in figure 11. Features of the apparatus shown in figure
11 may be combined with features of the apparatus shown in figure 10. The sensor
apparatus comprises a chamber 42 which has an opening (not shown) connected to the
atmosphere at one end and has an opening (not shown) connected to a pump 44 at the
other end. The opening connected to the pump may be larger than the opening connected
to the atmosphere, such that a vacuum is generated in the chamber when the pump is
operating. The sensor apparatus may be provided with a weather vane (not shown) and
may be rotatably mounted such that it turns towards the wind. The sensor apparatus may
include features described in Hirst JM (1951), An Automated Volumetric Spore Trap, Annals
of Applied Biology, 39(2), pp257-265, which is herein incorporated by reference.
The sensor apparatus includes a power supply unit 46 which comprises a power harvesting
system 48 (for example a solar panel or wind turbine) which charges a battery 50. The
battery 50 may be used to power electrical components of the sensor apparatus via a
DC/DC converter 52. Other forms of power supply unit may be used.
In addition to the pathogen sensor 1, the sensor apparatus 40 may be provided with one or
more additional ancillary sensors, for example in a meteorological unit 54. These may for
example include one or more of a temperature sensor 56, a humidity sensor 58, a wind
direction and wind speed sensor 60, a pressure sensor 62 and an ambient light sensor 64.
The sensor apparatus may be provided with control electronics 66, which may for example
comprise a CPU. The measurement electronics which are used to apply a potential step to
an electrode of the pathogen sensor 1 and to detect a resulting current may form part of the
control electronics 66 or may optionally be provided as a separate entity 68. In addition to
receiving data from the pathogen sensor, the control electronics 66 may receive data from
the additional ancillary sensors 54-64 (e.g. via a signal conditioner 65). The control
electronics 66 may include a memory which stores data as a function of time. The control
electronics may thus allow the quantity of the pathogen at the sensor to be tracked over a
period of time. Analysis electronics may be provided as part of the control electronics, the
analysis electronics being used to analyse data received from the pathogen sensor (and
optionally from other sensors).
The duty cycle of the pump 44 and other components of the sensor apparatus may be
actively managed by the control electronics 66, for example to take into account a power
budget arising from a battery 50 of the sensor apparatus.Although only one pathogen sensor 1 is shown in figure 8 , a plurality of pathogen sensors 1
may be provided in a single sensor apparatus 40. For example, more than one pathogen
sensor which is configured to detect a particular pathogen may be provided in the sensor
apparatus. Where this is done, a first pathogen sensor may be used to monitor for the
presence of the pathogen over a period of time until a supply of nutrients is exhausted or
close to being exhausted (and/or the pathogen sensor is dehydrated), whereupon operation
of a second pathogen sensor which is configured to detect the pathogen may be initiated.
This may for example be achieved by removing a film from the second pathogen sensor.
This may be an automated process performed by a sensor selector unit 69 which is
controlled for example by the control electronics 66, or may be performed manually.
Alternatively, the sensor apparatus 40 may be configured to expose a first pathogen sensor
to the atmosphere for a predetermined period of time (e.g. until a nutrient supply is
substantially exhausted and/or the pathogen sensor is dehydrated), then move a second
pathogen sensor from a sealed container such that it is exposed to the atmosphere. This
may be an automated process or may be performed manually. The first and second
pathogen sensors (and possibly additional pathogen sensors) may be provided in a
cartridge (not shown) which is removable from the sensor apparatus 40. This may be an
automated process performed by a sensor selector unit 69 which is controlled for example
by the control electronics 66, or may be performed manually. The cartridge may for
example comprise a disk which may be rotated to expose a selected pathogen sensor to
the atmosphere.
The measurement electronics 68 may monitor electrodes of a pathogen sensor 1 which is
newly exposed to the atmosphere, and may cease monitoring electrodes of a pathogen
sensor which has been replaced by the newly exposed pathogen sensor. This switch may
be controlled by the control electronics 66.
Additionally or alternatively, auxiliary pathogen sensors which are configured to detect the
presence of different pathogens may be provided in the sensor apparatus 40. The auxiliary
pathogen sensors may for example be capable of detecting proteins secreted by interfering
pathogens.
A wireless network may be provided which enables communication between the sensor
apparatus 40 (e.g. via a wireless transceiver 70) and remotely located system analysis and
control electronics (not shown). Alternatively, a wire-based network may be provided to
enable this communication. The remotely located system analysis and control electronicsmay for example be a CPU. The system analysis and control electronics may receive data
from a plurality of sensors apparatus. The system analysis and control electronics may
control a plurality of sensor apparatus 40 by sending control signals to the sensor apparatus
via the wireless network. The control signals may for example instruct that a pathogen
sensor 1 which has reached the end of its life is replaced by a new pathogen sensor.
Wireless communication between the sensor apparatus 40 and the system analysis and
control electronics may for example use local area wireless network (Wi-Fi) transmitters and
receivers and/or GSM transmitters and receivers. Communication may include one or more
relay nodes.
The system analysis and control electronics may analyse pathogen sensor data from
sensor apparatus spread over an area such as a field, a plurality of fields, a farm or some
other area. The data analysis may incorporate data from the additional ancillary sensors of
the sensor apparatus. The data analysis may identify progress of a pathogen across the
area, and may provide a forecast of the progress of the pathogen. The data received by the
system analysis and control electronics may include a degree of data-redundancy, and this
may be used to identify outlier pathogen sensor measurements which may indicate failure
or incorrect operation of a pathogen sensor. The data-redundancy may also facilitate
improved interpolation of pathogen ingress between pathogen sensors.
Data from a plurality of system analysis and control electronics may be collected at a central
data analysis system (for example collecting data from across a region, country or
internationally). The data may be merged with data from more traditional agronomy data
sources, such as meteorological data or crop data obtained by satellite imaging. The
central data analysis system may use the merged data to deliver ground-truthed real-time
maps of pathogen progress.
In the above described illustrated embodiments of the pathogen sensor, the nutrient liquid
8 , 108 (or gel) acts as a growth medium upon which and/or within which the pathogen may
germinate and grow, and provides nutrients which facilitate growth of the pathogen (the
nutrients thus sustaining the pathogen in a similar way to nutrients that the pathogen would
extract from a plant). Various properties of the pathogen sensor may be selected to mimic
a plant or mimic particular conditions, such that a pathogen may germinate and grow and
mediate an event which is to be detected. The pathogen may be S. sclerotiorum or may be
some other pathogen. Properties of the pathogen sensor may be selected to mimic part of
a plant (e.g. a leaf or a stem) upon which and/or within which the pathogen may grow.As explained above, the pathogen sensor may for example be configured to detect S.
sclerotiorum. Where this is the case the pathogen sensor provides a growth medium (e.g.
the nutrient liquid 8) upon which and/or within which S. sclerotiorum may grow, and
provides nutrients which nourish the S. sclerotiorum over a period of time which is sufficient
to allow the S. sclerotiorum to generate oxalic acid. In addition, the nutrients facilitate the
production of oxalic acid by the S. sclerotiorum. This facilitation of the production of oxalic
acid may be achieved for example by providing nutrients which facilitate growth of S.
sclerotiorum via metabolic pathways which provide more oxalic acid production than
alternative metabolic pathways (the alternative metabolic pathways producing less oxalic
acid). Selective fungicides, antibiotics or antimicrobials may be incorporated in the
pathogen sensor to inhibit the growth of other microorganisms which may inhibit S.
sclerotiorum growth and/or produce oxalic acid or some other interferent electroactive
species.
The pathogen sensor 1 may be configured to detect a pathogen other than S. sclerotiorum.
This may be achieved for example by providing nutrients in the growth medium which
nourish the pathogen to be detected and allow it to grow. For example, Sclerotinia other
than S. sclerotiorum may grow in a potato dextrose based medium. For example,
Sclerotinia homeocarpa may grow in a potato dextrose agar or a potato dextrose broth, and
may release oxalic acid as it grows - see Oxalic Acid Production by Sclerotinia
homoeocarpa: the Causal Agent of Dollar Spot" by R A Beaulieu; Senior Honors Thesis;
The Ohio State University; June 2008. For example, Sclerotinia minor may grow and
release oxalic acid in a variety of media, as described in Oxalic Acid Production and
Mycelial Biomass Yield of Sclerotinia minor for the Formulation Enhancement of a Granular
Turf Bioherbicide" by S C Briere, A K Watson and S G Hallett; Biocontrol Science and
Technology (2000) 10, 281-289, the disclosure of which is herein incorporated by reference.
The media mentioned in that paper include potato dextrose broth (PDB, Difco Laboratories,
Detroit, Michigan) at pH 6.0; PDB at pH 6.0 plus 56-mm sodium succinate (PDB-SS).
The nutrients may also facilitate the production of a detectable substance by the pathogen.
A supply of nutrients may be provided from a nutrient reservoir (e.g. via a one-way
membrane). The substance which is detected by the pathogen sensor 1 may be a chemical
or biological agent (including for example organic acids, nucleic acids, proteins (e.g.
enzymes), toxins, hormones, metabolites, peptides, carbohydrates or lipids).
The pathogen sensor may be considered to provide a two-step method of pathogen
detection. The first step is growth of the pathogen on and/or in the growth medium, and thesecond step is production of a detectable substance by the pathogen after some growth of
the pathogen has occurred.
Because it detects a substance produced by a pathogen (e.g. generation of oxalic acid in
the case of S. sclerotiorum ) , the pathogen sensor provides a real-time indication of the
presence of the pathogen as well as the viability of the pathogen. That is, the pathogen
sensor differentiates between an active pathogen and a dormant or dead pathogen.
Furthermore, in addition to detecting the presence of the pathogen, embodiments of the
invention may also provide an indication of the quantity of pathogen at the pathogen sensor.
An aspect of the pathogen sensor which may facilitate growth of the pathogen on and/or in
the growth medium is hydration of the growth medium. The growth medium may be kept
hydrated for example by delivering fluid to the growth medium from a fluid reservoir (e.g. via
a one-way membrane). The fluid reservoir may be separate from the growth medium (e.g.
located away from the growth medium as shown in figure 1).
An aspect of the pathogen sensor which may facilitate growth of the pathogen on and/or in
the growth medium is delivery of nutrients to the growth medium. Nutrients may be
delivered to the growth medium from a nutrient reservoir (e.g. via a one-way membrane).
The nutrient reservoir may be separate from the growth medium (e.g. located away from the
growth medium as shown in figure 1).
The nutrient reservoir and the fluid reservoir may be the same reservoir. The nutrient may
be provided in a fluid which keeps the pathogen hydrated.
The pathogen sensor may allow a pathogen to grow in a manner which is similar to the
manner in which the pathogen would grow on a plant. The pathogen sensor may for
example provide a favourable growth environment for the pathogen such that the pathogen
will grow on/in the growth medium at a speed which is faster than the speed of growth of the
pathogen on the plant (e.g. through incubation of the pathogen sensor). This allows the
plant to be protected through the application of a fungicide or other measures which will
prevent or restrict the growth of the pathogen. A crop which comprises the plant may for
example be protected in this manner.
The pathogen sensor 1 may be configured to detect a fungal pathogen, for example a
fungal pathogen which generates oxalic acid. This may be achieved for example by
providing nutrients in the growth medium which nourish the fungal pathogen to be detected.The nutrients may also facilitate generation of a detectable substance by the fungal
pathogen. Selective fungicides, antibiotics or antimicrobials may be incorporated in the
pathogen sensor to inhibit the growth of other fungicides or other microorganisms as
appropriate which may inhibit growth of the fungal pathogen or other microorganisms and/or
interfere with detection of a substance produced by the fungal pathogen (e.g. oxalic acid) or
other microorganisms.
As mentioned above, the substance which is detected by the pathogen sensor may be a
chemical or biological agent (including for example organic acids, nucleic acids, proteins
(e.g. enzymes), toxins, hormones, metabolites, peptides, carbohydrates or lipids). In this
context the term Organic acid' may be interpreted as meaning a molecule that contains a
carboxylic acid functional group. Embodiments of the invention detect the organic acid
using electrochemical transduction (as described above). Other chemical or biological
agents may also be detected using electrochemical transduction.
Embodiments of the invention include an enzyme with which a chemical or biological agent
released by a pathogen interacts, the interaction leading to an electronically detectable
signal. The interaction of the enzyme with the chemical or biological agent may comprise
the enzyme binding to and subsequently reacting with the chemical or biological agent. Any
suitable enzyme may be used. The interaction may lead to the generation of an
electroactive molecule which may then be detected using an electrode. The interaction may
lead to the generation of a molecule which is the substrate for subsequent interaction with
an enzyme (e.g. a different enzyme) or other reactive molecule. This subsequent
interaction may lead to the generation of an electroactive molecule which may then be
detected using an electrode. In this context, although the interaction of the chemical or
biological agent with the first enzyme does not directly generate an electroactive molecule it
leads towards generation of an electroactive molecule. The interaction may be considered
to lead indirectly to the generation of an electroactive molecule, and thus may be
considered to lead indirectly to an electronically detectable signal. One or more additional
enzyme interactions may take place before the electroactive molecule is generated. These
additional enzyme interactions may also be considered to lead indirectly to the generation of
an electroactive molecule.
The interaction of the chemical or biological agent released by a pathogen with the enzyme
may cause a conformational change in the enzyme which is recognised by other elements
in the pathogen sensor (e.g. other enzymes), and this may lead to the generation of an
electroactive molecule (either directly or indirectly). The conformational change may causethe enzyme to accept a substrate already present in the growth medium (the substrate
being something other than the chemical or biological agent). Interaction of this substrate to
the enzyme may lead to the generation of an electroactive molecule (either directly or
indirectly).
The electronic detection apparatus may detect the chemical or biological agent released by
a pathogen using some other form of transduction. The electronic detection apparatus may
detect the chemical or biological agent via enzymatic, immunoassay (antigen-antibody
binding), spectroscopic or other biosensing techniques. The electronic detection apparatus
may use the passage of the chemical or biological agent through a membrane (e.g. as
described above in relation to figure 3). Acid release from a pathogen may for example be
detected using an electronic detection apparatus which uses detection of swelling of a gel,
electrochemical sensing or detection of a refractive index change or colour change. The
electronic detection apparatus may for example detect protein secretions arising from
pathogen growth using antibody/antigen binding resulting in an optical refractive index
change, mass change on a surface acoustic wave device or resonant quartz crystal
microbalance, or electrochemical sensing.
As mentioned above, properties of the pathogen sensor may be selected to mimic a plant or
mimic particular conditions. Properties of the pathogen sensor may be selected to mimic
part of a plant (e.g. a leaf or a stem) upon which and/or within which the pathogen may
grow. One or more of lighting, humidity and/or moisture, pH conditions, orientation and
temperature may be selected to mimic a plant or part of a plant, or to mimic particular
conditions.
The pathogen sensor may be configured to take into account photo-inhibition or photo-
promotion of a pathogen. The natural lighting conditions which support pathogen
germination and growth may be mimicked at the growth medium of the pathogen sensor.
This may for example be through exposing the sensor surface to ambient light which has
passed through appropriate optical filters, through illuminating the sensor surface using a
photo-emitter such as a semiconductor or polymer, or through exposing the growth medium
to ambient lighting.
The pathogen sensor may be configured to take into account humidity and/or moisture
conditions. Appropriate humidity conditions and/or dew build up for an extended period
(e.g. 6-12 hours) may be necessary for an event mediated by the pathogen to take place.
The growth medium of the pathogen sensor may comprise a hydrophilic gel and/or polymerwhich provides moisture for the pathogen. Additionally or alternatively, the pathogen sensor
may include a one-way membrane configured to wick water from a reservoir to the growth
medium (e.g. in a manner analogous to that described above in relation to figure 1).
The pathogen sensor may be configured to take into account pH conditions which support
pathogen germination and growth. The pH of the growth medium of the pathogen sensor
may be selected via the inclusion of hydrophilic gels and buffers in the pathogen growth
medium. Additionally or alternatively, the pH of the growth medium may be controlled by
providing the pathogen sensor with a one-way membrane configured to wick a buffer from a
reservoir to the growth medium (e.g. in a manner analogous to that described above in
relation to figure 1).
The growth medium of the pathogen sensor may be oriented to take into account the effect
of gravity in supporting pathogen growth. For example, the growth medium may have an
orientation which corresponds with a likely orientation of a part of a plant on which the
pathogen will grow.
The growth medium of the pathogen sensor may be held at a temperature (or have a
temperature variation applied to it over time) which supports germination and growth of the
pathogen. The temperature of the growth medium may for example be controlled using a
Peltier-effect heat pump or any other suitable temperature control apparatus.
Selective fungicides, antibiotics or antimicrobials may be incorporated in the pathogen
sensor to inhibit the growth of other microorganisms which may inhibit growth of the
pathogen to be detected and/or interfere with detection of an event mediated by the
pathogen.
The sensor apparatus may incorporate air filtering, for example using a filter which is sized
to exclude larger interfering pathogens or other sources of interferents.
Although the description of embodiments of the invention has focussed on detection of
fungal pathogens, the invention may be used to detect other pathogens. Similarly, although
the description of embodiments of the invention has focussed on pathogens which grow on
plants, the invention may be used to detect pathogens which grow elsewhere (e.g. in the
human body, in an animal body, in foodstuffs, in water, etc). In an embodiment, the
pathogen sensor may be used to detect a pathogenic bacteria. In an embodiment the
pathogen sensor may be used to detect a pathogen from the Burkholderia genus, forexample Burkholderia glumae (e.g. in grain rot and seedling rot in rice), or Burkholderia
pseudomallei (e.g. which causes the disease melioidosis). Burkholderia releases oxalic
acid and may therefore be detected using the above described embodiments of the
pathogen sensor. In general, the pathogen sensor may be used to detect pathogens in a
variety of application areas, including for example: healthcare (e.g. Aspergillus niger, B.
pseudomallei, Saccharomyces cerevisiae), animal health (e.g. Aspergillus niger),
environmental monitoring (e.g. S. sclerotiorum, Fomitopsis palustris), food spoilage (e.g.
Botrytus cinera), post harvest grain storage (e.g. Burkholderia glumae, Botrytus cinera),
pre-harvest seedling storage (e.g. Burkholderia glumae, Botrytus cinera), materials
protection (e.g. Fomitopsis palustris) and bio-security (e.g. Burkholderia pseudomallei)".
Properties of the pathogen sensor may be selected to mimic an entity upon which and/or
within which the pathogen may grow.
Although embodiments of the invention have referred to the pathogen sensor being
provided in a crop which is growing or adjacent to a crop which is growing, the pathogen
sensor may be provided in other locations. For example, the pathogen sensor may be
provided in a storage area in which a crop is stored after the crop has been harvested (e.g.
a warehouse or barn).
References in this description to growth of the pathogen may be considered to include
germination of the pathogen (the pathogen is metabolically active during germination and
may thus be considered to be growing).
Detection of an electroactive species, as described in the above embodiments, is an
example of detection via an electrochemical change. Other electrochemical changes which
may be detected by embodiments of the invention may for example be a change of
capacitance, inductance or some other electrical property. Embodiments of the invention
may for example use antibody binding in conjunction with impedance spectroscopy
detection to monitor for an event mediated by the pathogen (the electrochemical change in
this case being a change of impedance).
Embodiments of the invention may be considered to use an enzyme system to mediate and
monitor an electrochemical change from a chemical agent which is electroactive at a high
applied potential (e.g. oxalic acid) to a chemical agent which is electroactive at a lower
applied potential (e.g. hydrogen peroxide).The term 'growth medium' as used in the above description may be interpreted as meaning
any medium upon which and/or within which a pathogen may grow (the structure being
sufficiently strong to support the pathogen). The growth medium may include any desired
level of porosity. The growth medium may be a nutrient liquid. The term growth medium
may be considered to mean an environment favourable to growth of a pathogen (the
environment may be a liquid or a solid).
Features of any embodiment of the invention may be combined with features of any other
embodiment of the invention.
Although some embodiments of the invention include a liquid nutrient medium, a solid
nutrient medium such as a gel may be used instead of a liquid nutrient medium. An
advantage of using a liquid nutrient medium is that diffusion of oxalic acid released by a
pathogen will take place more readily in a liquid than in a solid, thereby allowing the oxalic
acid to reach the electrode of the sensor more easily. A further advantage is that S.
sclerotiorum grows more readily in a liquid nutrient medium than in a solid nutrient medium.
Features from different embodiments of the invention may be combined with one another.CLAIMS:
1. A pathogen sensor comprising a growth medium upon which and/or within which a
pathogen may grow, the growth medium comprising nutrients which facilitate growth of the
5 pathogen, wherein the pathogen sensor further comprises an electronic detection apparatus
configured to detect an electrochemical change mediated by the pathogen.
2 . The pathogen sensor of claim 1, wherein the electrochemical change is caused by a
chemical or biological agent produced by the pathogen.
10
3 . The pathogen sensor of claim 2 , wherein the chemical or biological agent is one of
the following: an organic acid, a nucleic acid, a protein, an enzyme, a toxin, a hormone, a
metabolite, a peptide, a carbohydrate or a lipid.
15 4 . The pathogen sensor of claim 2 or claim 3 , wherein the chemical agent is oxalic
acid.
5 . The pathogen sensor of any of claims 2 to 4 , wherein the electronic detection
apparatus comprises an enzyme that interacts with the chemical or biological agent, the
20 interaction leading to an electronically detectable signal.
6 . The pathogen sensor of claim 5 , wherein the interaction generates an electroactive
species or leads to the generation of an electroactive species, and wherein the electronic
detection apparatus further comprises an electrode configured to detect the presence of the
25 electroactive species.
7 . The pathogen sensor of claims 6 , wherein the enzyme is immobilised on a surface
of the electrode.
30 8 . The pathogen sensor of claim 6 or claim 7 , wherein the enzyme is oxalate oxidase.
9 . The pathogen sensor of any of claims 6 to 8 , wherein the electrode is mediated with
ferric hexacyanoferrate.
35 10. The pathogen sensor of any of claims 6 to 9 , wherein the nutrients are separated
from the electrode by a barrier which is configured to be punctured when detection of the
electroactive species is to be performed.11. The pathogen sensor of any preceding claim, wherein the growth medium is a liquid
media which contains potato dextrose broth.
5 12. The pathogen sensor of any preceding claim, wherein the pathogen is a fungal
pathogen.
13. The pathogen sensor of any preceding claim, wherein the pathogen is from the
Sclerotinia species.
10
14. The pathogen sensor of claim 13, wherein the pathogen is Sclerotinia Sclerotiorum.
15. A sensor apparatus which comprises the pathogen sensor of any preceding claim
and further comprises measurement electronics configured to receive a signal from the
15 electronic detection apparatus and to generate an output if the signal is indicative of an
electrochemical change mediated by the pathogen.
16. The sensor apparatus of claim 15, wherein the sensor apparatus further comprises a
control apparatus which is configured to expose the pathogen sensor to the air, incubate the
20 pathogen sensor for a predetermined period of time, and then use the electronic detection
apparatus to monitor for the electrochemical change.
17. The sensor apparatus of claim 15 or claim 16, wherein the sensor apparatus further
comprises a puncturing apparatus configured to puncture a barrier which separates the
25 growth medium from the electrode.
18. A method of detecting a pathogen comprising providing nutrients which facilitate
growth of the pathogen on and/or in a growth medium for a period which is sufficiently long
to allow a pathogen to mediate an electrochemical change, then using an electronic
30 detection apparatus to detect the electrochemical change.
19. The method of claim 18, wherein the electrochemical change is caused by a
chemical or biological agent produced by the pathogen.
35 20. The method of claim 19, wherein the chemical agent is oxalic acid.2 1. The method of any of claims 18 to 20, wherein the electronic detection apparatus
comprises an enzyme which interacts with the chemical or biological agent, the interaction
leading to an electronically detectable signal.
22. The method of claim 2 1, wherein the enzyme is oxalate oxidase which catalyses the
production of hydrogen peroxide from the oxalic acid.
23. The method of any of claim 18 to 22, wherein the growth medium is a liquid media
which contains potato dextrose broth.
24. The method of any of claims 18 to 23, wherein the pathogen sensor is one of a
plurality of pathogen sensors distributed over an area, and wherein the method comprises
analysing outputs from the pathogen sensors to obtain information regarding the progress
of the pathogen through the area.
25. The method of any of claims 18 to 24, wherein analysis of information provided from
the pathogen sensor is combined with analysis of information provided from one or more
sensors which sense one or more of: temperature, humidity, wind direction, wind speed,
pressure sensor and ambient light.
26. Use of a pathogen sensor according to any of claims 1 to 16 or a sensor apparatus
according to any of claims 15 to 17 for detecting an electrochemical change in crops arising
from the presence of one or more of: fungi (including molds and yeasts), viruses,
oomycetes, bacteria, viroids, phytoplasmas, protozoa, nematodes and parasitic plants on
the crop.
27. Use of a pathogen sensor as described in relation to any of claims 1 to 16 or a
sensor apparatus according to claims 15 to 17 in the treatment of any one of oil seed rape,
canola, soybean, peanut, citrus, celery, coriander, melon, squash, tomato, lettuce,
cucumber, sunflower, beans, strawberries or peas.

WO 2012/076431
43
PCT/EP20111071686
21. The method of any of claims 18 to 20, wherein the electronic detection apparatus
comprises an enzyme which interacts with the chemical or biological agent, the interaction
leading to an electronically detectable signal.
5 22. The method of claim 21, wherein the enzyme is oxalate oxidase which catalyses the
production of hydrogen peroxide from the oxalic acid.
23. The method of any of claim 18 to 22, wherein the growth medium is a liquid media
which contains potato dextrose broth.
10
24. The method of any of claims 18 to 23, wherein the pathogen sensor is one of a
plurality of pathogen sensors distributed over an area, and wherein the method comprises
analysing outputs from the pathogen sensors to obtain information regarding the progress
of the pathogen through the area.
15
25. The method of any of claims 18 to 24, wherein analysis of information provided from
the pathogen sensor is combined with analysis of information provided from one or more
sensors which sense one or more of: temperature, humidity, wind direction, wind speed,
pressure sensor and ambient light.
20
26. Use of a pathogen sensor according to any of claims 1 to 16 or a sensor apparatus
according to any of claims 15 to 17 for detecting an electrochemical change in crops arising
from the presence of one or more of: fungi (including molds and yeasts), viruses,
oomycetes, bacteria, viroids, phytoplasmas, protozoa, nematodes and parasitic plants on
25 the crop.
27. Use of a pathogen sensor as described in relation to any of claims 1 to 16 or a
sensor apparatus according to claims 15 to 17 in the treatment of anyone of oil seed rape,
canola, soybean, peanut, citrus, celery, coriander, melon, squash, tomato, lettuce,
30 cucumber, sunflower, beans, strawberries or peas., ~
. ~
':J~ if
Dated this 13th Day of May 2013 Of Anand And Anand Advocates
Agent for the Applicant