Note: Descriptions are shown in the official language in which they were submitted.
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DIAGNOSTIC DEVICE FOR EVALUATING
MICROBIAL CONTENT OF A SAMPLE
CROSS-REFERENCE TO RELATED APPLICATION(S):
[0001] This application is being filed on 28 February 2014, as a PCT
International
Patent application and claims priority to U.S. Patent Application Serial No.
61/770,391
filed on 28 February 2013, the disclosure of which is incorporated herein by
reference
in its entirety.
BACKGROUND
[0002] One of the difficulties encountered in the treatment of issues
involving
microbes is that microbes are continually changing. Microbes include, for
example,
bacteria, fungi, viruses, nematodes, cell culture, and tissue. Microbes are
becoming
antimicrobial resistant. Antimicrobials include antibiotics, antivirals,
antifungals, or
parasiticides and include bacteriophage, mycoviruses, virophages,
nematophages, which
are viruses that attack bacteria, fungi, nematodes, respectively. With the
rise of
antibiotic resistant bacteria, for example, even if the type of bacteria is
properly
identified, a prior treatment thought to be effective against such bacteria
may no longer
be so. More specifically, the particular species of bacteria may have
developed a
resistance to the treatment, and therefore no longer be susceptible to that
treatment.
SUMMARY
[0003] In general terms, this disclosure is directed to a diagnostic
device that
evaluates microbial content of a sample. In one possible configuration and by
non-
limiting example, the diagnostic device performs one or more of: determining
antimicrobial sensitivity of microbes, identifying microbes, and counting
microbes.
Various aspects are described in this disclosure, which include, but are not
limited to,
the following aspects.
[0004] One aspect is a diagnostic device comprising: at least one sample
module
defining a sample cavity therein; at least four electrodes arranged in the
sample cavity;
and electronic circuitry operably connected to the electrodes, wherein the
electronic
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circuitry is operable in a first mode and a second mode, wherein when
operating in the
first mode, the electronic circuitry operates to determine a conductance of a
sample in
the sample cavity, and wherein when operating in the second mode, the
electronic
circuitry operates to determine an admittance of the sample in the sample
cavity.
[0005] Another aspect is an antimicrobial dispenser comprising: a sterile
carrier
material; and bacteriophage carried by the sterile carrier material.
[0006] A further aspect is a sample module comprising: at least one
substrate; at
least four electrodes; and a sample cavity formed in the at least one
substrate, the
sample cavity comprising: a sensing portion including the electrodes therein,
the
sensing portion having a shape configured to direct and focus electric fields
generated
by the electrodes within the sample cavity; and a chimney portion extending
from the
sensing portion and having a cross-sectional size that is less than a cross-
sectional size
of the sensing portion.
[0007] Yet another aspect is a diagnostic device comprising: a plurality
of sample
modules; electrodes arranged in the sample modules; a calibration fluid
disposed in a
calibration module of the sample modules; and electronic components coupled to
the
electrodes, wherein the electronic components are operable to measure a
conductivity of
the fluid in the calibration cell and to determine a temperature of the
calibration fluid
using the measured conductivity.
[0008] Additional aspects are disclosed herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 is schematic perspective view of an example diagnostic
device.
[0010] FIG. 2 is a block diagram of an example reader of the diagnostic
device
shown in FIG. 1.
[0011] FIG. 3 is a block diagram of an example diagnostic unit of the
diagnostic
device shown in FIG. 2.
[0012] FIG. 4 is a schematic perspective view of an example sensor
system of the
diagnostic unit shown in FIG. 3.
[0013] FIG. 5 is a schematic perspective view of an example sample cell of
the
sensor system shown in FIG. 4.
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[0014] FIG. 6 is a cross-sectional side view of the sample cell shown in
FIG. 5.
[0015] FIG. 7 is a top view of the sample cell shown in FIG. 5, also
illustrating an
electrode configuration and an example antimicrobial dispenser.
[0016] FIG. 8 is a top cross-sectional view of another example sample
cell and
another example electrode configuration.
[0017] FIG. 9 is a top cross-sectional view of another example sample
cell and
another example electrode configuration.
[0018] FIG. 10 is a cross-sectional top view of another example sample
cell and
another example electrode configuration.
[0019] FIG. 11 is a cross-sectional top view of another example sample cell
and
another example electrode configuration.
[0020] FIG. 12 is a cross-sectional side view of the example sample cell
and
example electrode configuration shown in FIG. 11.
[0021] FIG. 13 is a state diagram illustrating an example of the
operation of the
diagnostic device shown in FIG. 1.
[0022] FIG. 14 is a perspective view of an example antimicrobial
dispenser.
[0023] FIG. 15 is a perspective view of the example antimicrobial
dispenser shown
in FIG. 14, illustrating the dispensing of the antimicrobial into a fluid.
[0024] FIG. 16 is a schematic block diagram illustrating an exemplary
architecture
of a computing device that can be used to implement aspects of the present
disclosure.
[0025] FIG. 17 illustrates another exemplary architecture involving a
diagnostic
device.
[0026] FIG. 18 illustrates experimental data obtained using a diagnostic
device.
[0027] FIG. 19 illustrates additional experimental data obtained using a
diagnostic
device.
[0028] FIG. 20 illustrates additional experimental data obtained using a
diagnostic
device.
DETAILED DESCRIPTION
[0029] Various embodiments will be described in detail with reference to
the
drawings, wherein like reference numerals represent like parts and assemblies
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throughout the several views. Reference to various embodiments does not limit
the
scope of the claims attached hereto. Additionally, any examples set forth in
this
specification are not intended to be limiting and merely set forth some of the
many
possible embodiments for the appended claims.
[0030] In general terms, this disclosure is directed to a diagnostic device
that
evaluates microbial content of a sample. In some embodiments, the diagnostic
device
performs one or more of: determining antimicrobial sensitivity of microbes,
identifying
microbes, and counting microbes.
[0031] In some embodiments, the diagnostic device provides a rapid
(e.g., one hour)
antimicrobial sensitivity test. With the increase of antibiotic resistant
bacteria, the
antimicrobial sensitivity test enables medical professionals to prescribe the
correct
antibiotic the first time with less opportunity for antimicrobial resistant
microbes to
evolve. When a patient arrives at an emergency room with signs of sepsis,
those
patients who have an effective antimicrobial regime started within the first
hour have a
better outcome than patients who have delayed treatment. Additionally, the
diagnostic
device can, in some embodiments, identify and/or screen for bacteria that
require
special protocols (e.g. MRSA, vancomycin resistant bacteria, and other such
deadly
bacteria), to assist healthcare professionals in identifying and selecting
treatment
protocols that are effective, while reducing side effects of the treatment.
Counting
bacteria quantities can also be performed by the diagnostic device to quantify
the
severity of an infection.
[0032] In some embodiments, the diagnostic device operates to analyze
microbe
samples developed from human or animal samples of blood, urine, or the like,
by
measuring one or more electrical characteristics of the sample in the sample
cells.
Further, some embodiments of the diagnostic device operate to detect harmful
microbes
on food and to detect harmful corrosive microbes existing in pipelines of
sewers, oil,
gas and chemical plants, for example.
[0033] For each bacteria type there is a unique viral phage that attacks
and kills only
that specific bacterium. When bacteria are attacked by specially cultivated
bacteriophage, bacteria can be made to release ions as the bacteriophage
inject their
DNA into the bacteria thus causing an ionic flux that can be detected
electronically. In
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some embodiments, some of the sample cells in the array are loaded with
nutrient broth,
or with microbes plus nutrient broth, or with different antibiotics with
different viral
phages or antibiotics with one unique phage or antibiotic in each of at least
some of the
sample cells. Such bacteriophages can be embedded in a material that allows
for
controlled elution of the bacteriophage. Since each different viral phage
attacks only
one specific bacterium, the identity of the bacterium in the test can be
determined. In
some embodiments the bacterium identity is determined from analyzing the
difference
in electrical conductivity signatures between the sample cells that contain
only nutrient
broth; nutrient broth and microbes; and nutrient broth, microbes, and viral
phage. In
other embodiments the bacterium identity can be determined from a distinct
signal
generated when the bacteriophage attack their targeted bacteria, which occurs
within the
first fifteen minutes of the bacteria being introduced to the sample cell with
the
bacteriophage impregnated material. It is our hypothesis that bacteriophage
can be
cultivated with long shelf lives while impregnated in such material.
[0034] By performing calibration tests with known concentrations of
different
microbes, the value of the measured conductivity for sample cells that contain
nutrient
broth and microbes can be used to assess the concentration of microbes in the
sample
cell (e.g., count the bacteria in the sample cell).
[0035] Antimicrobials, such as antibiotics and bacteriophages, cause the
metabolism
to slow down or cease, yielding one or more detection mechanisms specific to a
given
antimicrobial agent's ability to eradicate the microbe. Monitoring the
microbes during
the first period of time (e.g., one hour) after the antimicrobial attack
provides a good
indication of the final outcome of the antimicrobial. Other sample cells in
the array
may be loaded with nutrient broth, microbes, and an array of unique
antimicrobial
agents in each of a plurality of the sample cells. Antimicrobial agents
include
antibiotics, antimicrobial peptides, bacteriophage and small molecule drugs,
for
example. In some embodiments, the effectiveness of different antimicrobial
agents in
killing the microbes, such as bacteria, fungi, viruses, nematodes, cell
culture, or tissue,
can be determined by comparing the electrical signal (conductance or
admittance)
signatures of the nutrient broth only; nutrient broth and microbes; and
nutrient broth,
microbes, and antimicrobials in the various sample cells. In other
embodiments, a
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distinct digital signal can be associated with each distinct antimicrobial and
its positive
or negative effect on the microbe's resistance to the distinct antimicrobial,
each
correlated signal thus becoming a digital signature. Comparison of the digital
signature
against a database of digital signatures can determine effectivity of the
antimicrobial.
When bacteria identity can be determined, this information also may optionally
be used
to determine antibiotic effectivity. This is especially important when 13-
lactamase
producing bacteria, such as staphylococcus spp, are identified or other
extended-
spectrum beta-lactamase (ESBL) bacteria are detected, for example.
[0036] In some embodiments, a mixture of bacteria in a sample can also
be detected
using the diagnostic device. For example, if a bacterial sample contains two
different
bacteria, growth of both bacteria will occur in multiple cells but a decrease
in growth
will be seen in more than one cell due to lysis of each of the different
bacteria with a
different bacteriophage.
[0037] Some embodiments of the diagnostic device utilize analog
impedimetric/
conductometric measurement instruments and techniques for the detection and
quantization of bacteria present in a broth culture. Some embodiments also
relate to
methods of manufacturing impedimetric measurement vessels.
[0038] FIG. 1 is schematic perspective view of an example diagnostic
device 100.
In some embodiments, the diagnostic device includes a reader 102, a diagnostic
unit
104, and an interface 106.
[0039] The diagnostic device 100 operates to evaluate microbial content
of a
sample. In some embodiments, the diagnostic device 100 is formed as a single
part.
However, in other embodiments the diagnostic device 100 is formed as at least
two
parts, as shown in FIG. 1, including a reader 102 and a diagnostic unit 104.
An
advantage of forming the diagnostic device 100 of at least two parts is that
the reader
102 can be configured as a reusable part, while the diagnostic unit 104 can be
configured as a disposable part. The sample is contained entirely within the
disposable
part, while at least most of the electronics are contained within the reader.
The interface
106 permits communication between the reader 102 and the diagnostic unit 104.
[0040] In one example, the reader 102 contains electronic components that
operate
in conjunction with the diagnostic unit 104 to evaluate microbial content of
the sample.
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In some embodiments, the reader 102 includes analog electronics that generate
alternating current (AC) signals that are provided to the diagnostic unit 104
for
interrogating the sample. The reader 102 also includes, in some embodiments,
sensing
electronics for detecting one or more characteristics of the sample during the
interrogation to evaluate the microbial content of the sample. Some
embodiments also
include a display device 110, or other output device, for conveying results of
the
microbial evaluation performed by the diagnostic device 100. An example of the
reader
102 is illustrated and described in more detail with reference to FIG. 2.
[0041] The diagnostic unit 104 includes one or more sample cells 112
where the
interrogation of the sample occurs. In this example, the diagnostic unit 104
also
includes a sample input port 114 and a cap 116. A sample is provided into the
input
port 114, and the cap 116 is secured onto the sample input port 114 to enclose
the
sample in the diagnostic unit 104. The sample is directed into the one or more
sample
cells 112. In some embodiments, the sample is directed into the sample cells
112 by the
action of securing the cap 116. Electrodes 118 arranged in the sample cells
are coupled
to the reader 102 through the interface 106. The reader 102 operates to
interrogate the
sample using the electrodes 118 in the sample cells. Examples of the
diagnostic unit
104 are illustrated and described in more detail with reference to FIGS. 3-12.
[0042] In some embodiments, the diagnostic unit 104 measures at least
one
characteristic of the sample. Examples of such characteristics include
electrical
characteristics, such as admittance, conductance, susceptance, and the like.
Changes in
one or more of characteristics of the sample over time are measured in some
embodiments.
[0043] In some embodiments, the diagnostic device 100 operates to
perform one or
more of the following: identify the quantity of a microbe present in a sample,
identify
the type of microbe present in the sample, and determine whether (and to what
extent)
microbes present in the sample are sensitive to an antimicrobial. Examples of
antimicrobials include an antibiotic, a bactericide, a peptide, a
bacteriophage, a
chemotherapeutic, or combinations thereof. Examples of microbes include
bacteria,
fungi, nematodes, cell cultures, and tissues.
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[0044] In order to determine whether the microbes are sensitive to an
antimicrobial,
in some embodiments the antimicrobial is included in at least one of the
sample cells
112. In some embodiments, multiple sample cells 112 each contain different
antimicrobials. In some embodiments, for the purpose of redundancy, which
improves
the accuracy of the diagnostic result, antimicrobials may exist in multiple
sample cells
112. If the microbes present in the sample are sensitive to the antimicrobial,
the
diagnostic unit 104 will detect changes one or more characteristics of the
sample in the
corresponding sample cell 112, such as by comparing it to a control cell that
contains a
sample but no antimicrobial, permitting the diagnostic unit 104 to determine
that the
microbe is sensitive to the antimicrobial in the sample cell 112.
[0045] In some embodiments, identifying a microbe aids in tracking the
source of
the infection, while microbe counting helps to quantify the severity of the
infection, for
example.
[0046] FIG. 2 is a block diagram of an example reader 102 of the
diagnostic device
100. In some embodiments the reader 102 includes a housing 132, electronic
components 134, and a diagnostic unit interface 106A. Some embodiments further
include a micropump 136. In this example, the electronic components 134
include a
power source 142, analog electronics 144 including an AC current source 146
and an
AC voltmeter 148, an AID converter 150, a digital signal processor 160, a
central
processing unit 162, a computer readable medium 164, a display processor 166,
a
display device 168, a communication device 170, a heater controller 172 (and
heating
element), and input device 174. Some embodiments further include a clock and
one or
more voltage meters. Other embodiments include more or fewer components.
[0047] The diagnostic unit interface 106A is a portion of the interface
106, which is
configured to couple the reader 102 to the diagnostic unit 104. As one
example, the
diagnostic unit interface 106A is a card slot configured to receive and
electrically
connect with the reader interface 106B of the diagnostic unit 104. In this
example, an
electrical connection is made between the reader 102 and the diagnostic unit
to permit
the communication of digital or analog electrical signals between the reader
102 and the
diagnostic unit 104. Other types of electrical or data communication are used
in other
embodiments. For example, some embodiments utilize wireless data
communication,
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such as using radio frequency, infrared, or inductive communication devices
and
signals.
[0048] The housing 132 provides a protective enclosure for the reader
102. In some
embodiments the housing 132 is formed of plastic. Other embodiments are formed
of
other materials. The housing 132 includes an interior space which houses at
least some
of the components of the reader 102, such as the micropump 136 and electronic
components 134. In some embodiments, one or more apertures are formed in the
housing 132, such as to permit passage of a conduit coupled to the micropump,
to
permit the diagnostic unit interface 106A to be coupled to the reader
interface 106B
(FIG. 3), and for a communication port of the communication device 170. In
some
embodiments, the communication device 170 includes a plurality of
communication sub
components. For example, in some embodiments the communication device 170
includes an RFID reader, which is used to communicate with the sensor system
192
(and more specifically, with the data storage device 208, all of which are
discussed in
more detail with reference to FIG. 3). The housing 132 can be formed of one or
more
materials. In some embodiments, at least a portion of the housing 132 is
transparent,
such as to permit viewing of the display device 168.
[0049] Some embodiments include a micropump system 136. The micropump
system 136 is connected through a conduit of the interface 106 (or a separate
interface)
to the fluidics system 190 of the diagnostic unit 104, and generates a
pressure
differential to move fluids within the diagnostic unit into the sample cells
112. As
discussed with reference to FIG. 3, some embodiments of the diagnostic unit
104 do not
include a fluidics system, and accordingly the micropump 136 is not needed in
such
embodiments. Alternatively, in some embodiments the diagnostic unit 104 itself
generates pressure differentials without utilizing the micropump 136.
[0050] The power source 142 stores and supplies power to the electronic
components 134. In some embodiments the power source 142 is a battery. Some
embodiments further include battery charging electronics. In other possible
embodiments, the power source 142 includes power supply electronics,
configured to
receive electrical energy from an external power source, such as mains power,
and to
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convert the electrical energy into a suitable form (such as into a relatively
low voltage
signal, such as 3, 12, or +/- 15V direct current).
[0051] The analog electronics 144 are coupled to the diagnostic unit
interface 106A.
In some embodiments, the analog electronics 144 include an AC current source
146 and
an AC voltmeter 148. The AC current source 146 generates AC signals that are
provided to a first set of electrodes in the sample cells 112 of the
diagnostic unit 104
through the diagnostic unit interface 106A. As one example, the AC current
source 146
generates and supplies a continuous AC current. In some embodiments, the AC
signal
is a sine wave having a frequency in a range from about 100 Hz to about 5 kHz.
In
some embodiments, the AC signal is a sine wave having a frequency in a range
from
about 100 Hz to about 10 kHz. Some embodiments have a frequency of about 40
kHz.
Some embodiments have a frequency of about 3 kHz.
[0052] The AC voltmeter 148 receives analog signals that are generated
on a second
set of electrodes in the sample cells 112 of the diagnostic unit 104, through
the
diagnostic unit interface 106A, and determines an AC voltage of the signal. In
some
embodiments, the AC voltmeter 148 determines a voltage across the second set
of
electrodes in the sample cells 112, for example. In some embodiments, the AC
voltmeter 148 can also be operated to determine a voltage across the first set
of
electrodes and the AC current source 146.
[0053] Some embodiments include a plurality of AC current sources 146
and/or a
plurality of AC voltmeters 148 that are directly electrically connected to the
electrodes
118, while in other embodiments one or more multiplexers 143 are arranged
between
the analog electronics 144 and the electrodes 118. The multiplexers 143 and
the analog
electronics 144 are controlled by the central processing unit 162.
[0054] In some embodiments the analog electronics 144 control the period,
frequency, voltage, and current optimized to measure and determine the
admittance, the
conductance, and the constant phase element of the sample in the sample cell
112, and
changes in same over time. The analog electronics 144 are controlled by the
central
processing unit 162 in some embodiments. The AID converter 150 converts the
sensed
values to digital values for further analysis by the digital signal processor
160.
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100551 The digital signal processor 160 executes algorithms to interpret
signals on
the electrodes 118, and is controlled by the central processing unit 162. In
some
embodiments, the digital signal processor 160 accesses a signature database
stored in
the computer readable storage medium 164, and compares the signals with the
signatures. In other embodiments, the digital signal processor 160 directly
interprets the
signals based on recognition algorithms, or a combination of both. Specific
algorithms
used by the digital signal processor 160 are determined by the type of sample
cell 112
which is determined by the diagnostic unit 104 model number, for example.
[0056] Some embodiments include a central processing unit 162. The
central
processing unit 162 is an example of a processing device. In some embodiments,
the
central processing unit 162 controls the overall operation of the diagnostic
device 100.
For example, the central processing unit 162 controls the operation of the
micropump
136 in some embodiments, selects a mode of operation of the electronic
components
134, and controls the electronic components 134 according to the selected mode
of
operation (as discussed in further detail with reference to FIG. 12), and
communicates
with external devices through the communication device 170.
[0057] The computer readable medium 164 is communicatively connected to,
or
part of, the central processing unit 162, and/or one or more of the other
processing
devices (e.g., digital signal processor 160 and display processor 166) of the
reader 102.
An example of a computer readable medium is a computer readable storage
device, as
discussed herein.
[0058] The display processor 166 operates to control the one or more
display
devices 168 to convey information in a visual form to a user. In some
embodiments, the
display processor 166 also acts as an input device when the display device is
a touch
sensitive display. In one example, the display device 168 is a plurality of
light sources,
such as light emitting diodes (LEDs). As another example, the display device
168 is a
two-dimensional display, such as a liquid crystal display (LCD), LED display,
and the
like.
[0059] A communication device 170 is provided in some embodiments to
permit
communication between the reader 102 and another device, such as a computing
device,
an RFID storage medium, or a data communication network. In some embodiments,
the
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communication device 170 includes multiple communication devices. In some
embodiments the communication device 170 includes a communication port for
connection with a communication cable, such as a USB cable or an Ethernet
cable. In
other embodiments, the communication device is a wireless communication
device,
such as an RFID reader, a Wi-Fi communication device, a cellular communication
device, or a Bluetooth communication device.
[0060] Some embodiments further include a heater controller 172 and
heating
element that is configured to apply heat to the diagnostic unit 104 to achieve
and
maintain a temperature conducive to microbial growth. In some embodiments, the
heating element is arranged in a heating pad (i.e., an incubator warmer),
which can be
external, or partially external, from the housing of the reader 102, and
arranged so that
at least a portion of the diagnostic unit abuts the heating pad. In some
embodiments, the
heating pad is a silicone heating pad. In some embodiments, the heating
element is
formed of tungsten or nichrome wire. In some embodiments a thermocouple, or
other
temperature detecting device is provided in the heating pad, in the reader
102, or in the
diagnostic unit 104, to provide feedback to the reader 102 to permit the
reader 102 to
maintain a desired temperature, or range of temperatures, within the sample
cells 112.
In some embodiments the thermocouple is inserted directly into the
electrolytic
solution.
[0061] Some embodiments include one or more input devices 174. The input
devices 174 can include buttons, switches, touch-sensitive displays, and the
like. Other
interface devices can also be used, such as an audio (e.g., voice) interface.
The input
devices can be used to turn the diagnostic device 100 on or off, and to adjust
a mode of
operation of the device, such as to adjust the device between the first and
second states
of operation, as described herein with reference to FIG. 11. Other inputs are
provided
in other embodiments.
[0062] The exemplary components of the reader 102 are provided by way of
example only. Other embodiments can include more or fewer components. Further,
in
some implementations, some of the components can be combined into a single
component.
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[0063] In some embodiments, the reader 102 is formed of two or more
parts. For
example, in some embodiments the reader 102 includes an integral cellular
telephone.
In another possible embodiment, the reader 102 is configured to receive and
cooperate
with a cellular telephone. In another embodiment, the reader 102 includes a
computing
device, such as a mobile computing device (e.g., smart phone, laptop computer,
tablet
computer, etc.), a desktop computer, or other computing devices. The computing
device can be integrated into the reader 102, or external from and in data
communication with the reader 102, for example.
[0064] Other reader 102 configurations are also possible. For example,
another
possible configuration of a reader 102 including multiple parts includes a
first part and a
second part. The first part of the reader includes a housing which contains at
least some
of the electronic components 134. The second part of the reader has its own
housing
and forms a warming cradle, including at least the heating element of the
heater
controller 172. The first part connects with the second part through a first
interface,
which permits communication between the sensor and the warming cradle. The
second
part connects with the diagnostic unit 104 through the diagnostic unit
interface 106A.
[0065] FIG. 3 is a block diagram of an example diagnostic unit 104 of
the
diagnostic device 100 shown in FIG. 1. In this example, the diagnostic unit
104
includes a housing 188, a fluidics system 190, and a sensor system 192.
[0066] Also in this example, the input to the fluidics system 190 includes
a sample
input port 114 and a cap 116. The example fluidics system 190 includes a
filtration
system 202, including a fluid source 203, and a sample distribution system
204,
including a manifold 206. The example sensor system 192 includes sample cells
112
and electrodes 118.
[0067] In some embodiments, the diagnostic unit 104 receives a sample
through the
sample input port 114. In some embodiments, the diagnostic unit 104 further
includes a
sample input receptacle having an internal volume suitable to temporarily
store part or
all of the sample as it is received.
[0068] A wide variety of samples can be used in various embodiments. In
some
embodiments, samples are obtained from a subject suspected of having an
infection
with a microbe, from a food or water sample, a soil sample, or from a surface
or other
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environmental source. Samples obtained from a subject can include or be
obtained
from urine, blood, sweat, mucus, saliva, semen, vaginal secretion, vomit,
tears, sebum,
pleural fluid, peritoneal fluid, gastric juice, earwax, cerebrospinal fluid,
breast milk,
endolymph, perilymph, aqueous humor, vitreous humor, biomass, mucous
membranes,
stool sample, infected cells or tissues, lung lavage, cell extracts, biopsies
and
combinations thereof, for example. Samples can further include sources for
yeast,
fungi, viruses, nematodes, cell culture, or tissue.
100691 Once the sample has been received into the sample input port 114,
in some
embodiments a cap 116 is provided, which can be secured onto the sample input
port
114. In some embodiments the cap 116 is a locking cap, which includes a
locking
feature that resists removal of the cap after the cap has been secured to the
sample input
port 114. In this way, the sample is contained within the housing 188 of the
diagnostic
unit 104. In some embodiments the housing 188 (including cap 116) forms a
sealed
enclosure. In some embodiments the sealed housing permits the diagnostic unit
104 to
be discarded while continuing to contain the biological materials, which may
be
considered a biohazard, within the sealed enclosure of the housing 188.
Therefore, in
some embodiments the diagnostic unit 104 is a single-use disposable unit.
[0070] In some embodiments the cap 116 drives a mechanical cam attached
to a
plunger that creates different pressures within the diagnostic unit 104, which
then drives
the automation of the fluidics system 190. In other embodiments the automation
of the
sample handler is driven by a micropump 136 contained in the reader 102.
100711 In some embodiments, the received sample is delivered to the
sensor system
192 by the fluidics system 190. In other embodiments, however, the fluidics
system
190 is omitted, and manually processed sample that has been re-suspended in
the
nutrient broth designed to work with the sensor system 192, and may be input
by the
user directly into the sensor system 192 along with the antimicrobial
dispenser 282.
Some such embodiments give users the flexibility to customize the device for
their
specific application. For example, in some embodiments the reader 102 includes
a
customizing application which permits the users to identify the customizations
made to
the diagnostic device 100.
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[0072] In some embodiments, the fluidics system 190 transfers the
received sample
from the sample input port 114 (or sample input receptacle) to the sensor
system 192
after filtering and mixing the sample with nutrient broth from the fluid
source 203. In
some embodiments the fluidics system 190 is driven by the micropump 136 of the
reader 102, shown in FIG. 2. In other embodiments, the filtration system 202
is driven
by another source, such as by pressures formed by the insertion and rotation
of locking
cap 116. In some embodiments, the cap 116 is attached to a cam system wherein
turning of the cap causes a plunger to create pressure differentials to drive
the fluidics
system 190.
[0073] In some embodiments, the fluidics system 190 includes a filtration
system
202 that filters the received sample. As one example, the filtration system
202 is
specialized to handle urine and includes one or multiple filtering stages,
such as
including a first stage and a second stage. In the case of a urine sample, the
first stage
of the filtration system 202 may be provided to remove blood and protein from
the urine
sample. In the second stage, the microbes are removed from the urine sample.
The
microbes may then be removed from the second stage for further evaluation by
the
diagnostic unit 104. The remaining urine including wild bacteriophages are
passed to a
waste receptacle.
[0074] In some embodiments, a sample is processed through the filtration
system
202 before placing the samples in the sample cells. In some embodiments, for
example,
the samples are filtered to remove larger particles, cells, and cell debris.
In some
embodiments, a filtration system 202 is provided that passes the sample
through a filter.
The filter can have apertures measuring about 5 microns, for example. The
filter allows
the passage of the microbes while retaining larger particles. The filtration
system can
further include a secondary filter. The secondary filter can be used to remove
unwanted
medium in the sample (e.g. urine), such as using a smaller sized filter (e.g.,
having
apertures measuring about 0.45 microns) leaving the bacteria on the surface of
the filter
so it can be removed and suspended in the nutrient solution for further
testing by the
diagnostic device 100. In some embodiments, an additional filtering stage is
provided
between the first and second filtering stages discussed above to capture wild
bacteriophage. For example, a filter having 0.22 micron apertures can be used.
After a
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period of time these wild phage can then be reintroduced into the sample cells
containing antimicrobials to detect remaining live bacteria since
bacteriophage will only
attack live bacteria. All bacteria have a host of wild phages in any sample.
[0075] Some embodiments of the filtration system 202 also include a
fluid source
and a mixing device. The fluid source 203 provides a source of a fluid that
can be
mixed with the sample for use within the sensor system 192. The fluid may be a
single
fluid or a combination of fluids. An example of a fluid is an electrolytic
solution, such
as including an electrolyte. One example of an electrolytic solution is a
culturing broth.
Another example is a culturing broth combined with one or more other culturing
broths
or other fluids. The electrolyte or nutrient broth should support the growth
of the
microbe being tested. A sample, as used herein, refers generally to any fluid
containing
at least a portion of the biological fluid (or any other fluid, material, or
other input)
received in the sample input port 114, including before or after filtering
and/or mixing
with another fluid.
[0076] To obtain accurate repeatable results, it is desirable that the
ionic makeup be
tightly controlled within predefined ranges. Further, because real-time
monitoring of
microbe life signs is desired, the electrolytic solution must support and even
stimulate
the microbe growth.
[0077] The sample distribution system 204 is configured to distribute
the received
sample to the sample cells 112 in the sensor system 192. In some embodiments,
the
sample distribution system 204 includes a manifold 206 that evenly mixes and
delivers
a homogenous sample to at least some of the sample cells 112. Some embodiments
include a manifold 206 that does not deliver the sample to all of the sample
cells 112, to
permit one or more of the sample cells 112 to be used as a control cell. In
some
embodiments the sample distribution system 204 includes a metering device that
provides a substantially equal quantity of the sample to the sample cells 112.
In another
possible embodiment, fill level sensors on the sample cells 112 operate to
provide
feedback to the fluidics system 190 to obtain appropriate fill levels of the
sample in the
sample cells 112.
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[0078] The sensor system 192 includes one or more sample cells 112 and
electrodes
118 arranged within the sample cells. An example of the sensor system 192 is
illustrated and described in more detail with reference to FIG. 4.
[0079] In some embodiments, the sensor system 192 (or elsewhere in the
diagnostic
unit) includes a data storage device 208 for storing data, such as patient
information,
diagnostic results, diagnostic unit model number, diagnostic unit serial
number, or
combinations thereof In some embodiments, the data storage device 208 is a
passive
read-write RFID device, for example. In some embodiments, the data storage
device
208 can be written to and read by an RFID reader of the communication device
170
(FIG. 2) of the reader 102.
[0080] A reader interface 106B is provided in some embodiments to permit
electrical or data communication between the diagnostic unit 104 and the
reader 102
(FIG. 2). As one example, the reader interface 106B includes a card-type
interface
having a plurality of electrical contact pins that is insertable into a
corresponding card
slot of the diagnostic unit interface 106A of the reader 102. Other interfaces
are used in
other embodiments, such as a data communication port (e.g., USB, serial, etc.)
or a
wireless communication device. The reader interface 106B permits communication
between the reader 102 and the electrodes 118 for interrogation of the sample
in the
sample cells 112.
[0081] In some embodiments, the diagnostic unit 104 is coupled to the
reader 102
(FIG. 2) to conduct a diagnostic evaluation for a period of time of at least 1
minute or
less, 5 minutes or less, 10 minutes or less, 15 minutes or less, 20 minutes or
less, 30
minutes or less, 45 minutes or less, or 60 minutes or less.
[0082] FIG. 4 is a schematic perspective view of an example sensor
system 192 of
the diagnostic unit shown in FIG. 3. In this example, the sensor system 192
includes a
base substrate 222, a plurality of sample cells 112 (including sample cells
112A to
112X), and electrodes 118.
[0083] In this example, the sensor system 192 includes a base substrate
222. An
example of a base substrate 222 is a circuit board, such as a printed circuit
board.
Another example of the base substrate 222 is a flexible substrate, such as a
flex circuit.
The base substrate can be formed of one or more layers and includes at least
one
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insulating layer. One or more conductive layers are provided in some
embodiments,
such as a ground plane or one or more layers including electrical traces. In
some
embodiments, the base substrate 222 includes electrical conductors between the
electrodes and the reader interface 106B (not visible in FIG. 4).
[0084] The sample cells 112 are arranged on and supported by the base
substrate
222. In some embodiments the sample cells 112 are formed of a single piece of
material, while in other embodiments the sample cells 112 are individual
pieces. In yet
other embodiments, a subset of the sample cells 112 are formed of a single
piece (e.g.,
each row of sample cells 112 can be formed of a single piece of material). In
one
example embodiment, the sample cells 112 are made of plastic, such as molded
plastic
or injection molded plastic. The sample cell 112 material can be constructed
out of a
medically approved insulating material. It is preferred that the material does
not have
an adverse effect on the growth of microbes in the sample. The sample cells
112 are
coupled to the base substrate 222 by a fastener, such as adhesive or other
bonding layer
or material. Further, some embodiments include one or more materials between
the
sample cells 112 and the base substrate 222, such as a gasket layer. In some
embodiments, the sample cells 112 are bonded to the base substrate 222. The
one or
more fasteners that connect the sample cells to the base substrate 222 are
preferably
configured to inhibit leakage of the sample or other fluid out of and between
the sample
cells 112. In some embodiments, the sample cells 112 are molded around
electrodes in
a lead frame.
[0085] In some embodiments, the sensor system 192 includes a plurality
of sample
cells 112. In the illustrated embodiment, the sensor system 192 includes an
arrangement of 24 sample cells 112A-X. In some embodiments the sample cells
are
arranged in a grid of rows and columns. In this example, the sample cells are
arranged
in four rows and six columns. In other embodiments, the sensor system 192
includes a
plurality of sample cells 112 in a range from 2 to 50, or 2 to 48, or 2 to 36,
or 2 to 24.
The sample cells can be arranged in one or more rows (e.g., 1, 2, 3, 4, 5, 6,
7, 8, or more
rows). The sample cells can have cubed, cylindrical, or rectangular shapes,
for
example, and can also have other configurations, such as hexagonal shapes,
etc.
Further, in some embodiments the sample cells 112 are all formed of a single
piece of
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material. For example, the sample cells 112 are formed of a single piece of
plastic
material that is molded around a lead frame which forms the electrodes, in
some
embodiments.
[0086] The sample cells 112 include therein a sample chamber. Electrodes
118 are
arranged within the sample cells 112, to interact with the sample for
determining the
one or more characteristics of the sample, as discussed in further detail
herein. More
specific examples of the sample cell 112 are illustrated and described in more
detail
with reference to FIGS. 5-10 herein.
[0087] In some embodiments, the shape of the sample chamber is tuned to
provide
more accurate measurements of the characteristics, including positioning the
electrodes
such that the shapes of the electric fields that are generated by the
electrical signals
applied to the electrodes are optimized for signal-to-noise performance.
[0088] In some embodiments, the sample cells 112 are manufactured so
that the size
and shape of the sample chambers are substantially the same. In this way, the
sample
cells have similar dimensional constants, permitting the diagnostic device to
make
comparisons between measured characteristics of one or more sample cells as
compared
with the measured characteristics of one or more other cells, as discussed in
further
detail below.
[0089] In some embodiments, the electrodes 118 are formed on the base
substrate
222. To improve the seal between the sample cells 112 and the base substrate
222, the
electrodes can be formed within recessed regions formed in the surface of the
base
substrate 222, such that the surfaces of the electrodes are flush with the
surface of the
base substrate 222. In some embodiments the recessed regions are nanowells. In
another possible embodiment, the electrodes are formed on the surface of the
substrate
222. In yet other possible embodiments, the electrodes can be arranged in
other
locations, such as on the walls of the sample chamber of the sample cells 112.
In a
further embodiment, the sample cells 112 include a bottom surface, and the
electrodes
118 are arranged on the interior side of the bottom surface, or side walls
near the bottom
of the sample cells 112.
[0090] The electrodes 118 can be made from one or more of a variety of
materials,
such as any noble metal, a metal coated with graphene or graphenol-like
substances, or
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combinations of one or more of these (e.g., metal alloys). As one example, the
electrodes 118 are formed of metal pins. In another possible embodiment, the
electrodes 118 are gold plated. In some embodiments, the electrodes are gold
plated
electrodes patterned onto the base substrate 222. In some embodiments, the
electrodes
118 are coated with a graphenol-like substance applied directly to copper
traces on the
base substrate 222 before or after the solder-mask is applied (in the case of
a printed
circuit board, for example). Other embodiments use gold or graphene directly
printed
onto a flexible plastic substrate to make flex circuits. Other embodiments
utilize
electrodes formed from the exposed tips of a lead frame molded into the
plastic
diagnostic unit 104.
[0091] In some embodiments, the size of each of the electrodes and the
distance
apart of each of the electrodes is precisely controlled. In embodiments, the
size of the
electrodes is substantially the same, that is, the electrodes have a
difference in size of
less than 5%, 1%, 0.1%, 0.01%, or .001%. In some embodiments, the distance
between
the electrodes in different sample cells is substantially the same, that is,
having a
difference in distance between electrodes of less than 5%, 1%, 0.1%, 0.01%, or
0.001%.
In some embodiments, the electrode sizes and distances between electrodes are
controlled to a difference of 1% of less.
[0092] In some embodiments, the sample cells further include
antimicrobial
dispensers 282, as illustrated and described in more detail with reference to
FIGS. 7 and
12-13.
[0093] FIGS. 5-7 illustrate an example of a sample cell 112.
[0094] FIG. 5 is a schematic perspective view of the example sample cell
112.
Portions of the sample cell 112 are depicted as transparent to illustrate the
interior
structure of the sample cell 112. In this example, the sample cell 112
includes a body
240, an input opening 242, a sample chamber 244, and electrodes 118. In some
embodiments the sample chamber 244 includes a chimney 246 and an interrogation
region 248.
[0095] A sample is received through the opening 242. In some
embodiments, the
input opening 242 is coupled to the manifold 206 of the sample distribution
system 204
shown in FIG. 3. The opening 242 includes a coupling port in some embodiments,
such
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as configured to be connected to a fluid delivery conduit, such as tubing, to
connect the
opening 242 with the manifold 206. The sample distribution system 204 delivers
the
sample through the manifold and into the input opening 242. In another
possible
embodiment, the sample is provided directly by a user or another device into
the input
opening 242, such as using a pipette or other sample container or fluid
delivery conduit.
[0096] After the sample has passed through the input opening 242, the
sample then
passed through the chimney 246 of the sample chamber 244, and then into the
interrogation region 248.
[0097] Electrodes 118 arranged within the interrogation region 248 of
the sample
chamber 244 are electrically coupled to electronic circuitry, such as the
analog
electronics 144 of the reader 102 (shown in FIG. 2), which operate to generate
electrical
signals at one or more of the electrodes 118, and to detect electrical signals
at one or
more of the other electrodes 118. The detected electrical signals are then
used to
evaluate one or more characteristics of the sample.
[0098] FIG. 6 is a schematic side elevational view of the example sample
cell 112,
shown in FIG. 5. In this example, the sample cell 112 includes the body 240,
the input
opening 242, the sample chamber 244, and electrodes 118.
[0099] In this example, the sample chamber 244 includes both the
interrogation
region 248 and the chimney 246. The interrogation region 248 is sized to hold
a precise
volume of the sample. It is preferred that the interrogation region be
entirely filled
before interrogating the sample, to provide uniform results among the sample
cells 112.
When electrical signals are applied to one or more of the electrodes 118,
electrical
currents as well as electric fields are generated within the sample. If the
interrogation
region is not entirely filled, the currents and electric fields produced
within the sample
are modified, potentially resulting in a change in the one or more measured
characteristics of the sample. Therefore, for a given type of sample (e.g.,
blood, urine,
etc.), the volume of the interrogation region 248 is selected to be small
enough that it
can be filled by the sample based on sample volumes that can typically be
obtained for
that given type of sample. In some embodiments, the volume of the
interrogation
region 248 is in a range from about 0.1mL to about 10mL, or from about 0.5mL
to
about 2mL, or from about lmL to about 1.5mL.
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[00100] The chimney 246 extends from the interrogation region and is provided
in
some embodiments to contain an additional volume of the sample, in addition to
the
volume of the interrogation region 248. In this way, the volume of the sample
does not
have to be precisely measured to match the volume of the interrogation region
248
exactly, but rather can be somewhat greater than the volume of the
interrogation region
248¨up to the combined volume of the interrogation region 248 and the volume
of the
chimney 246. In some embodiments the volume of the chimney is in a range from
about 0.01mL to about 2mL, or from about 0.1mL to about 0.2mL, or about
0.14mL. In
some embodiments the volume of the chimney is in a range from about 5% to
about
20% of the volume of the interrogation region, or from about 1% to about 10%,
or about
10%.
[0100] The configuration of the chimney 246 permits some variation in
the sample
volume without significantly affecting the measured characteristics of the
sample. For
example, the chimney 246 has a cross-sectional dimension (W2) that is much
less than
the cross-sectional dimensions (W1) of the interrogation region 248.
Additionally, the
chimney 246 extends away from the interrogation region 248, and does not
provide a
return path for current to flow through the chimney 246. As a result, when an
electrical
signal is applied to one or more of the electrodes 118, very little electrical
current is
conducted through any portion of the sample that is within the chimney.
Therefore,
whether the level of the sample is at or near the top of the chimney 246
(i.e., at opening
242), at or near the bottom of the chimney 246, or somewhere in between, the
one or
more measured characteristics of the sample are not significantly changed. The
chimney 246 therefore provides a sample volume buffer that permits variations
in the
volume of the sample up to the total volume of the chimney 246.
[0101] In this example, the chimney has a width (W2) and an equal depth
(D2, not
shown), and a height (H2). The volume of the chimney is W2 x D2 X H2. The
volume
can therefore by adjusted by increasing or decreasing any of these dimensions.
For
example, the volume can be increased or decreased by adjusting the height (H2)
of the
chimney. In one example, the width W2 is in a range from about lmm to about
20mm,
or from about 2mm to about 6mm, or from about 4mm to about 5mm, or about
4.5mm.
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In this example, the height H2 is in a range from about lmm to about 50mm, or
from
about 5mm to about lOmm, or from about 6mm to about 8mm, or about 7mm.
[0102] In some embodiments, the interrogation region 248 includes a
central region
262 and radially extending arms 264 (including arms 264A to 264D). As best
shown in
FIG. 7, in some embodiments the interrogation region 248 has a cross-sectional
shape
of a plus, a cross, or an
[0103] In this example, the central region 262 has a square horizontal
cross section
and a rectangular vertical cross section. For example, the central region 262
has a width
(W2), an equal depth (D2) (not shown in FIG. 6), and a height (H3+H4). In some
embodiments, the width (W2) is the same as the width (W2) of the chimney. In
other
embodiments, the width of the central region 262 is different than the width
of the
chimney. In one example, the height (H3+H4) of the central region 262 is in a
range
from about 2mm to about 35mm, or from about 5mm to about 20mm, or from about
lOmm to about 14mm, or about 12mm.
[0104] Four arms 264 extend radially from the central region 262. Each of
the arms
has a straight region 266 and terminates in a semi-cylindrical shaped region
268. The
straight region 266 has a tapered height that varies from H4 to (F13+H4). The
semi-
cylindrical shaped regions 268 have a diameter equal to the depth (D3, not
shown) of
the straight region 266, and a height (H4). In one example, the length of the
straight
region 266 is in a range from about lmm to about 20mm, or from about 2mm to
about
6mm, or from about 4mm to about 5mm, or about 4.5mm. The length can be greater
than or less than the length W2 of the central region 262. In this example,
the
diameter of the semi-cylindrical shaped regions 268 are in a range from about
lmm to
about 20mm, or from about 2mm to about 6mm, or from about 4 mm to about 5 mm,
or
about 4.5 mm. The height (H4) of the semi-cylindrical region is in a range
from about
2mm to about 30mm, or from about 5mm to about 20mm, or from about 8mm to about
12mm, or about 9.6mm.
[0105] In some embodiments, an upper portion 250 of the interrogation
region 248
has a tapered shape. If bubbles are present in the sample within the
interrogation region
248, such bubbles could potentially change the one or more measured
characteristics of
the sample. The tapered shape of the upper portion 250 collects the bubbles as
they rise
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to the top of the interrogation region 248 and directs the bubbles toward and
into the
chimney 246. The bubbles then rise through the chimney 246 to the surface of
the
sample and exit the sample. The accuracy of the sample measurements are
therefore
improved. In this example, the upper portion 250 has a taper angle Al. In some
embodiments the taper angle Al is in a range from about 10 degrees to about 80
degrees, or from about 10 degrees to about 45 degrees, or from about 10
degrees to
about 20 degrees. Some embodiments have an angle Al of about 15 degrees.
Although
this example illustrates the tapered upper portions 250 terminating before the
semi-
cylindrical shaped regions 268, in other possible embodiments the tapered
upper portion
250 extends to the ends of the arms 264.
[0106] The exemplary dimensions described herein are provided by way of
example
only. Other embodiments can have dimensions that are greater or less than the
dimensions discussed herein. Additionally, the overall dimensions of the
sample cell
112 can be any desired dimensions greater than (or equal to) the dimensions of
the
sample chamber 244.
[0107] FIG. 7 is a schematic top plan of the example sample cell 112,
shown in
FIG. 5. FIG. 7 also illustrates the base substrate 222, electrical conductors
280
(including conductors 280A-D), and an example antimicrobial dispenser 282, as
well as
the AC current source 146 and AC voltmeter 148 of the reader 102 (shown in
FIG. 2).
[0108] As previously described, this example of the sample cell 112
includes a body
240 and a sample chamber 244. The sample chamber 244 includes an opening 242,
a
chimney 246, and an interrogation region 248. The interrogation region 248
includes a
central region 262 and arms 264 (including arms 264A-D). The sample cell 112
is
arranged on a base substrate 222 in some embodiments, and electrodes 118
(including
electrodes 118A-D) and the antimicrobial dispenser 282 are arranged thereon.
[0109] The cross-sectional shape of the example sample chamber 244 is
shown in
FIG. 7, which has the general shape of a plus, cross, or "X", in which the
arms 264
extend radially from the central region 262 and extend at right angles to
adjacent arms.
For example, arms 264A and 264C extend perpendicular to arms 264B and 264D,
while
arm 264A extends parallel with arm 264C, and arm 264B extends parallel with
arm
264D.
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[0110] In this example, electrodes 118 are arranged on the base
substrate 222 at one
end of each of the arms 264. Each of the electrodes 118A-D is electrically
coupled to
an electrical conductor 280A-D, respectively. The electrical conductors 280
are
coupled to the analog electronics 144 of the reader 102. For example,
electrodes 118A
and 118B are electrically coupled to the AC current source 146, and electrodes
118C
and 118D are electrically coupled to the AC voltmeter 148. Electrode 118A
operates as
a low current (LcuR) terminal. Electrode 118B operates as a high current
(HcuR)
terminal. Electrode 118C operates as a high potential (HpoT) terminal.
Electrode 118D
operates as a low potential (LpoT) terminal. In some embodiments, additional
electrical
connections are possible, such as by using a multiplexer, as discussed herein.
In some
embodiments, the AC voltmeter 148 is capable of reading voltages across
electrodes
118A and 118B, as well as across electrodes 118C and 118D, or other
combinations of
the electrodes.
[0111] In some embodiments, the measurement of conductance is
insensitive to the
capacitive reactance at the driven or forced electrodes (i.e., 118A-B). The
measurement
is also insensitive to capacitive reactance at the voltage sensing electrodes
(i.e., 118C-
D) because the reactance is insignificant compared to the input impedance of
the
voltage sensing instrument at frequencies at or above a few hundred Hz. Low
frequency performance is improved by the use of larger electrodes 118 to
produce more
capacitance from the electrode polarization.
[0112] In some embodiments, the four electrode sample cell (including
any one of
the examples shown in FIGS. 5-12) provides direct measurement of conductivity
scaled
by a geometry constant. Scaling the conductivity of the sample cell 112
contents by the
geometry constant yields the measured conductance.
[0113] Geometry constant () as defined herein relates conductance (G) and
conductivity (lc) as:
Go
[0114] where Go and ico are reference values at a particular
temperature.
[0115] The geometry constant can be computed from the conductance at a
temperature G(T) with knowledge of the temperature coefficient (c) as:
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G(T)
= Kna+m-1.0)) =
[0116] An average of a group of individual geometry constants is thus:
¨ =
[0117] If the group of cells contain a variety of electrolytic contents,
the average
geometry constant can be indicated as an effective value (e) under the
assumption that
the conductivities and temperature coefficients are reasonably matched. Then,
a unique
scaling constant can be derived for each conductance curve as a function of
time, so that
the scaled curves appear as they would if the cells had well matched geometry
constants
near the average of the actual geometry-constant values. A set of scaling
constants can
be obtained from the above geometry constants as:
= , C, = , C =
C .
1 6;
[0118] Under the above assumptions, KO cancels out of the scaling
constants. For
example:
14- __________________________ 4-= -4- __
Cl
Gig 0) G CT o) G(T)
= = Gr,(To)
1+ (T9¨T0)
[0119] These equations applied over an entire set of data produces a C,,
set for each
point in time.
[0120] The inability to measure temperature accurately in the presence
of thermal
gradients, such as during a rapid warm-up period, confounds the ability to
compute
accurate G(To) values and hence the above scaling constants. However,
averaging the
scaling constant of each cell over a period of time following the warm-up
yields a
single, useful value. Each conductance at temperature G(T) function of time
can be
multiplied by the corresponding scaling constant (C,,) to get the scaled
function:
G(T)= CGõ(T) .
[0121] For the case with all the test cells at the same temperature, the
scaling
constant equation from the above example simplifies to.
r G1 Gi
- ¨ =
[0122] Accurate conductivity measurements of electrolytic solutions
facilitate
quantitative comparisons of the ionic content within multiple sample cells
112. If an
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electrolytic solution includes a nutrient media (sometimes alternatively
referred to
herein as broth) with the addition of living microbes, the presence of the
microbe is
detectable as an effective increase in ion content due to metabolic activity
of the
microbe on the broth components. The increase in conductivity caused by the
presence
of live bacteria can be expressed as the difference in conductivity
measurements
obtained with a sample cell containing broth with bacteria (Cell 1) and a
sample cell
containing broth only (Cell 0). In this example, the sample cells have four
electrodes
each (118A-D), one pair (118A-B) to deliver electrical current ("forced
electrodes")
from the AC current source 146, and one pair (118C-D) to sense the voltage
developed
inside the sample cell in response to the current ("sensed electrodes"), as
measured by
the AC voltmeter 148. If two sample cells are dimensionally well matched, a
scaled
difference in conductivity is obtained from the difference in conductance
measurements
as:
_______________________________________________ /fo
KB G1 GO
Vs Vso
where Ics is the proportion of the conductivity in Cell 1 attributable to
bacterial activity,
G1 is the conductance of Cell 1, Go is the conductance of Cell 0, and ko are
the
respective dimensional constants of Cell 1 and Cell 0, If1 and Ifo are the
respective
currents flowing in Cell 1 and Cell 0, and Vs1 and Vso are the respective
sensed
voltages of Cell 1 and Cell 0 . If the cells are not well matched, the
respective values k
and ko are applied to Cell 1 and Cell 0:
Go
KB = K1. KO
71
where K1. and Ko are the respective conductivities of the electrolytic
solutions in Cell 1
and Cell 0.
101231 Since the magnitude of KB increases monotonically with the number
of
bacteria colony forming units (CFU) present in sample cell 1, it provides a
basis for
counting (or quantifying) bacterial concentration and real-time monitoring its
change
across time.
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[0124] By the same method used to obtain Ks , another sample cell
containing
broth, bacteria and an antimicrobial agent (Cell 3) has a portion of the
conductivity (KA )
attributable to bacterial activity countered by the antimicrobial agent
provided by the
antimicrobial dispenser 282:
G3 Go
KA = = K13
[0125] Relationships between Ka , KB and KA can be analyzed in real time
to detect
increasing and decreasing bacterial metabolic activity. From these data
relationships,
the effectiveness of the given antimicrobial agent can be evaluated.
[0126] In some embodiments, temperature compensation is beneficial when
quantifying conductance measurements of microbial broth solutions, or when
comparing results from separate tests. Some broth recipes yield conductivities
with
linear coefficients of temperature typically around 20,000 ppm/ C. Rapid test
results
require mixing and transferring broth cultures into sample cells and not
waiting for
thermal equilibrium conditions before beginning the test. Microbial broth
cultures are
incubated to a normal human body temperature, such as 35 C, 37 C , or in a
range
from about 35 C to 37 C (normal human body temperature) to promote growth, so
some control of the temperature is necessary, but fast and highly accurate
feedback
control of the incubator temperature would add cost and complexity to the
measurement
system. Compensation can be applied to test data if the temperature of the
broth is
monitored during testing, but accurate direct monitoring may add cost and
complexity
particularly undesirable for a single-use disposable sensor. An indirect
method of
temperature compensation that does not require control or monitoring of the
temperature would be advantageous in cost and performance. The following
describes
such a method appropriate when testing a plurality of sample cells in unison
that is used
in some embodiments.
[0127] A control cell containing only broth (Cell 0 above) can be used
to indicate
temperature if the conductivity at a reference temperature and the temperature
coefficient are known:
T
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where T is the broth temperature at which the conductivity Cc (T)is measured,
To is the
reference temperature at which the broth conductivity KO CFO) is known and is
the
temperature coefficient.
[0128] If another cell containing broth with bacteria (Cell 1 above) is
measured at
the same temperature T as Cell 0, the conductivity at the reference
temperature can then
be expressed as:
(To) = irt (T) ¨'(T¨T0)
[0129] Combining these last two equations gives:
i(T0) = (T) ¨ Ko(T) -1- Ko (To)
[0130] Assumptions using this method are: the microbial concentration added
to the
broth has negligible effect on the temperature coefficient, and the sample
cells differ in
temperature by a negligible amount. Notice that no measurement of temperature
is
necessary during the test, and that the temperature coefficient need not be
known.
[0131] An alternative method can be used to express the conductance of a
sample
cell, or the conductivity of its contents, at an arbitrary temperature value
within the
temperature range occurring over the test duration. Using again Cell 0 and
Cell 1 as
defined above and with the same assumptions, the measured broth conductance in
Cell
0 at an arbitrary test point can be defined as GoA. A set of correction ratios
(Rn) can
then be generated at each test point as:
Rn= GOA/GOn
[0132] Then, if each measured conductance value, Gron, is multiplied by
the
appropriate value of Rn all Gon conductivity values will be corrected to the
Go n value.
[0133] This same set of correction ratios, R, applied to the conductance
of Cell 1
will result in removing the temperature dependence of the broth from the broth
plus
bacteria conductance values, for example.
Temperature Compensated Gi n G1 comp Rn* G n
[0134] Remaining differences between the broth only and broth plus
bacteria
conductance will then be due entirely to the presence of the bacteria.
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[0135] Note that GoA need not be an actual data-point measurement. In a
preferred
embodiment GoA may be defined as the mean average value of the measured Go n
data
set. Then, U1 comp represents the data as though the temperature had been held
constant
at the average value attained over the test duration.
[0136] Bacteria, when attacked by bacteriophages, admit quantities of ions
which
can be detected by this measurement technique even against the background
conductivity of the medium. Bacteriophages can be cultivated so that they will
attack
one and only one bacteria species or sub-species. Additionally, the
bacteriophages can
be selected so that it causes the bacteria to release ions during the initial
attack. When
using a bacteriophage that attacks one and only one bacteria, identification
of the
bacteria is possible by observing the ionic surge and the eventual reduction
in live
bacteria which occurs during the period (i.e., the first five to fifteen
minutes) after the
introduction of the cultivated bacteriophage and the target bacteria enabling
rapid
identification of the bacteria.
[0137] Ultimately, antimicrobials¨specifically antibiotics and
bacteriophages¨
cause the metabolism to slow down or cease, yielding one or more detection
mechanisms specific to a given antimicrobial agent's ability to eradicate the
microbe.
Monitoring the microbes during a period (i.e., the first hour) after the
antimicrobial
attack gives a good indication of the final outcome of the antimicrobial.
[0138] Algorithms to identify bacteria include monitoring the electrical
properties
and thermal properties while the bacteria are in the presence of
antimicrobials including
bacteriophage and in some embodiment's simultaneously testing in separate test
cells
the bacteria's reaction to antibiotics. Combined analysis across sample cells
give
increased accuracy. Furthermore, adding redundant identification test cells
increase
accuracy of the identification.
[0139] Algorithms to identify bacteria sensitivity to antibiotics can
also include
monitoring electrical and thermal properties taking into account results from
bacterial
identification tests and using redundancy to statistically improve the
accuracy of the
antimicrobial sensitivity test results.
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[0140] In this embodiment, antimicrobials (including antibiotics or
bacteriophages)
are impregnated into a fibrous substrate designed to store a premeasured
concentration
of the antimicrobial in a moisture free environment and further designed to
eluent the
antimicrobial into the nutrient solution when the two come into contact with
each other.
[0141] An experimental setup was performed. The performance of the
experimental setup was tested using a work process that counted the bacteria
using
standard plating techniques. Bacteria were counted (by making culture plates
of three
bracketing dilutions of the bacteria used in the experiment or the source
bacteria) before
the test began. Source antimicrobials were tested against source bacteria
using
overnight culturing techniques. Post experiment each cell was plated to show
that the
broth was/wasn't contaminated during the experiment; the antimicrobial
did/didn't
work against the bacteria; and the exact growth of the control bacterial (by
making
culture plates from two bracketing dilutions made from the contents of the
bacteria-only
cell). In general, each experiment used four cells: broth only, microbe only,
antimicrobial to generate a true positive, antimicrobial to generate a true
negative.
[0142] FIG. 8 is a cross-sectional top view of another example sample
cell 112 and
another example electrode configuration. The sample cell 112 includes a body
240
having a sample chamber 244 formed therein, including an interrogation region
248.
Electrodes 118A-D are arranged in the sample chamber 244, as well as the
antimicrobial dispenser 282. Electrical conductors 280A-D provide electrical
connections to the electrodes 118A-D, to couple the electrodes 118A-D to the
analog
electronics 144 of the reader 102.
[0143] The electrodes 118 include electrode 118A, which operates as the
low
current (LcuR) terminal, and is coupled to the AC current source 146 through
the
electrical conductor 280A. The electrode 118B operates as the high current
(HcuR)
terminal, and is coupled to the AC current source 146 through the electrical
conductor
280B. The electrode 118C operates as the high potential (HpoT) terminal, and
is
coupled to the AC voltmeter 148 through the electrical conductor 280C. The
electrode
118D operates as the low potential (LpoT) terminal, and is coupled to the AC
voltmeter
148 through the electrical conductor 280D.
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[0144] The sample chamber 244 includes an interrogation region 248 where
interrogation of the sample occurs. In this example, the interrogation region
248 has an
elongated shape including longitudinal sidewalls 302 and 304 and semi-circular
ends
306 and 308. In some embodiments, the elongated interrogation region 248
includes
recesses 310 and 312 formed at the the sidewalls 302 and 304, which provide
additional
space in the interrogation region 248 for the antimicrobial dispenser 282.
[0145] The driven or forced electrodes 118A and 118B are arranged within
the
elongated interrogation region, which when energized by the AC current source
146,
generate an AC current that flows through the elongated interrogation region
from the
high current electrode 118B to the low current electrode 118A.
[0146] The sample chamber 244 also includes sensing regions 314 and 316.
The
sensing regions 314 and 316 both extend from a common sidewall 304 of the
elongated
interrogation region 248. The sensing regions 314 include narrowed arm
portions that
extend perpendicular to the sidewall 304 and terminate in a larger circular
region at the
ends of the narrowed arm portions. The sensed electrodes 118C and 118D are
arranged
in the larger circular regions of the sensing regions 314 and 316,
respectively. In some
embodiments, the sample chamber 244 is symmetrical about a central axis
extending
between the recesses 310 and 312.
[0147] FIG. 9 is a top cross-sectional view of another example sample
cell 112 and
another example electrode configuration. The sample cell 112 includes body 240
and
sample chamber 244, including an interrogation region 248.
[0148] The interrogation region 248 includes a straight elongated region
having
longitudinal sidewalls 322 and 324 and flat end walls 326 and 328. Recesses
330 and
332 are formed at the sidewalls 322 and 324 in some embodiments, adjacent the
location of the antimicrobial dispenser 282.
[0149] Forced regions 334 and 336 extend from opposite ends of the
sidewall 322
in a common direction. The forced regions 334 and 336 include narrowed arm
portions
that extend from the sidewall 322. A portion of each of the narrowed portions
of the
forced regions 334 and 336 shares a common wall with the flat end walls 326
and 328,
respectively. The forced regions 334 and 336 terminate in larger circular end
regions.
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[0150] The forced electrodes 118B and 118A are arranged in the larger
circular end
regions of the forced regions 334 and 336. The high current electrode 118B is
arranged
in the forced region 334 and the low current electrode 118A is arranged in the
forced
region 336, for example. The high current electrode 118B and the low current
electrode
118A are coupled to the AC current source 146 through electrical conductors
280B and
280A, respectively.
[0151] Sensed regions 338 and 340 similarly extend from opposite ends of
the
sidewall 324 in a common direction, parallel to but opposite the direction of
the forced
regions 334 and 336. The sensed regions 338 and 340 include narrowed arm
portions
that extend from the sidewall 324. A portion of each of the narrowed portions
of the
sensed regions 338 and 340 shares a common wall with the flat end walls 326
and 328,
respectively. The sensed regions 338 and 340 terminate in larger circular end
regions.
[0152] The sensed electrodes 118C and 118D are arranged in the larger
circular end
regions of the sensed regions 338 and 340. The high potential electrode 118C
is
arranged in the sensed region 338 and the low potential electrode 118D is
arranged in
the sensed region 340, for example. The high potential electrode 118C and the
low
potential electrode 118D are coupled to the AC voltmeter 148 through
electrical
conductors 280C and 280D, respectively.
[0153] In this example, the interrogation region 248 is symmetrical
about central
axes extending through the end walls 326 and 328, and extending through the
recesses
330 and 332. Accordingly, the functions of the electrodes can be swapped
accordingly
without modifying the operation of the sample cell 112.
[0154] FIG. 10 is a cross-sectional top view of another example sample
cell 112 and
another example electrode configuration. The sample cell 112 includes a sample
chamber 244 having an interrogation region 248.
[0155] In this example, the interrogation region 248 has a cylindrical
shape having a
single sidewall 342. All of the electrodes 118A-118D are arranged at the
bottom of the
sample cell 112 within the cylindrical interrogation region 248. The
antimicrobial
dispenser 282 is also arranged within the sample cell 112.
[0156] FIGS. 11 and 12 illustrate another example sample cell 112 and
another
example electrode configuration. FIG. 11 is a cross-sectional top view and
FIG. 12 is a
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cross-sectional side view. This example sample cell 112 includes a sample
chamber
244 (FIG. 12) having an interrogation region 248.
[0157] In this example, the interrogation region 248 has a cylindrical
shape having a
single sidewall. All of the electrodes 118A-118D are arranged at the bottom of
the
sample cell 112 within the cylindrical interrogation region 248. A chimney 246
having
a cylindrical shape extends from input opening 242 to interrogation region
248. The
antimicrobial dispenser 282 is arranged in a horizontal position on top of an
antimicrobial dispenser support 284, which extends across the chimney 246,
having the
shape of a cross or X-shape.
[0158] FIG. 13 is a state diagram 350 illustrating an example of the
operation of the
diagnostic device 100, and also illustrates a method of operating a diagnostic
device
100. In this example, the diagnostic device 100 operates in states 352, 354,
and 356.
[0159] The diagnostic device 100 begins at state 352 when the diagnostic
device
100 is turned on. Prior to being turned on, the diagnostic device 100 is
prepared for
interrogating a sample, such as by adding a suitable quantity of the sample
into the
sample input port 114 (FIG. 3) of the diagnostic unit 104. Once turned on, the
diagnostic device 100 transitions to one of the states 354 or 356.
[0160] When the diagnostic device operates in the state 352, the
diagnostic device
utilizes all four electrodes 118 to perform measurements on the sample. In
some
embodiments, a first set of the forced electrodes (e.g., 118B and 118A) are
energized by
the AC current source 146 to generate a current flow through the sample. The
second
set of sensed electrodes (e.g., 118C and 118D) are then used by the AC
voltmeter 148 to
detect one or more characteristics of the sample.
[0161] For example, in some embodiments the diagnostic device 100
operates to
measure conductance of the sample. The conductance measurement is then used to
count the quantity of bacteria present in the sample. In some embodiments the
quantity
of bacteria are determined as a quantity of colony forming units (CFU).
[0162] As another example, in some embodiments the diagnostic device 100
operates to identify a type of bacteria present in the sample. To identify the
type of
bacteria, the diagnostic device 100 monitors the conductance of the sample
over time
across a plurality of the sample cells. At least some of the sample cells
include
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antimicrobial dispensers including different antimicrobials. For example,
identification
of the bacteria is possible by observing the ionic surge (and corresponding
increase in
conductance) and the eventual reduction in live bacteria (and corresponding
reduction
in conductance) which occurs during the period (i.e., the first five to
fifteen minutes)
after the introduction of an antimicrobial that is effective at attacking the
bacteria
present in the sample cell 112. The diagnostic device 100 can therefore
monitor for the
changes in conductance that occur in sample cells having an effective
antimicrobial, and
can similarly determine that little to no change in conductance occurs in
other sample
cells that do not have an effective antimicrobial. Additionally, the
conductance can be
compared to one or more other control cells, such as a control cell containing
a control
fluid absent any of the sample, and/or a control cell containing a control
fluid and the
sample but no antimicrobial.
[0163] When the diagnostic device operates in the second mode 356, two
or more of
the electrodes in one or more of the sample cells 112 to measure one or more
characteristics of the sample. For example, in some embodiments, diagnostic
device
100 operates to measure the admittance of the sample. Admittance can be
computed,
for example, as the forced current divided by the voltage between the forced
electrodes.
[0164] In some embodiments, the second mode 356 utilizes only two of the
electrodes 118 within a sample cell. Alternatively, the electrodes can be
operated in
pairs to utilize four electrodes for the admittance measurement.
[0165] In some embodiments, the electrodes are controlled using the
electronic
components of the reader 102 (shown in FIG. 2).
[0166] The second state 356 can be used to take impedimetric
measurements of the
sample to monitor chemical processes and biological activity. In particular,
in some
embodiments the reader 102 includes electronic components including
impedimetric-
based electronics for detection and real-time monitoring and quantification of
bacteria
in nutrient solution (broth culture). The impedimetric electronics rely on an
admittance
change within a microbial culture, resulting from a change in ionic content,
produced by
metabolization of compounds by microorganisms within the culture media.
Impedimetric measurements offer advantages of convenience and rapid results
over
other techniques, such as plating techniques for microbial colony counts.
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[0167] The admittance measurement can be made with an AC signal. In
general,
the admittance of the sample cell 112 is complex. A susceptive component of
the
admittance is apparent due to a capacitance that is generated from a charge
double-
layer, also known as electrode polarization, which forms at each electrode-
sample
interface. The bulk conductivity of the electrolytic solution contained within
the
particular geometry of a sample cell 112 determines the conductive component
of the
admittance. Changes in the concentration and or type of ions in the
electrolytic solution
produce changes in both the susceptive and conductive components. For
admittance/impedance modeling, the cell can be represented as a capacitor and
resistor
connected in series. More sophisticated models, such as those that apply a
constant
phase element to include distributed effects, are beneficial for a detailed
analysis.
[0168] The efficaciousness of the constant phase element in modeling the
cell
admittance as a function of frequency may be indicative of a distribution of
relaxation
times or ionization energies within a cell, resulting in random electrical
noise that varies
inversely as a power of frequency. The constant phase element also provides
insight to
variability attributed to electrode surface roughness that can confound cell-
to-cell
repeatability. The charge double-layer that embodies the cell capacitance, and
varies
according to ion concentration and ion type and temperature, can also respond
to
problematic influences such as precipitates, biofilms, or bubbles at the
electrodes.
[0169] Furthermore, Van der Waals forces at the electrode surfaces support
film
growth that diminishes the capacitive response to changes in ion concentration
within
the electrolytic solution. This results in an additional change in the
admittance, and a
reduction in responsivity, as a function of time during a measurement
sequence. The
random variables, noise and film growth, limit the signal-to-noise ratio
available for
measurement of microbial colony forming units (CFU) per unit volume of
electrolytic
solution.
[0170] To avoid much of the limitations imposed by measuring
predominantly the
capacitance at low frequency, measurements can be performed at higher
frequency so
that the capacitive susceptance contributes less to the total admittance. The
conductance of the bulk electrolyte then dominates the measured cell
admittance.
However, the distributed nature of the cell admittance confounds applying the
lumped
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resistor-capacitor (series RC) model that would allow sufficient isolation of
these
parameters by merely changing frequency. Indeed, this measured conductance is
observed to vary in relation with the capacitance. Also, for a given
fractional change in
ion concentration, observations show the resulting fractional change in
capacitance is
typically greater than the fractional change in conductance by more than one
order of
magnitude. So the "conductance" parameter exhibits less noise compared to the
"capacitance" parameter, but also develops less response to changes in ion
concentration.
[0171] Therefore, while the second state 356 can be used to measure
admittance and
evaluate microbe sensitivity, the first state 354 can be used to obtain
extended accuracy
and dynamic range beyond what is available through the second state 356.
[0172] In some embodiments, application of the four-terminal techniques
used
during the first state 354 involve two terminals supplying an electrical
current through a
test sample, and two terminals with which the subsequent voltage drop therein
is
sensed. Embodiments of the four-terminal cell operating during state 354 are
less
sensitive to effects at the forced/driven electrodes than when operating in
the second
state 356 by excluding effects of electrode polarization instead of merely
excluding
series impedance of terminals and interconnects. Additionally, the first state
can also
provide a direct measurement of the electrolytic solution conductivity scaled
by a
geometry dependent sample cell factor.
[0173] The diagnostic device 100 can use the admittance measurements,
for
example, to determine antibiotic sensitivity of the microbial present in the
sample. For
example, the diagnostic device 100 can determine that the microbial has a low,
moderate, or high sensitivity to the antimicrobial present in the sample cell.
[0174] Some embodiments include one or more additional states, not shown in
FIG.
11. For example, some embodiments include a fluid processing state, during
which the
fluidics system 190 is activated to process and distribute the received sample
into the
sample cells 112.
[0175] FIGS. 14-15 illustrate an example of an antimicrobial dispenser
282.
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[0176] FIG. 14 is a perspective view of the example antimicrobial
dispenser 282. In
this example, the antimicrobial dispenser 282 includes a carrier material 370
and an
antimicrobial 372.
[0177] The carrier material 370 is a piece of material such as paper,
cloth, and the
like. In some embodiments the carrier material 370 includes a fastener
configured for
attaching the carrier material 370 inside of a sample cell 112 (e.g., to the
interior of the
sample cell 112 itself, or to the base substrate 222, such as shown in FIG.
4).
[0178] In some embodiments the carrier material 370 is a thin sheet of
material,
having a thickness that is much less than (e.g., < 10% of, or <1% of) its
length and
width. This provides increased surface area for interaction with the sample.
[0179] The antimicrobial 372 is carried by the carrier material 370. In
some
embodiments, the antimicrobial 372 is applied to the outside of the carrier
material 370.
In some embodiments, the antimicrobial 372 is also contained within the
carrier
material 370. The antimicrobial dispenser (including the antimicrobial 372 and
carrier
material 370) are dry prior to use.
[0180] This antimicrobial dispenser 282 can have various possible shapes
in
different embodiments, including rectangular, circular, cylindrical, square,
triangular, or
other shapes. In some embodiments, the face surfaces of the carrier material
370 of the
antimicrobial dispenser 282 are slightly hardened against moisture and the
edges give
access to a fibrous material that easily wicks moisture thus forcing
premeasured
antimicrobials 372 to eluent into the surrounding liquid. One embodiment of
the
antimicrobial dispenser 282 uses a specific bacteriophage or specifically
design
bacteriophage cocktail as the antimicrobial.
[0181] One example of an antimicrobial dispenser is the SENSIDISCTM
susceptibility test discs available from Becton, Dickinson and Company, of
Franklin
Lakes, NJ, containing an antibiotic drug.
[0182] In other possible embodiments, the antimicrobial dispenser 282
includes a
bacteriophage or bacteriophage cocktail. The bacteriophage is a virus that
infects and
replicates within bacteria. For example, for detection of urinary tract
infections,
bacteriophage are selected that are specific for that set of bacteria which
can include E.
coli, Staphylococcus aureaus, Klebsiella, Proteus, Pseudomonas, and
Enterobacter.
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Examples of bacteriophages that can be used include phages TI, T4 57, VD13,
92, PB-
1, or other specially cultivated bacteriophage of interest used alone or in
combination.
A concentration of bacteriophages can be identified by plaque forming units
(PFU) per
milliliter.
[0183] In some embodiments the bacteriophage have one or more, or all, of
the
following features: is cultivated and isolated so it attacks one and only one
species;
inserts DNA through a hole in the bacteria's cell wall so potassium ions are
rapidly
released; has a long shelf life when lyophilized (e.g., dried shelf life of 2
years or more);
revives rapidly when rehydrated; if targets a sub-species, co-exists in a
cocktail
targeting the species; will attack bacteria regardless of initial bacterial
concentration and
whether bacteria is in exponential growth phase; has a rapid life cycle (e.g.,
less than 30
minutes to lysis or shorter); and is a comprehensive blend of phage to
minimize any
resistant bacteria masking an effective attack¨has enough different phage
targeting
multiple subspecies of a single species to eliminate virtually all bacteria in
a sample.
These features can be found in Caudovirales phages, for example.
[0184] FIG. 15 is a perspective view of the example antimicrobial
dispenser 282
shown in FIG. 14 arranged inside of a sample chamber 244 of a sample cell 112.
The
sample chamber 244 includes a sample 380. FIG. 15 also illustrates the
dispensing of
the antimicrobial 372 into the sample 380.
[0185] In this example, the antimicrobial dispenser 282 is arranged within
the
sample chamber 244 of a sample cell 112. In some embodiments, the
antimicrobial
dispenser 282 is fastened inside of the sample chamber 244 in a vertical
orientation, as
shown. The vertical orientation increases the surface area of the carrier
material 370
that is exposed within the sample chamber 244. In other possible embodiments,
the
antimicrobial dispenser 282 is fastened horizontally. In some embodiments, the
antimicrobial dispenser 282 is positioned within the chimney 246 (FIG. 5 and
FIGS. 11-
12) of the sample cell.
[0186] When the sample 380 is provided into the sample chamber 244 or
chimney
246, the sample wets the antimicrobial dispenser 282. When wetted, the
antimicrobial
372 is released from the carrier material and is disbursed into the sample
380. Due to
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the large surface area and relatively small internal volume, a large
proportion of the
antimicrobial 372 is quickly dispensed into the sample 380.
[0187] In some embodiments, a plurality of sample cells 112 include
different
antimicrobial dispensers 282 containing different antimicrobials. For example,
in some
embodiments at least 10 sample cells 112 each contain an antimicrobial
dispenser 282
for dispensing a different antimicrobial. As one example, the antimicrobials
are at least
different bacteriophages, that lyses a certain species of bacteria. The
diagnostic
device 100 can then operate to monitor the 10 sample cells to determine
whether the
microbial present in the sample cell is affected by the antimicrobial, and if
so, the
10 identity of the microbial can be determined, for example. In some
embodiments, at
least one or more of the sample cells 112 include an antimicrobial dispenser
282 that
dispenses an antibiotic. Typically at least two sample cells 112 serve as
controls, in
which case the sample cells 112 may not include an antimicrobial dispenser
282, or
alternatively may include an antimicrobial dispenser 282 carrier material 370
without an
antimicrobial 372. In some embodiments, a first control sample cell contains
an
electrolyte solution and does not contain the sample 380 (and microbes
contained
therein) or an antimicrobial 372. A second control sample cell contains an
electrolyte
solution and the sample (and microbes contained therein), but does not include
an
antimicrobial 372. Additional control sample cells are present in some
embodiments,
such as containing an electrolyte and antimicrobial dispenser including a
bacteriophage
or an electrolyte and an antimicrobial dispenser including an antimicrobial
other than a
bacteriophage. Many different embodiments with differing numbers of sample
cells to
perform different antimicrobial sensitivity tests, microbial identification
tests or
microbial counting are possible.
[0188] FIG. 16 is a schematic block diagram illustrating an exemplary
architecture
of a computing device 410 that can be used to implement aspects of the present
disclosure. For example, the computing device 410 can be coupled to the
diagnostic
device 100 through the communication device 170 of the reader 102 (FIG. 2). In
another possible embodiment, the computing device 410 is part of the reader
102 (such
as to provide the CPU 162, computer readable medium 164, display processor
166,
display device 168, communication device 170, and power source 142). By way of
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example, the computing device will be described below as a separate computing
device
410.
[0189] The computing device 410 includes, in some embodiments, at least
one
processing device 420, such as a central processing unit (CPU). A variety of
processing
devices are available from a variety of manufacturers, for example, Intel or
Advanced
Micro Devices. In this example, the computing device 410 also includes a
system
memory 422, and a system bus 424 that couples various system components
including
the system memory 422 to the processing device 420. The system bus 424 is one
of any
number of types of bus structures including a memory bus, or memory
controller; a
peripheral bus; and a local bus using any of a variety of bus architectures.
[0190] Examples of computing devices suitable for the computing device
410
include a desktop computer, a laptop computer, a tablet computer, a mobile
computing
device (such as a smart phone, an iPod0 or iPad0 mobile digital device, or
other
mobile devices), or other devices configured to process digital instructions.
[0191] The system memory 422 includes read only memory 426 and random
access
memory 428. A basic input/output system 430 containing the basic routines that
act to
transfer information within computing device 410, such as during start up, is
typically
stored in the read only memory 426.
[0192] The computing device 410 also includes a secondary storage device
432 in
some embodiments, such as a hard disk drive, for storing digital data. The
secondary
storage device 432 is connected to the system bus 424 by a secondary storage
interface
434. The secondary storage devices 432 and their associated computer readable
media
provide nonvolatile storage of computer readable instructions (including
application
programs and program modules), data structures, and other data for the
computing
device 410.
[0193] Although the exemplary environment described herein employs a
hard disk
drive as a secondary storage device, other types of computer readable storage
media are
used in other embodiments. Examples of these other types of computer readable
storage media include magnetic cassettes, flash memory cards, digital video
disks,
Bernoulli cartridges, compact disc read only memories, digital versatile disk
read only
memories, random access memories, or read only memories. Some embodiments
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include non-transitory media. Additionally, such computer readable storage
media can
include local storage or cloud-based storage.
[0194] A number of program modules can be stored in secondary storage
device
432 or memory 422, including an operating system 436, one or more application
programs 438, other program modules 440 (such as the software engines
described
herein), and program data 442. The computing device 410 can utilize any
suitable
operating system, such as Microsoft WindowsTM, Google ChromeTM, Apple OS,
Google
DroidTM, Google Ice CreamTM, and any other operating system suitable for a
computing
device.
[0195] In some embodiments, a user provides inputs to the computing device
410
through one or more input devices 444. Examples of input devices 444 include a
keyboard 446, mouse 448, microphone 450, and touch sensor 452 (such as a
touchpad
or touch sensitive display). Other embodiments include other input devices
444. The
input devices are often connected to the processing device 420 through an
input/output
interface 454 that is coupled to the system bus 424. These input devices 444
can be
connected by any number of input/output interfaces, such as a parallel port,
serial port,
game port, or a universal serial bus. Wireless communication between input
devices
and the interface 454 is possible as well, and includes infrared, BLUETOOTHO
wireless technology, 802.11a/b/g/n, cellular, or other radio frequency
communication
systems in some possible embodiments.
[0196] In this example embodiment, a display device 456, such as a
monitor, liquid
crystal display device, projector, or touch sensitive display device, is also
connected to
the system bus 424 via an interface, such as a video adapter 458. In addition
to the
display device 456, the computing device 410 can include various other
peripheral
devices (not shown), such as speakers or a printer.
[0197] When used in a local area networking environment, a wide area
networking
environment (such as the Internet), or a personal area network, the computing
device
410 is typically connected to the network 462 through a network interface 460,
such as
an Ethernet interface or wirelessly, such as using any one or more of the
wireless
communication devices noted above. The network interface 460 can interface
with
many different kinds of networks, in some embodiments. Other possible
embodiments
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use other communication devices. For example, some embodiments of the
computing
device 410 include a modem for communicating across the network 462 (such as
the
internet or a cellular network, for example).
[0198] For example, in some embodiments an application program 438
operates to
transfer patient information for storage in the data storage medium 208 (FIG.
3), and
similarly to receive diagnostic results from the diagnostic device 100 and
transfer such
results across the network to another computing device.
[0199] In some embodiments, the computing device 410 transfers
diagnostic results
from the diagnostic device 100 to the network for storage in a cloud data
storage
system. Similarly, the computing device 410 operates in some embodiments to
transfer
digital data to the cloud data storage device and for further analytic
processing, such as
when the analytic processing required is too intensive for the computing
device 410 or
the diagnostic device 100.
[0200] The computing device 410 typically includes at least some form of
computer
readable media. Computer readable media includes any available media that can
be
accessed by the computing device 410. By way of example, computer readable
media
include computer readable storage media and computer readable communication
media.
[0201] Computer readable storage media includes volatile and
nonvolatile,
removable and non-removable media implemented in any device configured to
store
information such as computer readable instructions, data structures, program
modules or
other data. Computer readable storage media includes, but is not limited to,
random
access memory, read only memory, electrically erasable programmable read only
memory, flash memory or other memory technology, compact disc read only
memory,
digital versatile disks or other optical storage, magnetic cassettes, magnetic
tape,
magnetic disk storage or other magnetic storage devices, or any other medium
that can
be used to store the desired information and that can be accessed by the
computing
device 410. Computer readable storage media does not include computer readable
communication media.
[0202] Computer readable communication media typically embodies computer
readable instructions, data structures, program modules or other data in a
modulated
data signal such as a carrier wave or other transport mechanism and includes
any
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information delivery media. The term "modulated data signal" refers to a
signal that
has one or more of its characteristics set or changed in such a manner as to
encode
information in the signal. By way of example, computer readable communication
media includes wired media such as a wired network or direct-wired connection,
and
wireless media such as acoustic, radio frequency, infrared, and other wireless
media.
Combinations of any of the above are also included within the scope of
computer
readable media.
[0203] The computing device illustrated in FIG. 16 is also an example of
programmable electronics, which may include one or more such computing
devices,
and when multiple computing devices are included, such computing devices can
be
coupled together with a suitable data communication network so as to
collectively
perform the various functions, methods, or operations disclosed herein.
[0204] FIG. 17 illustrates another example architecture involving a
diagnostic
device 100. In this example, the diagnostic device 100 includes a fluidics
system 190, a
sensor system 192, a reader computing unit 102A, a reader analog unit 102B, a
computing device 410 including interface applications 438, a data
communication
network, and a cloud server computing device 510.
[0205] In this example, the fluidics system 190 receives a sample from a
healthcare
worker. The reader computing unit 102A receives inputs from the healthcare
worker,
such as to select a mode of operation, or other inputs.
[0206] The fluidics system 190 and sensor system 192 operate under the
control of
the reader computing unit 102A and the sample is evaluated by the sensor
system 192
and the reader analog unit 102B.
[0207] Data communication occurs between the reader computing unit 102A
and
the computing device 410. Data communication also occurs between the computing
device 410 and a cloud server 510 across a data communication network.
Examples of
such data communication are discussed herein.
[0208] FIGS. 17-20 illustrate experimental data obtained using a
diagnostic device
100.
[0209] The present disclosure uses the word "cell" in at least two
contexts. One
context is a biological "cell" and another context is a "sample cell." To
avoid
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confusion, a sample cell can alternatively be referred to as a sample unit, a
test cell, a
test unit, a sample module, or the like.
[0210] Some embodiments include one or more of the following:
[0211] An impedimetric measurement device for the monitoring of microbes
present in a liquid medium comprising: an electrolytic solution, a containment
vessel,
two electrodes driven with an electric stimulus (forced electrodes) and two
electrodes
sensing an electric signal (sensed electrodes): a. For the use in monitoring
the count of
live microbes as a function of time; b. For the use in determining antibiotic
sensitivity
of the microbes; and/or c. For the use in identifying microbes using selected
antimicrobials
[0212] A device with the electric stimulus being an ac voltage or
current.
[0213] A device with the sensed electric signal being an ac voltage.
[0214] A device with the sensed electrodes located along the path of
electrical
signal current that flows between the forced electrodes.
[0215] A device with electrodes arranged in a four-cornered geometrical
shape so
that the forced electrodes are adjacent along the geometrical shape boundary
and the
sensed electrodes are adjacent along the geometrical shape boundary.
[0216] A device with the shape of the electrolyte containment vessel
boundary
generalized to encompass the electrodes while directing the electric field for
optimal
performance.
[0217] A device with the electrodes fabricated on a planar substrate
comprising the
bottom of the electrolytic cell.
[0218] A device with a printed circuit board comprising the planar
substrate.
[0219] A device of claim 6 with the electrodes installed within the side
walls of the
electrolytic cell, and at or adjacent a bottom of the electrolytic cell.
[0220] A device wherein a volume is defined to locate an antimicrobial
impregnated
material for the purpose of introducing a measured amount of one or a
plurality of
antimicrobial agents into the cell, when the cell is filled with an
electrolytic solution.
[0221] A device calibrated to provide indications of microbial
concentrations.
[0222] An algorithm for detection of increasing and decreasing microbial
concentrations as a function of time.
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[0223] An algorithm with temperature compensation of measurement data
from
multiple cells and referenced to cell containing only an electrolytic
solution.
[0224] An algorithm with capability to compare data from individual
cells, for the
purpose of indicating relative biological activity within such cells.
[0225] An algorithm with capability of discriminating the effectiveness of
an
antimicrobial agent present within a particular cell.
[0226] Any of the algorithms described herein, wherein the algorithm is
performed
by or using a computing device.
[0227] A device that has a volume defined that holds a material
impregnated with a
measured concentration of one or more bacteriophage which when wetted releases
the
bacteriophage into the electrolytic solution.
[0228] A device that has a volume defined that holds a material
impregnated with
one or more antimicrobials which when wetted releases the antimicrobial into
the
electrolytic solution.
[0229] A method wherein the sensed voltage is used to calculate conductance
or
admittance.
[0230] A device whose shape alleviates cell factor dependence on the
containment
vessels fill factor.
[0231] A device with a containment vessel comprising: a. at least one
substrate; b.
at least four electrodes; and c. a sample cavity formed in the at least one
substrate, the
sample cavity comprising: i. a sensing portion including the electrodes
therein, the
sensing portion having a shape configured to direct and focus electric fields
generated
by the electrodes within the sample cavity; and ii. a shape extending from the
sensing
portion and having a cross-sectional size that is less than a cross-sectional
size of the
sensing portion, the shape extending having a volume, wherein a volume of the
extending portion permits a volume of the sample to vary without substantially
affecting
electrical measurements from the electrodes.
[0232] A method of using the conductance of the broth only sample cavity
to
correct all of the signatures for temperature variation during the duration of
the test.
[0233] Means for keeping the liquid in all of the sample cavities at the
same
temperature during the test.
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[0234] Means for heating the sample holder to a temperature of 35
degrees C if that
is required to achieve more robust admittance and conductance signatures.
[0235] Using an A/D converter to reduce the analog AC currents and
voltages to a
digital format that can be used to more easily calculate and compare the
admittance and
conductance signatures.
[0236] A diagnostic device including one of the various possible sample
cavity and
electrode geometries and the required mechanical tolerances of electrode size
and
spacing and how those affect the variation in the calibration factor for the
plurality of
sample cavities in a sample holder.
[0237] A method of using the four-terminal measurement of conductance to
achieve
temperature compensation from the broth only sample cavity and to avoid the
negative
effects of biofilm growth on the electrodes.
[0238] A method of operating a diagnostic device by defining the limits
for the
applied alternating current and/or voltage to avoid plating effects and
electrolysis at the
electrode surfaces.
[0239] A method of defining biocompatible materials used in the
construction of the
antimicrobial dispenser.
[0240] A method of providing viral phages that are specific to each type
bacteria
and using the viral phages to identify the bacteria.
[0241] A method of utilizing the unique conductance signature of an
effective
phage attack.
[0242] A diagnostic device utilizing a nutrient broth that promotes
bacterial growth
and has a controlled conductivity and temperature coefficient.
[0243] A method of identifying the microbes present in a sample
involving
identifying the ratio of admittance and/or conductance signatures between the
broth +
bacteria, and broth + bacteria + viral phage sample cavities.
[0244] A method of identifying an effective antimicrobial for the
identified
bacterium involving determining the ratio of admittance and/or conductance
signatures
between the broth + bacteria and broth + bacteria + antimicrobial sample
cavities.
[0245] A method of determining the CFU concentration of the bacteria in a
sample
involving identifying a conductance signature of the broth + bacteria sample
cavity.
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[0246] Means for heating the sample holder to a temperature of 35 degrees
C.
[0247] The various embodiments described above are provided by way of
illustration only and should not be construed to limit the claims attached
hereto. Those
skilled in the art will readily recognize various modifications and changes
that may be
made without following the example embodiments and applications illustrated
and
described herein, and without departing from the true spirit and scope of the
following
claims.
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