U.S. patent application number 14/097048 was filed with the patent office on 2014-06-12 for device and method for identifying microbes and counting microbes and determining antimicrobial sensitivity.
This patent application is currently assigned to Texas State University. The applicant listed for this patent is Telemedicine Up Close, Inc. dba DxUpClose, Texas State University. Invention is credited to Gary M. ARON, James R. BIARD, Ray G. COOK, Jeanette HILL, Daniel M. JUSTISS, Andrei M. MANOLIU, Cynthia S. NICKEL, Clois E. POWELL, William A. STAPLETON, Frederick J. STRIETER.
Application Number | 20140162308 14/097048 |
Document ID | / |
Family ID | 47108270 |
Filed Date | 2014-06-12 |
United States Patent
Application |
20140162308 |
Kind Code |
A1 |
NICKEL; Cynthia S. ; et
al. |
June 12, 2014 |
DEVICE AND METHOD FOR IDENTIFYING MICROBES AND COUNTING MICROBES
AND DETERMINING ANTIMICROBIAL SENSITIVITY
Abstract
A method of determining antimicrobial activity of an agent can
include providing a well, wherein the well contains at least one
antimicrobial agent, the well further including at least two
electrodes. A sample of a microbe can be added into the well and a
voltage pulsed between the electrodes. An electrical property can
be sampled and recorded. In another aspect, a method of identifying
at least one microbe includes taking a sample containing the at
least one microbe, isolating the at least one microbe from the
sample, dividing the at least one microbe into at least one well,
wherein each well contains at least one antimicrobial agent and at
least two electrodes. A voltage is pulsed between the at least two
electrodes, an electrical property is sampled during the pulsing
and recorded. In another aspect, a diagnostic device for detecting
at least one microbe is presented.
Inventors: |
NICKEL; Cynthia S.; (Frisco,
TX) ; POWELL; Clois E.; (Seguin, TX) ; BIARD;
James R.; (Richardson, TX) ; STAPLETON; William
A.; (San Marcos, TX) ; ARON; Gary M.; (San
Marcos, TX) ; HILL; Jeanette; (Manor, TX) ;
COOK; Ray G.; (San Marcos, TX) ; JUSTISS; Daniel
M.; (Austin, TX) ; STRIETER; Frederick J.;
(Dallas, TX) ; MANOLIU; Andrei M.; (Atherton,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Texas State University
Telemedicine Up Close, Inc. dba DxUpClose |
San Marcos
Frisco |
TX
TX |
US
US |
|
|
Assignee: |
Texas State University
San Marcos
TX
Telemedicine Up Close, Inc. dba DxUpClose
Frisco
TX
|
Family ID: |
47108270 |
Appl. No.: |
14/097048 |
Filed: |
December 4, 2013 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13464936 |
May 4, 2012 |
8637233 |
|
|
14097048 |
|
|
|
|
61493152 |
Jun 3, 2011 |
|
|
|
61482569 |
May 4, 2011 |
|
|
|
Current U.S.
Class: |
435/32 ;
435/287.1; 435/29 |
Current CPC
Class: |
B01L 2300/0636 20130101;
C12Q 1/02 20130101; B01L 3/5085 20130101; C12Q 1/04 20130101; C12Q
1/18 20130101; B01L 2300/0645 20130101; G01N 33/48735 20130101;
G01N 15/0656 20130101 |
Class at
Publication: |
435/32 ; 435/29;
435/287.1 |
International
Class: |
C12Q 1/18 20060101
C12Q001/18; C12Q 1/02 20060101 C12Q001/02 |
Claims
1. A method of determining a count of microbes in a sample, the
method comprising: filtering the sample to separate the at least
one microbe from the sample, immersing the at least one microbe in
an analyte to form an immersion, incubating the immersion for a
specific time, dividing the immersion into at least one wells,
measuring an electrical property in the wells for a
sample-duration, and correlating the electrical property to a count
as a function of time.
2. The method according to claim 1, further comprising adding at
least one bacteriophage to at least one of the wells before
measuring the electrical property.
3. The method according to claim 1, wherein the sample-duration is
at is about 1 second, about 5 seconds, about 10 seconds, about 20
seconds, about 30 seconds, about 40 seconds, about 50 seconds,
about 60 seconds.
4. The method according to claim 1, wherein the specific time for
the incubating is between 1 millisecond and 5 minutes, between 0.5
second and 2 minutes, between 1 second and 1 minute, between 1 hour
and 8 hours.
5. The method according to claim 1, wherein the method determines a
first count for a first microbe and a second count for a second
microbe.
6. A method of determining antimicrobial resistance of a microbe,
comprising: adding a sample of at least one microbe into a well
containing at least one antimicrobial, and adding a sample of at
least one microbe into a control well measuring an electrical
property in the well for a sample-duration as a function of
time.
7. The method according to claim 6 where the sample-duration is at
least one hour and not more than six hours.
8. The method according to claim 6, wherein the at least one
microbe is selected from Aerobacter, Bacillus, Bordetella,
Brucella, Campylobacter, Chlamydia, Chromobacterium, Clostridium,
Corynebacterium, Enterobacter, Escherichia, Haemophilus,
Klebsiella, Listeria, Mycobacterium, Mycoplasma, Neisseria,
Pneumococcus, Proteus, Pseudomonas, Providencia, Salmonella,
Serratia, Shigella, Staphylococcus, Streptococcus, Vibrio,
Yersinia, Acinetobacter, Bacteroides, Bifidubacterium, E. kenella
corrodens, Gardnerella vaginalis, Mobiluncus, Proteobacteria,
Desulfobacterales, Desulfovibrionales, Syntrophobacterales,
Thermodesulfobacteria, Nitrospirae, gram positive Peptococcaceae,
Archaea, Archaeoglobus, or any combinations thereof.
9. The method according to claim 6, wherein the at least one agent
is selected from Actinomyces phages, Bacillus phage .PHI.29,
bacteriophage M102, bacteriophage e10, bacteriophage f1,
bacteriophage .lamda., bacteriophage PI, spherical phage PhiX174,
spherical phage G4, spherical phage S13, bacteriophage T1,
bacteriophage T2, bacteriophage T3, bacteriophage T4, bacteriophage
T5, bacteriophage T6, bacteriophage T7, ssRNA bacteriophages MS2,
ssRNA bacteriophages R17, ssRNA bacteriophages f2, ssRNA
bacteriophages Q beta, S. mutans phages, and any combinations
thereof.
10. The method according to any one of the claim 6, wherein the
well has a holding capacity between about 10 .mu.L to about 2
mL.
11. The method according to claim 6, wherein the electrical
property is selected from conductance, resistance, voltage,
amperage, capacitance, impedance, inductance, and any combinations
thereof.
12. The method according to claim 6, wherein the sample is taken
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 and any
combinations thereof.
13. A diagnostic device of detecting at least one microbe,
comprising: a first unit and a second unit; the first unit is
stackable into the second unit; wherein the first unit is a
diagnostic unit comprising at least one well, the at least one well
having electrodes contacting the inside and the outside of the at
least one well; wherein the second unit is a reader unit comprising
a connector section for the electrodes of the diagnostic unit; a
fluidic system consisting of one-way valves and a port for
pressurizing the fluidic system; wherein the second unit is a
reader unit comprising a connector section for the
pressurizing.
14. The diagnostic device according to claim 13, wherein the first
unit further comprising a sample holder and filter unit, the sample
holder and filter unit being in fluidic communication.
15. The diagnostic devices according to claim 13, wherein the
electrodes comprise a non-oxidizing material.
16. The diagnostic devices according to claim 15, wherein the
non-oxidizing material is selected from metals, nonmetals,
polymers, composites, resists, resins, carbon nano-tubes, plastics,
or any combinations thereof.
17. The diagnostic devices according to claim 13, wherein the
electrodes comprises copper covered with graphene.
18. The diagnostic devices according to claim 16, wherein the
electrodes comprise copper, gold, nickel, or any combination
thereof.
19. The diagnostic device according to claim 13, wherein the at
least one antimicrobial is selected from aminoglycosides,
amphenicols, ansamycins, beta-lactams, lincosamides, macrolides,
polypeptide antibiotics, tetracyclines, cycloserine, mupirocin,
tuberin, 2,4-diaminopyrimidines, nitrofurans, quinolones,
sulfonamides, sulfones, clofoctol, hexedine, methenamine,
nitroxoline, taurolidine, and xibernol.
20. The diagnostic device according to claim 13, wherein the at
least one antimicrobial is selected from amikacin, azlocillin,
carbencillin, cefaclor, cefemandole, cefonicid, cefotaxime,
cefoperazone, cefoxitin, ceftizoxime, ceftriaxzone, ciprofloxacin,
clindamycin, gatifloxacin, gemifloxacin, gentamicin, kanamycin,
linezolid, mecillinam, meropenem, methicillin, metronidazole,
mezlocillin, minocyclin, moxifloxacin, nafcillin, netilmycin,
oxacillin, penicillin, piperacillin, quinupristin-dalfopristin,
sparfloxacin, sulbactam, tazobactam, teicoplanin, tetracyclines,
tobramycin, trimethoprim, trospectomycin and vancomycin.
Description
CROSS-REFERENCE TO RELATED APPLICATION(S)
[0001] This application is a divisional of and claims priority
under 35 U.S.C .sctn.120 to U.S. patent application Ser. No.
13/464,936 entitled "DEVICE AND METHOD FOR IDENTIFYING MICROBES AND
COUNTING MICROBES AND DETERMINING ANTIMICROBIAL SENSITIVITY," by
Cynthia Nickel et al., which claims priority from U.S. Provisional
Patent Applications No. 61/482,569, filed May 4, 2011 and
61/493,152, filed Jun. 3, 2011 both entitled "DEVICE AND METHOD FOR
IDENTIFYING MICROBES AND COUNTING MICROBES AND DETERMINING
ANTIMICROBIAL SENSITIVITY," both naming inventors Cynthia Nickel et
al., which all applications are incorporated by reference herein in
its entirety.
FIELD OF THE DISCLOSURE
[0002] The disclosure, in general, relates to microbial diagnostics
applicable to the fields of human health care, animal medicine,
animal care, clinical laboratories, biomedical and biological
research, food control, and all industries impacted by
microbes.
BACKGROUND
[0003] In 1942, for the first time, the life of a patient was saved
by treatment with penicillin. Yet, the battle against infectious
diseases and pathogenic bacteria continues. In 2006, the Infectious
Disease Society of America reported that each year, 90,000 of the 2
million people who acquire a hospital bacterial infection will die.
That is a 4.5% mortality rate resulting from just visiting a
hospital. Multi-drug resistance bacterial strains are a major
problem and one that has been increasing very rapidly every year
during the last few decades. Beside the need for new antibiotics,
there is also a need to quickly identify and quantify a bacterial
infection in order to embank the spread of the infection into an
epidemic.
[0004] In the food industry, pasteurization involves heating liquid
food products like milk, juices, etc. to kill pathogenic organisms
such as viruses, bacteria, molds, and yeast. However, some amount
of microbes may survive the pasteurization process or may be
inadvertently introduced during further processing. Such microbes
typically cause spoilage of food products causing an economic loss
exceeding $1 billion each year. Moreover, if the surviving microbes
are pathogenic, outbreaks of food borne illnesses may occur among
consumers. It has been estimated that approximately 76 million food
borne illnesses occur per year in the U.S. alone, of which up to
5000 cases result in death, thereby affecting the economic loss
even further.
[0005] Therefore, detecting and quantifying microbes that survive
treatments such as pasteurization is important for assuring food
quality and food safety and further for complying with standards
set by government agencies or trade organizations. For example, the
U.S. Pasteurized Milk Ordinance requires that "Grade A" pasteurized
milk has a total microbial count of not more than 20,000 colony
forming unit (CFU)/ml and a coliform count of not more than 10
CFU/ml. Food producers and/or market food distributors have to
perform microbiological tests to fulfill the regulatory standards.
It is important to their economic operation that they do so with
the least possible expenditure of material and labor.
[0006] There are presently several ways to detect microbes in
clinical or food samples. Broadly categorized, there are (i)
traditional methods such as plate cultures and biochemical assays,
(ii) DNA and antibody based methods, often involving micro/nano
particles and fluorescence, and (iii) other "automated" techniques
that rely on monitoring the effects of bacterial metabolism on the
medium. Of these, traditional methods are the most extensively
used, and often serve as the standard to which other techniques are
compared. However, such traditional methods are tedious, labor
intensive, and require very long times to detect microbes, which
can range from overnight to weeks depending on the type of the
organism and medium used.
[0007] The foregoing are solely two examples how microbes affect
people's daily life and the economy. It is well known how
widespread the impact of such microbes is, spanning from the health
care and pharmaceutical sectors, over the food and livestock
sectors, into municipal and rural population, even into the oil and
gas industries, and industries served with pipelines or storage
tanks are corroded by microorganisms present. Therefore, in a broad
area of economic fields, there is a need to provide an improved
method and device to detect, identify, quantify, viable microbes in
a sample.
SUMMARY OF THE INVENTION
[0008] In one aspect, a method for monitoring the viability of
microbes includes placing a sample of the microbes in a well, the
well is configured with at least two electrodes. A voltage is
pulsed between the two electrodes and an electrical property is
sampled during the voltage pulse. The electrical property is
recorded as a function of time and analyzed to determine microbial
growth.
[0009] In another aspect, a method for identifying bacteria
includes taking a sample of the bacteria, isolating the bacteria
from the sample and dividing the bacteria into a number of wells,
wherein each well is configured with two electrodes. The method
further comprises adding bacteriophages specific to the bacteria
being identified to at least one of the wells. A voltage is pulsed
between the two electrodes and an electrical property is sampled
during the voltage pulse. The electrical property is recorded as a
function of time and analyzed looking for a distinct digital
signature of a successful bacteriophage attack.
[0010] In another aspect, a method for determining the count of
microbes in a sample includes filtering the sample to separate the
microbes from the sample, and immersing the microbes in a life
supporting medium (henceforth called analyte) to form an immersion.
The immersion is divided into wells, and a voltage is pulsed
between the two electrodes and an electrical property is sampled
during the voltage pulse. The electrical property is recorded as a
function of time and analyzed. The electrical property is
correlated to a count.
[0011] In one other aspect, a method for determining antimicrobial
resistance of microbes, includes adding a sample of microbes into a
well containing at least one antimicrobial, and measuring the
viability or growth rate of the microbes by placing a sample of the
microbes in a well, the well is configured with at least two
electrodes. A voltage is pulsed between the two electrodes and an
electrical property is sampled during the voltage pulse. The
electrical property is recorded as a function of time and analyzed
to determine microbial reaction to the antimicrobial.
[0012] In yet another aspect, a diagnostic device for detecting
viability of microbes includes a set of stackable units. The first
unit is a diagnostic unit having a series of wells. The wells have
electrodes contacting the inside and the outside of the wells. The
first unit also has a connection mechanism to facilitate control of
the automated sample preparation. The second unit is a reader unit.
The reader unit includes a connector section for the electrodes and
the automated sample preparation.
[0013] In even another aspect, a diagnostic device for identifying
microbes in a sample, includes a first unit and a second unit,
wherein the first unit is stackable into the second unit. The first
unit is a diagnostic unit including wells, the wells having
electrodes contacting the inside and the outside of the well. The
first unit also has a connection mechanism to facilitate control of
the automated sample preparation. The diagnostic unit also includes
bacteriophage. The second unit is a reader unit and includes a
connector section for the electrodes of the diagnostic unit and the
automated sample preparation.
[0014] In one further aspect, a diagnostic device for determining
the count of microbes in a sample includes a first unit and a
second unit, wherein the first unit is stackable into the second
unit. The first unit is a diagnostic unit including wells, the
wells having electrodes contacting the inside and the outside of
the well. The first unit also has a connection mechanism to
facilitate control of the automated sample preparation. The second
unit is a reader unit and includes a connector section for the
electrodes of the diagnostic unit and the automated sample
preparation, and the reader unit includes a memory chip containing
correlation data.
[0015] In another aspect, a diagnostic device for determining
antimicrobial resistance microbes in a sample includes a first unit
and a second unit; the first unit is stackable into the second
unit. The first unit is a diagnostic unit including wells, the
wells having electrodes contacting the inside and the outside of
each well and an automated sample preparation system. The
diagnostic unit also includes antimicrobials. The second unit is a
reader unit and includes a connector section for the electrodes of
the diagnostic unit and mechanisms for driving the first unit's
automated sample preparation system.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] So that the manner in which the features and advantages of
the embodiments are attained and can be understood in more detail,
a more particular description of the embodiments briefly summarized
above may be had by reference to the appended drawings. However,
the drawings illustrate only some embodiments and therefore are not
to be considered limiting of the scope of the invention which may
admit to other equally effective embodiments.
[0017] FIG. 1 includes a plot of voltages pulses. Further support
for voltage pulse selection is found in FIG. 9.
[0018] FIG. 2 includes an illustration of an embodiment of a
diagnostic device.
[0019] FIG. 3 includes an illustration of an embodiment of the
diagnostic unit of the diagnostic device.
[0020] FIG. 4 includes an illustration of an embodiment of the
reader unit of the diagnostic device.
[0021] FIGS. 5A-5B include a plot of signatures at various
microbial counts of colony forming units per mL.
[0022] FIG. 6 includes plots of signatures showing a phage attack
on bacteria, the second signature showing the control, bacteria in
the absence of a phage.
[0023] FIGS. 7A-7D include plots of signatures for microbes being
treated with different antimicrobial agents.
[0024] FIGS. 8A-8C include an illustration of a technical set-up of
a reader unit.
[0025] FIGS. 9A-9B include a method for determining the correct
voltage, pulse length, and sampling given a particular analyte
[0026] FIGS. 10A-10B include an illustration of a multiplexed
sensor with a 4.times.4 sensor configuration as an extension of
FIG. 8c.
DETAILED DESCRIPTION OF THE INVENTION
[0027] As used herein, the terms "comprises," "comprising,"
"includes," "including," "has," "having" or any other variation
thereof, are intended to cover a non-exclusive inclusion. For
example, a process, method, article, or apparatus that comprises a
list of features is not necessarily limited only to those features
but may include other features not expressly listed or inherent to
such process, method, article, or apparatus. Further, unless
expressly stated to the contrary, "or" refers to an inclusive-or
and not to an exclusive-or. For example, a condition A or B is
satisfied by any one of the following: A is true (or present) and B
is false (or not present), A is false (or not present) and B is
true (or present), and both A and B are true (or present).
[0028] Also, the use of "a" or "an" are employed to describe
elements and components described herein. This is done merely for
convenience and to give a general sense of the scope of the
invention. This description should be read to include one or at
least one and the singular also includes the plural unless it is
obvious that it is meant otherwise.
[0029] Benefits, other advantages, and solutions to problems have
been described above with regard to specific embodiments. However,
the benefits, advantages, solutions to problems, and any feature(s)
that may cause any benefit, advantage, or solution to occur or
become more pronounced are not to be construed as a critical,
required, or essential feature of any or all the claims.
[0030] After reading the specification, skilled artisans will
appreciate that certain features are, for clarity, described herein
in the context of separate embodiments, may also be provided in
combination in a single embodiment. Conversely, various features
that are, for brevity, described in the context of a single
embodiment, may also be provided separately or in any
subcombination. Further, references to values stated in ranges
include each and every value within that range.
[0031] The metabolism of microbes in a medium results in the
release of electrolytes such as carbonates, organic acids, and
salts of sodium, potassium and magnesium into the biomass, i.e., as
a colony of microbes is growing, the electrolytes are being
exchanged with the medium and certain life-events change the
electrolytes in a predictable fashion. For example when
bacteriophages attack a bacterium, up to 10.sup.8 potassium ions
are released.
[0032] On a molecular level, conductance in a medium is a direct
result of mobility of molecules and microbes can be viewed as a
complex of molecules and impacts conductance accordingly. As the
colony of microbes grow, the conductance increases in a predictable
fashion according to Ohm's Law and making use of
equivalent-conductance relations (law of Kohlrausch) and the
Debye-Hueckel theory. As a consequence of life events of the
microbes, the electric property of the medium changes similar to
conductometric titrations.
[0033] The principle for detection relies on the easily
quantifiable and measurable change in electrical properties as a
function of ion content. For example, conductometric titration is a
well-established example of the utility of the method. Ion
conductivity in water is a function of ion mobility in water. For
example, examination of ions produced from the metals from the
first column in the Periodic Table indicate that lithium is the
smallest ion, sodium next larger in size, potassium even larger,
etc. However, lithium ion has the lowest conductivity of all the
metal ions found in the first column of the Periodic Table. This is
because lithium ion is very hydrophilic and builds a large water of
hydration structure around it. The conductive response of these
ions is very specific and, as a consequence, their concentrations
(and changes in concentration) are easily measured. As the change
in concentration of the potassium ions increase with a specific
phage attack of specific harmful microbes, the change in
concentration of potassium ion can easily be measured by changes in
conductivity and conversely resistance and capacitance of the
solution. Therefore, as a result, if one would monitor the change
of electrical properties of a growing colony of microbes over time,
one would observe a decrease of resistance component of total
impedance. This change of an observable electrical property, here
resistance and capacitance, over time forms a signature which
individually or together have characteristics attributable to the
concentration or count of the microbes. When some or all of the
electrical properties are used, various life signs of bacteria may
be detected.
[0034] Furthermore, if a colony has reached its maximum growth and
stagnate in its population, the signature would indicate no change
in the electrical property. Taking this concept further, if a
colony begins to decline in population, their bodies decay into
electrically inert pieces and do not participate in the
conductivity of the medium and the resistance in the medium
increases and can conclude the colony is dying.
[0035] This concept provides the basis for a method that identifies
viability of a microbial colony, providing a positive signature for
a growing colony because the resistance part of impedance is
decreasing over time, a constant signature for a stagnating
population because the resistance part of impedance is constant and
a negative signature for a depleting colony because the resistance
part of impedance is increasing.
[0036] The concept can be even further refined if one applies
agents that target specific microbial species. For example, if one
adds an antimicrobial to the medium and the antimicrobial is active
against the growing colony, the result would be an observation of a
negative signature, or for slow acting antimicrobials a constant
signature for a stagnating population. Likewise, the addition of an
inactive antimicrobial to the medium would result in a signature
representing continued growth of the colony. Furthermore, slight
changes in the signature, would give information as to the
sensitivity of the microbes towards the applied antimicrobial.
[0037] Moreover, the concept can be even tuned to identifying one
specific species of bacteria by applying bacteriophages or phages
(also considered to be an antimicrobial). Phages are viruses that
infect and kill bacteria. Generally, phages are lytic and cause
lysis of the bacteria resulting in a distinctive signature.
Furthermore, the vast number of phages available allow for methods
to identify a single species of bacteria, a class of bacteria, or
even a mixture of bacteria.
[0038] When phages attack a bacteria 10.sup.8 potassium ions are
released into the medium decreasing the resistance part of total
impedance momentarily until the bacteria then reabsorb the ions
during their recovery cycle. The change in resistance of the medium
is immediate and the recovery occurs across a five minute time
span. So without waiting for the bacteria to lysis, the phage
attack can be determined.
[0039] The viability of microbes in a medium can be measured by
monitoring changes in the electrical properties in the medium.
Change of an electrical property over time is defined as a
signature. Electrical properties can be measured by at least two
electrodes present in a well, such as a sample well, containing the
microbes in a supportive medium, such as LB broth.
[0040] Measuring the electrical property should be done in such a
way that the microbes in the aqueous medium are not or minimally
affected by voltages or currents applied to the sample well. One
way to minimize this is by a procedure called sampling.
[0041] 1. Sampling
[0042] FIG. 1 depicts the concept of sampling. A voltage is pulsed
between two electrodes in the well containing a microbe sample in a
supportive medium. The voltage pulse comprises an on-period and an
off-period. The sampling-period is defined by the total length of
time that voltage is applied plus the time that the voltage is
removed, i.e., the on-period+the off-period.
[0043] In embodiments, the measuring circuit comprises a sample
cell containing the microbes in a supportive growth medium of LB
Broth, at least two electrodes for applying a voltage and measuring
conductance. A constant voltage, or reference voltage is applied to
one electrode and the other electrode is connected to a source of
DC voltage that is applied at intervals to create a current
measuring circuit capable of measuring total impedance including
resistance and conductance. The current measuring circuit includes
a low-noise amplifier with a feedback resister; the reference
voltage can be 0.0V or any other DC voltage that is suitable for
ease of implementation of the low-noise amplifier. Then DC voltage
is applied to the other electrode using a circuit with a low-noise
amplifier and as the voltage is applied, the current is measured
according to the clock device. In some cases, it is advantageous
for the voltage applied to each current measuring circuit to have
opposite polarity from one sampling-period to the next.
[0044] In some embodiments, it can be advantageous to measure
resistance, capacitance, and inductance, or total impedance, for
which an alternating current (AC) can be applied during the
sampling-period instead of a direct current (DC).
[0045] In other embodiments, a thermistor or similar device can be
added to the measuring circuit used to capture temperature during
the sampling-period. In yet other embodiments, a pH electrode or pH
probe can be added to the circuit to capture pH and changes of the
pH during the sampling.
[0046] In embodiments, the applied on-period of voltage is at least
about 1 millisecond, at least about 2 milliseconds, at least about
3 milliseconds, at least about 5 milliseconds, at least about 10
milliseconds, at least about 15 milliseconds, at least about 20
milliseconds, at least about 50 milliseconds, at least about 100
milliseconds, at least about 200 milliseconds, or at least about
500 milliseconds.
[0047] In other embodiments, the on-period is not greater than
about 500 milliseconds, not greater than about 200 milliseconds,
not greater than about 100 milliseconds, not greater than about 50
milliseconds, not greater than about 20 milliseconds, not greater
than about 10 milliseconds, not greater than about 5
milliseconds
[0048] In other embodiments, the off-period is at least about 100
milliseconds, at least about 200 milliseconds, at least about 500
milliseconds, at least about 1 second, at least about 2 seconds, at
least about 3 seconds, at least about 5 seconds, at least about 10
seconds, at least about 20 seconds, at least about 40 seconds, or
at least about 50 seconds, or at least about 1 minute.
[0049] In yet other embodiments, the off-period is not greater than
about 60 seconds, not greater than about 30 seconds, not greater
than about 10 seconds, not greater than about 5 seconds, not
greater than about 2 seconds, not greater than about 1 second, not
greater than about 500 milliseconds, not greater than about 200
milliseconds, not greater than about 100 milliseconds, or not
greater than about 50 milliseconds.
[0050] In yet other embodiments, the sum of on period is between
one second and one minute. For example, the on-period can be 50
milliseconds and the off-period can be 950 milliseconds. In other
examples, the on-period can be 5 milliseconds and the off-period
can be 995 milliseconds. From these examples, it can be seen that
the on-period comprises a relative short fraction of the sampling
period, while the off-period comprises the majority of the sampling
period. Accordingly, during the monitoring, the sample is exposed
to a voltage and current only for a brief duration.
[0051] In embodiments, the voltage applied to a sample is DC
voltage can be at least about 0.0005 V, at least about 0.001 V, at
least about 0.002 V, at least about 0.005 V, at least about 0.01 V,
at least about 0.02 V, at least about 0.05 V, at least about 0.1 V,
at least about 0.2 V, 5 V, at least about 1.0 V, at least about
2.00 V, at least about 5.0 V, or at least about 10.0 V.
[0052] In yet other embodiments, the voltage is not greater than
about 5.0 V, not greater than about 2.0 V, not greater than about
1.0 V, not greater than about 0.5 V, not greater than about 0.2 V,
or not greater than about 0.1 V. For example, the voltage can be
applied between 50 mV to 1.24 volts and still be below the
electrolysis of water or other ingredients of the sample well.
[0053] In embodiments, the sampling-duration is defined by the
total number of sampling-periods. The sampling-duration varies by
the diagnostic function being implemented. For example, bacterial
identification's sampling-duration can be from 2 minutes to 10
minutes. Yet in other embodiments, the sampling duration can be
from 2 minutes to 30 minutes, or even 60 minutes. In another
example, antimicrobial sensitivity test sampling-duration can be
from 40 minutes to 4 hours. Yet in other embodiments, the
antimicrobial sensitivity test sampling can be longer than 4 hours.
In yet another example, the colony counter can have a sampling
duration of one sampling-period. Thus, the colony counting can be
achieved in as little as one minute.
[0054] In embodiments, testing or monitoring the viability of
microbes can take not longer than about 360 minutes, not longer
than about 180 minutes, not longer than about 120 minutes, or not
longer than about 90 minutes. In yet other embodiments, testing or
monitoring the viability of microbes can take not longer than about
60 minutes, not longer than about 45 minutes, or not longer than
about 30 minutes. In even further embodiments, testing or
monitoring viability of microbes can take not longer than about 20
minutes, not longer than about 10 minutes, not longer than about 5
minutes, or not longer than about 2 minutes.
[0055] The method according to any one of the preceding claims,
wherein the monitoring of the viability is between about 15 seconds
and about 60 minutes, between about 15 seconds and about 45
minutes, between about 15 seconds and about 20 minutes, between
about 15 seconds and about 10 minutes, between about 1 minute and
about 20 minutes, between about 2 minutes and about 20 minutes,
between about 5 minutes and about 20 minutes, between about 5
minutes and about 10 minutes, or between about 10 minutes and about
20 minutes.
[0056] Refinement of Signal Fidelity (FIGS. 9B-9B) and (FIGS.
10A-10B)
[0057] In order to improve fidelity of the signal for a particular
analyte, multiple electrical properties may be sampled, and the
voltage strength and pulse on and pulse off-period must be
carefully tuned to the analyte. In one embodiment of the invention
resistance, capacitance and the electrical time constant was
sampled and the system can be characterized by a RC circuit
model.
[0058] In this embodiment, circuitry was constructed to provide a
low impedance voltage buffer to drive the cell under test, with a
transimpedance amplifier to convert the resulting cell current into
a voltage signal. This allowed characterization of the cell
response to proceed with a good signal-to-noise ratio and high
repeatability. A signal generator was used that output a positive
pulse at several pulse amplitudes to refine the signal generation
and sampling.
[0059] The 4.times.4 test cells, FIG. 10A, that were tested under
the above conditions with LB broth show an ionic solution about
resistance of 550 OHMs and an electrode capacitance of 0.04 uF.
With a 50 mV positive pulse, the initial test cell current (peak)
is +91 uA. This current produces a peak transimpedance amplifier
output voltage of -91 mV. During the positive voltage pulse the
test cell current decreases exponentially as the electrode
capacitance charges. This exponential current trace has time
constant of 22 us.
[0060] Verification that the cell responds as a linear network, at
low signal levels, and measurement of the resistance and
capacitance that comprise this equivalent series RC circuit,
provided a foundation for comparing different analytes. At higher
signal levels nonlinear effects take over as the system loses
scaling and time invariance, making comparisons more difficult.
Operating well into the nonlinear regime can cause permanent
changes in the response, often with visible signs of electrode
corrosion.
[0061] An important part of this work was identifying the boundary
separating the linear and nonlinear regimes. To remain below the
threshold of departure from the linear model, maximum test signal
amplitudes should be characterized for the analyte solution and
operating below the threshold is required.
[0062] 4.times.4 Sensor (FIGS. 10A-10B)
[0063] One embodiment of the sensor was made by placing copper
traces on a circuit board, then using solder mask to provide at
least two electrodes of diameter 1 mm with centers 5 mm apart in
each cell. A standard electroless nickel-gold plating process was
then used to coat the traces with gold. The gold thickness in the
IPC-4552, Specification for Electroless Nickel/Immersion Gold
(ENIG) Plating for Printed Circuit Boards is 1.97 microinches
minimum. The nickel thickness is 118.1 to 236.2 microinches thick.
A solder mask was carefully applied leaving two circular electrode
patterns exposed and carefully spaced at the appropriate area in
each cell. It is important to size the ending square pads of each
electrode so they are large enough to accommodate the inaccuracy of
solder mask application. The functioning of the sensor is dependent
on the relationship of the diameter of the opening in the solder
mask, the distance between the openings and the size of the cell
holding the solution. In this embodiment, sixteen 1 cm diameter
holes were bored in a Delrin block. Then the Delrin block was
attached to the circuit board using an epoxy adhesive. In this
embodiment each cell holds approximately 1 ml of liquid. In this
embodiment a Van Der Pauw cell was added to aid in process control
during manufacturing.
[0064] Other embodiments can be made using injection mold
techniques using a wiring harness.
[0065] Another embodiment could coat the copper traces on the
circuit board or wires in the wiring harness with graphene or other
conductive materials instead of gold.
[0066] 2. Diagnostic System
[0067] FIG. 2 displays an implementation of the diagnostic system
200 in its assembled configuration and this configuration is
optimized for testing of liquid samples, such as urine. The
diagnostic system comprises two stackable units, the diagnostic
unit 202 and the reader unit 204. The diagnostic system 200 has an
outside length l, width w, and height h in its stacked
configuration. The length can be between about 2.5 and about 4.5
inches, preferably, between about 3.0 and about 4.0 inches, more
preferably about 3.5 inches. The width w of the reader unit 204 and
can range from about 3.5 to about 5.5 inches, preferable between
about 4.0 and about 5.0 inches, more preferably, about 4.4 inches
The heights h is depending from the size of the diagnostic unit 202
and can range from about 3.5 to about 5.0 inches, preferable
between about 4.0 and about 4.5 inches, more preferably, about 4.2
inches. In other embodiments, the stackable units can stack in a
side by side configuration. In other embodiments, the stackable
units can be miniaturized to accommodate lessor volumes required
when testing other sample types. In other embodiments, the
stackable units can be formed into one integrated unit.
[0068] FIG. 3 depicts the interior of the diagnostic unit 202. The
interior of the diagnostic unit has an assembly of tubes, liquid
compartments, filters, and wells. The flows of a sample and other
liquids can be regulated by pressure applied in series to chambers
312, 306, 308. Valves 314 can be one-way valves, i.e., allowing
flow only in one direction. The valves can control flows into and
from 320, 322, 330, and into 332, and also into and from manifold
316 and further into wells 318. In other embodiments, pressure can
be independently regulated by an electronic mechanism present in
reader unit 204 and further discussed below.
[0069] A sample holder 302 receives a sample from a patient. The
samples can be taken 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 figured
in such way that a positive pressure is created inside the sample
holder during the tightening of the cap.
[0070] Some embodiments can have the reader unit 204 provide
pressure to the fluidic system of the diagnostic unit using an
electronic pressurizing system instead of the screw cap pressure
system. The reader unit's pressurizing system would then connect to
the diagnostic unit via at least one pressure port.
[0071] While the sample remains in sample holder 302, pressure
forces liquid from chamber 312 into the manifold 316 and then
evenly into wells 318. Antimicrobial or bacteriophage can be stored
dry separately in some or all wells. Liquid that flows into wells
318 dissolve or emulsify these antimicrobials or bacteriophages.
The liquid will depend on best practices for dissolving the
antimicrobials or bacteriophages. Some embodiments will have more
than one chamber such as chamber 312. For example, when different
dried materials require different liquids for reconstituting,
dissolving, or preparing antimicrobials or bacteriophages,
additional chambers such as chamber 312 will hold the necessary
liquid. Some embodiments divide each chamber 318 into two halves
and the dissolved or emulsified antimicrobial flow into one half
then through a micron filter and one-way valve into the other half.
Some of the wells receive the microbial sample after the sample has
been filtered and prepared. The volume of this liquid can range
between 4.0 ml to 12 ml depending on donor sample type.
[0072] 3044 displays a tube that connects liquid compartment 306
with the sample holder 302. Liquid compartment 306 can contain an
aqueous liquid, deionized water, buffer, or broth. The liquid in
306 can serve various purposes. For example, in some embodiments,
the liquid in compartment 306 can dilute a sample. In other
embodiments, the liquid in compartment 306 can adjust the pH of a
sample. In yet other embodiments, the liquid in compartment 306 can
contain sterilized broth that facilitates growth of microbes
present in the sample. The volume capacity of compartment 306 can
range from about 1 mL to about 24 mL, preferably from about 3 mL to
about 5 mL, more preferably, about 4 mL.
[0073] In embodiments where pressure is applied to chamber 306, the
liquid there is forced into sample holder 302 and forces the sample
to discharge through 3044 and then through a one-way valve 314. The
sample holder 302 discharges its content via tube 3042 into
filtration unit 310. Filtration unit 310 comprises of several
chambers separated by filters and configured with one-way valves
314 assuring no backflow of liquid after filtration. The content of
sample holder 302 is discharged into receiving chamber 320.
Adjacent to receiving chamber 320 is the microbe chamber 322.
Chambers 320 and 322 are separated by a filter 324. Filter 324 has
a filter size selected in such way that microbes can pass through
the filter into chamber 322, while insoluble material, particles,
human or animal cells, and biological matter larger than the filter
size remain in receiving chamber 320. The filter material can be
any suitable material. For example, the filter material can be
cellulose, polymer, or glass fiber. For example, in embodiments,
the filter material can be polyvinylidene fluoride (PVDF) membrane.
The PVDF membrane can have a cellulose ester (RW06) prefilter
layer. In embodiments, filter 324 can have a filter size of not
greater than 0.45 microns, not greater than 0.5 microns, or not
greater than 0.6 microns. In other embodiments, filter sizes for
filter 324 that are not greater than 0.8 microns, or 1.0 microns,
or even 2.0 microns are contemplated.
[0074] Adjacent to microbe chamber 322 is phage chamber 330, which
is separated by filter 326. Contrary to filter 324, filter 326 has
a filter size selected in such way that microbes do not pass
through the filter into chamber 330, while material smaller than
the filter size flow from chamber 322 into chamber 330. Such
material includes wild-type phage present in the sample. The filter
material of filter 326 can be any suitable material. For example,
the filter material can be cellulose, polymer, or glass fiber. For
example, in embodiments, the filter material can be polyvinylidene
fluoride (PVDF) membrane. The PVDF membrane can have a cellulose
ester (RW06) prefilter layer. In embodiments, filter 326 can have a
filter size of not greater than 0.1 microns, not greater than 0.2
microns, or not greater than 0.45 microns. In other embodiments,
filter sizes for filter 324 that are not greater than 0.5 microns,
or 0.6 microns, or even 0.7 microns are contemplated
[0075] Adjacent to phage chamber 330 is located waste chamber 332
separated by filter 328. Contrary to filters 324 and 328, filter
328 has a filter size selected in such way that wild-type phage do
not pass through the filter into chamber 332, while material
smaller than the filter size flow from chamber 330 into chamber
332. The filter material of filter 328 can be any suitable
material. For example, the filter material can be cellulose,
polymer, or glass fiber. For example, in embodiments, the filter
material can be polyvinylidene fluoride (PVDF) membrane. The PVDF
membrane can have a cellulose ester (RW06) prefilter layer. In
embodiments, filter 328 can have a filter size of not greater than
0.05 microns, not greater than 0.1 microns, or not greater than 0.2
microns. In other embodiments, filter sizes for filter 328 are not
greater than 0.3 microns, or 0.4 microns or even 0.45 microns are
contemplated.
[0076] Although not depicted in FIG. 3, the flow of liquid from
chamber 320 through filter 324 into chamber 322 or the flow of
liquid from chamber 322 through filter 326 into chamber 330, as
well as the flow of liquid from chamber 330 through filter 328 into
chamber 332 can be regulated by one-way valves, to avoid backflow
of liquids into the previous chamber.
[0077] Analyte chamber 308 contains an analyte solution and is in
connection with microbe chamber 322. The analyte solution is the
supportive of the microbes, once mixed it forms the microbe sample
which will be analyzed for identity, count, or antimicrobial
resistance. The analyte solution flows directed by one-way valve
314 then into chamber 322 and thereby immersing microbes present
from the filtration of the donor sample. The analyte solution
includes ingredients that support the viability of the microbes.
For example, the analyte solution can contain a broth, a diluted
broth, a buffer, or a buffer mixed with a broth. The same solution
can also be contained in chamber 312.
[0078] Upon immersion of microbes in chamber 322 by the analyte
solution to form the microbe sample, the microbe sample flows from
chamber 322 into manifold 316, which distributes the sample evenly
over a number of wells 318. The number of wells 318 can be between
2 to 24, preferably, 8 to 20, preferably about 18. Regardless of
the total number of wells, at least one well does not receive the
microbe sample but receives analyte solution from chamber 312. This
well is designated the control well. Each well 318 is equipped with
at least two electrodes, the electrodes are connected to a
stackable interface 320, which connects with reader unit 204.
[0079] The electrodes in the wells can be made of any known
electrode material. In embodiments, the electrode material can be
coated with a material that increases the sensitivity of the
electrode. In embodiments, the electrode material can be coated
with noble metals such as gold, platinum, or palladium. The
electrodes should be made from non-oxidizing material and may
consist of several metal and non-metal materials. In other
embodiments, the electrode material is copper coated with a special
formulation of graphene. The copper coating and graphene creates a
non-oxidizing, highly conductive electrode.
[0080] In one embodiment, an aqueous dispersion of graphene can be
prepared by catalytic hydrogenation of humic acid. Humic acid can
be extracted from leonardite (Agro-Lig) and then catalytically
hydrogenated. Catalytic hydrogenation can be done using various
catalysts in a Parr reactor at 150.degree. C. The catalysts can be
palladium or platinum metal or palladium on charcoal or platinum on
charcoal. The dispersion can be passed through a strong acid ion
exchange column to remove excess cations. The aqueous dispersion of
graphene can be applied to electrodes, such as copper electrodes,
gold electrodes, or silver electrodes. In embodiments, the graphene
content of the aqueous dispersion can be 0.5% by weight. In another
embodiment, the graphene content might be 1% by weight. And yet
another embodiment the graphene content might be 2% by weight. In
yet other embodiments, the graphene content can be about 0.1% by
weight, about 0.2% by weight, about 0.5% by weight, about 0.8% by
weight, about 0.9% by weight, about 1.0% by weight, about 1.5% by
weight, about 1.8% by weight, about 2.0% by weight, or about 2.5%
by weight.
[0081] Graphene prepared by this method has functional groups, such
as hydroxyl groups bonded to the graphene. These functional groups
have an affinity to bind the graphene to a metal and thus, improve
the coating of the electrode with graphene. A graphene coated
electrode improves resistance of the electrode to oxidation and
also improves the conductivity property of the electrodes.
[0082] In some embodiments, the diagnostic device further includes
a heating component, for heating the unit or compartments thereof.
For example, a heating component, such as a heating coil, can be
placed around microbe chamber 322, to control the temperature of
the microbes sample.
[0083] In embodiments, the diagnostic unit, including a sample
holder and a filtration unit can be used in combination with any
analytical reader unit. For example, the diagnostic unit can be
adjusted as sample preparation device for antimicrobial analysis,
where the analysis is conducted by conventional methods, such as
enzyme assays or fluorescent assay.
[0084] FIG. 4 displays the reader unit 204. The reader unit has a
card connector slot 402 to receive the stackable interface 320. The
interior includes various electronic components. The interior
includes an analog to digital (A/D) converter 404, a digital signal
processor 406, a display processor 408, and a memory component 410.
The reader has a clock unit which is not shown in diagram for
controlling the sample-period and sample-duration. The reader can
receive an input using communication port represented by 412 so the
data being collected into memory component 410 can be associated
with the patient. In some embodiments, the input mechanism can be
integrated to the reader device. Some or all of these electronic
components 404 through 410 can be consolidated into one component.
The reader unit further includes a power source 414, such as a
battery and a port 412 for transmitting data to another computing
device where it can be stored as a database or further processed.
The transmission through port 412 can occur directly through a wire
into a computing device where it can be stored in a database or
further processed. Alternatively, the transmission through port 412
can occur wirelessly through a wireless network, cellular network
or the World Wide Web to reach an application for further
processing, storing in a database or presented to a user. In
embodiments, data obtained by the diagnostic device can be
transmitted as a secured email or SMS to at least one user, such as
a doctor or nurse. In other embodiments, data obtained from the
diagnostic unit can be transmitted to other users or databases such
as health care facilities, centers for disease control, or
insurance companies. In other embodiments, port 412 may receive
input directly associated with the patient to be stored in memory
component 410 where it is used to associate the data to the
patient.
[0085] The reader unit further includes a display, the display can
be a well indicator field 416 which display, which well has
signature associated with an attack or inhibition by an
antimicrobial. Further the display can be associated with positive
or negative bacteria identification. The well indicator field can
be correlated with a template envisioned to be applied as a label
to the first stackable unit. The indicator field might be lights
but other embodiments are being contemplated.
[0086] The reader unit further can include a count output 418 that
displays the measured count. The count can be displayed as a figure
or as a meter light, which, for examples, shows more, bars the
higher the count is. Further, there are displays to indicate that
self-test diagnostics functioned properly and further that the
battery is active. There is also an on/off button contained in area
418.
[0087] Upon inserting a biological sample into sample holder 302 of
the diagnostic unit 202, the unit is inserted into card reader slot
402. In embodiments, the reader unit can additionally be equipped
with a micro pump system that controls the flow of liquids through
the diagnostic unit 202. In other embodiments, the reader unit
controls one-way valves 314 to direct the flow of liquids through
the diagnostic unit 202. In yet other embodiments, the reader unit
controls the flow of liquids through the diagnostic with the help
of a micro pump and individual control over one-way valves 314. In
some embodiments, the card slot 402 can be located to facilitate
side by side arrangement of 204 and 202. In some embodiments, 202
and 204 can be integrated into one unit and miniaturized according
to the sample type.
[0088] Upon pretreatment of the biological sample to obtain a
microbes sample in chamber 322, the microbes sample is distributed
into a number of wells 318, while at least one well serves as a
control and contains only analyte solution form compartment 312. At
this point, the diagnostic device can sample data in accordance
with the sampling method described herein.
[0089] In embodiments, where a count is determined, the reader unit
samples an electrical property such as conductance, resistance,
voltage, amperage, capacitance, impedance, inductance, or any
combinations thereof to create a digital signature.
[0090] In embodiments where identity of a microbe in a microbial
sample is determined, the wells 318 of the diagnostic unit contain
bacteriophages, mycoviruses, virophages, or nematophages. These
phages are specifying in attacking bacteria, fungi, viruses, or
nematodes, respectively. The phages are present in the wells before
a biological sample is applied to the diagnostic unit.
[0091] In embodiments, each well that receives bacteria sample
contains a different bacteriophage. Bacteriophages only attack
bacteria having the appropriate binding sites. Accordingly,
bacteriophages can be chosen for specific bacterium so that it will
only attack that bacterium. Once a bacteriophage attacks bacteria,
a signal in the sampling can be observed. Therefore, by measuring
bacteriophage attacks of one or more bacteriophages onto the
bacteria sample, one can obtain conclusive data as to the
percentage of bacteria species present in the sample and the
identity of such bacteria.
[0092] Some embodiments might use other types of phages to identify
other types of microbes. For example, if the microbe is a fungus, a
mycovirus can be used to identify the microbe. In other
embodiments, virophages or nematophages can be used to identify
viruses or nematodes.
[0093] In embodiments, the bacterial identification feature can be
implemented with only one well, but may consist of more wells for
increased accuracy. At least one well contains the sample with
bacteriophage specific to the bacterium being identified.
Bacteriophage can be selected to attack one-and-only-one bacterium.
In yet other embodiments, bacteriophages may be combined to create
a phage-cocktail which can be used to identify a group of
bacteria.
[0094] In embodiments, where antibiotic resistance or antimicrobial
resistance of a microbes sample is determined, the wells 318 of the
diagnostic unit contain antibiotics or antimicrobials and at least
one well contains the microbial sample as a control well. These
antibiotics or antimicrobials are present in the wells before a
biological sample is applied to the diagnostic unit. In
embodiments, each well that receives microbes sample contains a
different antibiotic or antimicrobial. Antibiotics or
antimicrobials work differently for different strains or species of
microbes. Once a microbe colony stagnates or dies from an
antibiotic or antimicrobial present in the well, a signal of this
particular well in the sampling can be observed. Therefore, by
measuring antibiotic or antimicrobial activity of one or more
antibiotics or antimicrobial onto the microbial sample, one obtains
conclusive data as to the antibiotic or antimicrobial
susceptibility of the microbial species present in the sample. On
the other hand, for wells where there is no antibiotic or
antimicrobial activity, and the control well shows microbial
viability, then one observes antibiotic resistance or antimicrobial
resistance of the microbe sample.
[0095] In embodiments, the antimicrobial sensitivity test requires
at least two sensor-wells. At least one sensor-well contains a
control sample consisting of the microbial sample, i.e. microbes
immersed in analyte solution. The other sensor contains the sample
with the antibiotic or antimicrobial being tested for
effectiveness. At the end of the sample-duration, the colony count
of the control sample will be compared against the beginning colony
count and if there were microbes the count will have increased by
the end of the sample-duration. When there is no growth of microbes
in the control sample, then the results from the sample with the
antibiotic or antimicrobial will be suppressed to support the
observation that the microbes was not active and therefore not in
need of antibiotic or antimicrobial treatment. Otherwise, the
results from comparing the antimicrobial sample's digital signature
created during the sample-duration will be analyzed and reported.
Digital signature is further described herein.
[0096] In embodiments, two cells both containing samples from the
same source can be compared to quantify the effectiveness of an
antimicrobial by placing antimicrobial in one cell along with the
sample and after a time period releasing bacteriophage also into
that cell (Cell AP) and measuring the signal in the sampling and
comparing it against the sampling signal in another cell with only
bacteriophage released. (Cell P). The difference in the signals of
the cells divided by the signal of the sample signal of the second
cell will indicated the percentage of bacteria killed by the
antimicrobial. (Cell P-Cell AP)/Cell P; where Cell P is the signal
strength of the cell after adding bacteriophage to the sample; Cell
AP is the signal strength determined when the bacteriophage are
added after waiting a time period after the antibiotic have been
added.
[0097] In yet other embodiments, the diagnostic system may consist
of one or any combination of the features: bacterial
identification, microbial colony counter, or antimicrobial
sensitivity test. For example, some wells may contain no additives,
such as phage or antimicrobials, some wells may contain phages, and
some wells may contain antimicrobials or antibiotics. Such assembly
facilitates the analysis for count, identity, and treatment with an
antibiotic or antimicrobial of a microbial sample.
[0098] 3. Digital Signature
[0099] A digital signature consists of data captured during the
sample-duration and is a distinctive curve. Digital signal
processing pattern matches across the distinctive digital signature
to arrive at a pattern match. The distinctive digital signatures
are recorded in a database based on earlier characterization. In
embodiments, digital signatures are based on capturing changes in
electrical properties such as the average resistance of the test
sample as a function of time. In some embodiments the digital
signature can be determined without pattern matching, but using
other functional analysis.
[0100] In general, the reader unit is able to detect the change in
concentration of live microbes based on changes in the resistance
of the microbial sample, i.e. microbes immersed in the analyte
solution. In embodiments, there are other distinctive digital
signatures. For example when a bacteriophage attacks a bacterium,
up to 10.sup.8 potassium ions can be released from the bacteria
followed by reabsorption of the ions by the bacteria, which creates
a distinctive change in measured resistance with time.
[0101] Each life event of the microbes has a distinctive pattern.
For example, growing microbes multiply and create less resistance
because they emit protons and ions of potassium, calcium and sodium
as part of their natural respiration and metabolic process. For
example, bacteria multiply every twenty minutes and this life event
can be detected by constantly decreasing resistance
measurements.
[0102] 4. Microbial Count
[0103] FIG. 5A depicts resistance measured of samples containing
various concentrations of colony forming units (CFU) of E. Coli B
suspended in LB Broth. FIG. 5B depicts resistance measured of
samples containing various concentrations of colony forming units
(CFU) of E. Coli B suspended in artificial urine. Samples have the
following concentration: 10.sup.2 CFU/mL, 10.sup.4 CFU/mL, 10.sup.5
CFU/mL, 10.sup.6 CFU/mL, 10.sup.7 CFU/mL and 10.sup.9 CFU/mL FIGS.
5A-5B clearly show that samples have distinctive resistance
component of total impedance over time. Samples can be
distinguished by their average resistance value. In embodiments,
this feature is employed to determine the concentration of a
microbial sample and correlate such concentration under
consideration of the volume of the biological sample placed in the
sample holder to a count or concentration present in the biological
sample. This feature is also employed to determine microbial
viability.
[0104] 5. Bacterial Identity
[0105] FIG. 6 depicts the signature of a sample of 10.sup.8 CFU/ml
E. Coli B in an analyte of LB Broth for a final volume of 0.9 mL
being attacked by 0.1 mL concentration of 1.62.times.10.sup.11 T4
phage and a control sample in the absence of a phage. Signature 602
displays the course of a control sample not containing a phage.
Initially, the sample rapidly reduces in resistance due to a fast
growth of colonies in the sample, which contains some broth. The
signal was captured just as the bacteria slow their growth. The
resistance component of total impedance value levels off into a
linear course as the bacteria growth begins to slow down due to the
limited resources of nutrients in the sample. The control sample
maintains the linear slope for at least more than 20 minutes (or
1200 seconds). The last section of signature, starting at about
1401 seconds shows a slight increase in resistance, indicating that
the bacteria population reduces in numbers.
[0106] On the other hand, signature 604 of FIG. 6 shows the
signature of a microbial colony of E. Coli B under phage attack.
Phage attack is initiated by adding phage before the data are being
collected and at the point indicated on the graph. The phage attack
is instantaneous and is already in progress before the sample
duration was initiated and the bacteria have each released up to
10.sup.8 potassium ions causing a rapid drop in resistance of the
analyte. Within the next 200 seconds following the phage attack,
there is a rise in resistance component of total impedance,
indicating that the colony is attempting to recover from the attack
by reabsorbing the potassium ions that the phage attack caused the
bacteria to release. After about 200 seconds, the phage attack is
over and the signature levels off. During the course of the next 20
to 25 minutes, a positive slope of the signature can be observed,
indicating that the bacteria colony is inhibited in its growth.
Furthermore, an increase in the slope of the signature can be
observed at about 1400 seconds. This is approximately the time at
which phages have replicated and begin to lyse the bacteria
colony.
[0107] Comparing signatures 602 and 604 in FIG. 6, a sample
containing a known phage specific to a certain strain or species of
bacteria can be used to identify whether the sample contains this
specific bacteria colony. Likewise, if a sample contains two types
of bacteria distinct in strain or species, an assay of sampling
these bacteria against different phages can identify the presence
of each strain or species, independently.
[0108] 6. Antimicrobial Resistance
[0109] FIGS. 7A-7D depict signatures of microbial sample of 0.9 mL
of a concentration of 10.sup.3 CFU/mL of two-hour E. Coli B treated
with 0.1 mL of antibiotics or antimicrobials and control samples in
the absence thereof. What is common to all FIGS. 7A-7D is the slope
for signatures with antibiotics or antimicrobials become positive,
i.e., the resistance component of total impedance of the microbial
sample increases, upon addition of the antibiotic or antimicrobial.
The slope of the control samples remains negative.
[0110] In further detail, a difference in the degree of the slope
change for the signature can be observed. Such differences are due
to the type of antibiotic or antimicrobial, and its mechanism of
action. For example, sulfamethoxazole, FIG. 7C, causes inhibition
of DNA synthesis in the cell, thereby aiming essential cell
functions. In contrast, azithromycin, FIG. 7B, inhibits protein
inhibition in a bacterium, thereby acting on latent cell functions.
As a result, the slope of the signature for sulfamethoxazole is
steeper than for azithromycin, because the microbial colony treated
with the former is expected to die faster.
[0111] 7. Technical Preparation of a Reader Unit
[0112] FIGS. 8A-8C displays an implementation of a reader unit
having two leads. The leads are connected to the electrodes of the
sample well. The reader unit as shown in FIGS. 8A and 8B controls
the sampling and recording of data. In embodiments, a sample well
or sample tube as for example depicted in FIG. 8C, includes a
container equipped with electrodes (not visible in FIG. 8C) and
contacts on the outside of the tube to which the reader unit can be
connected. In one embodiment, the electrodes can be copper wires
coated with graphene from a 2 wt % graphene solution to form a
non-corrosive yet conducting surface on the copper wire. A control
well contains the same ingredients as the sample well with the
exception of the microbe and both wells are connected to the leads
depicted in FIG. 8a as sensors #1 and #2, respectively. Data is
collected by way of the sampling method described herein. The
reader unit includes a feedback resistor and low noise amplifier, a
display with a display controller and a central processing unit
(CPU) which also may house an analog to digital (A/D) converter.
The communication port can transmit data to another computer for
analysis.
[0113] In embodiments, the communication port is a POTS phone
connection. Another embodiment implemented was a USB connection.
Another embodiment is envisioned as a connection to other direct
wired ports used by other computing devices which may be laptops,
desktops, notebooks, tablets, mini-PC, mini-tablets, and cell
phones or other such devices.
[0114] Another embodiment is contemplated to be a wireless
connection. Yet another embodiment is envisioned to be a cellular
connection. Both embodiments are envisioned as a connection to
other wireless ports used by other computing devices which may be
laptops, desktops, notebooks, tablets, mini-PC, mini-tablets, and
cell phones or other such devices.
[0115] Other embodiments may communicate with a Smartphone
providing final results. Yet other embodiments might stream data to
a Smartphone so the Smartphone might analyze the data and send
resulting data to user via secured email or SMS messages.
[0116] Other embodiments may communicate with a Smartphone
providing parameter inputs to the diagnostic device. Additionally
other embodiment might download patient information to be stored
with results for later transmission by diagnostic device along with
the data or results from the diagnostic reader unit.
[0117] Another embodiment is envisioned to connect to the World
Wide Web with an application on a server to analyze the data and
store it in a database. It is envisioned that the application can
further interface to other applications as a software-as-a-service
application. In this embodiment a fee-for-service can provide
healthcare workers and antibiotic manufacturers with regional
demographics of antibiotic resistant microbes and antibiotic use
collected from statistics using depersonalized results of
accumulated diagnostic results. Another embodiment will place
antibiotic advertising on the labeling of the diagnostic kit.
[0118] Another embodiment might receive input commands from a
computing device via the communication port. Additionally other
embodiment might download patient information to be stored with
results for later transmission by diagnostic device along with the
data or results from the diagnostic reader unit.
[0119] Many different aspects and embodiments are possible. Some
aspects and embodiments are described below. After reading this
specification, skilled artisans will appreciate that those aspects
and embodiments are only illustrative and do not limit the scope of
the present invention.
[0120] In one aspect, a method for determining antimicrobial
activity of an agent includes providing a well, wherein the well
contains one or more antimicrobial agents. The well further
includes at least two electrodes. The method further includes
adding a sample of at least one microbe into the well, pulsing
voltage between the electrodes, sampling an electrical property
during the pulsing, and recording the electrical property.
[0121] In embodiments, the method further includes repeating the
pulsing, the sampling and the recording, and plotting the
recordings versus time to form a signature. In other embodiments,
the method further includes that the microbes are selected from
bacteria, fungi, viruses, or nematodes. In other embodiments, the
method may include one or more antimicrobial agents selected from
bacteriophages, mycoviruses, virophages, nematophages, antibiotics,
antimicrobials, antivirals, antifungals, or parasiticides.
Bacteriophages, mycoviruses, virophages, nematophages are viruses
that attack bacteria, fungi, viruses, and nematodes, respectively.
In yet other embodiments, the method further comprises measuring a
temperature inside the well. The temperature can be measured with a
thermistor.
[0122] The pulsing of the method includes an on-period and an
off-period, the sum of the on-period and the off-period comprises a
sample-period. In embodiments, the on-period is at least about 1
millisecond, at least about 2 milliseconds, at least about 3
milliseconds, at least about 5 milliseconds, at least about 10
milliseconds, at least about 15 milliseconds, at least about 20
milliseconds, at least about 50 milliseconds, at least about 100
milliseconds, at least about 200 milliseconds, or at least about
500 milliseconds. In other embodiments, the off-period is at least
about 100 milliseconds, at least about 200 milliseconds, at least
about 500 milliseconds, at least about 1 second, at least about 2
seconds, at least about 3 seconds, at least about 5 seconds, at
least about 10 seconds, at least about 20 seconds, at least about
40 seconds, at least about 50 seconds, or at least about 60
seconds. In other embodiments, the on-period is not greater than
about 500 milliseconds, not greater than about 200 milliseconds,
not greater than about 100 milliseconds, not greater than about 50
milliseconds, not greater than about 20 milliseconds, not greater
than about 10 milliseconds, not greater than about 5 milliseconds.
In yet other embodiments. the off-period is not greater than about
60 seconds, not greater than about 30 seconds, not greater than
about 10 seconds, not greater than about 5 seconds, not greater
than about 2 seconds, not greater than about 1 second, not greater
than about 500 milliseconds, not greater than about 200
milliseconds, not greater than about 100 milliseconds, not greater
than about 50 milliseconds. In yet other embodiments, the
sample-period is about 1 second, about 5 seconds, about 10 seconds,
about 20 seconds, about 30 seconds, about 40 seconds, about 50
seconds, about 60 seconds.
[0123] In embodiments, the voltage is at least about 0.0005 V, at
least about 0.001 V, at least about 0.002 V, at least about 0.005
V, at least about 0.01 V, at least about 0.02 V, at least about
0.05 V, at least about 0.1 V, at least about 0.2 V, at least about
0.5 V, at least about 1.0 V, at least about 2.00 V, at least about
5.0 V, or at least about 10.0 V. In other embodiments, the voltage
is not greater than about 2.0 V, not greater than about 1.0 V, not
greater than about 0.5 V, not greater than about 0.2 V, or not
greater than about 0.1 V. In further embodiments, the voltage is
ranging from about 0.0005 V to about 2.0 V, from about 0.0005 V to
about 1.0 V, from about 0.001 V to about 1.0 V, from about 0.05 V
to about 1.0 V, from about 0.05 V to about 0.5 V, or from about
0.05 V to about 0.1 V.
[0124] In embodiments, the sampling of the electrical property
occurs during the sample-period. Yet, in other embodiments, the
sampling of the electrical property is at least about 0.5
milliseconds, at least about 1 millisecond, at least about 2
milliseconds, at least about 3 milliseconds, at least about 5
milliseconds, at least about 10 milliseconds, at least about 15
milliseconds, at least about 20 milliseconds, at least about 50
milliseconds, at least about 100 milliseconds, at least about 200
milliseconds, or at least about 500 milliseconds. In further
embodiments, the sampling is not longer than about 360 minutes, not
longer than about 180 minutes, not longer than about 120 minutes,
not longer than about 90 minutes, not longer than about 60 minutes,
not longer than about 45 minutes, not longer than about 30 minutes,
not longer than about 20 minutes, not longer than about 10 minutes,
not longer than about 5 minutes, or not longer than about 2
minutes. In yet other embodiments, the sampling is between about 15
seconds and about 60 minutes, between about 15 seconds and about 45
minutes, between about 15 seconds and about 20 minutes, between
about 15 seconds and about 10 minutes, between about 1 minute and
about 20 minutes, between about 2 minutes and about 20 minutes,
between about 5 minutes and about 20 minutes, between about 5
minutes and about 10 minutes, or between about 10 minutes and about
20 minutes.
[0125] In another aspect, a method for identifying at least one
microbe includes taking a sample containing the at least one
microbe, isolating the at least one microbe from the sample,
dividing the at least one microbe into a number of wells, wherein
each well contains at least one antimicrobial agent and at least
two electrodes. The method further includes pulsing a voltage
between the at least two electrodes, sampling an electrical
property during the pulsing; and recording the electrical property
for a sample-duration.
[0126] In embodiments, the isolating of the method further includes
filtering the sample to separate the at least one microbe from the
sample, and immersing the at least one microbe in an analyte. The
analyte is selected from water, buffer, saline, broth, or any
combination thereof. The microbes are selected from bacteria,
fungi, viruses, or nematodes. The antimicrobial agents are selected
from bacteriophages, mycoviruses, virophages, nematophages,
antibiotics, antimicrobials, antivirals, antifungals, or
parasiticides.
[0127] In another aspect, a method for determining a count of
microbes in a sample includes filtering the sample to separate the
at least one microbe from the sample, immersing the at least one
microbe in an analyte to form an immersion, incubating the
immersion for a specific time, dividing the immersion into a number
of wells, measuring an electrical property in the wells for a
sample-duration, and correlating the electrical property to a
count. In embodiments, the method further comprises adding at least
one bacteriophage to at least one of the wells before measuring the
electrical property.
[0128] In embodiments, the sample-duration is at is about 1 second,
about 5 seconds, about 10 seconds, about 20 seconds, about 30
seconds, about 40 seconds, about 50 seconds, about 60 seconds. In
other embodiments, the specific time for the incubating is at least
about 0.5 seconds, at least about 1 second, at least about 30
seconds, at least about 1 minute, or at least about 2 minutes. In
other embodiments, the specific time for the incubating is not
longer than about 1 millisecond, not longer than about 1 minute,
not longer than about 2 minutes, not longer than about 5 minutes.
In yet other embodiments, the specific time for the incubating is
between 1 millisecond and 5 minutes, between 0.5 second and 2
minutes, between 1 second and 1 minute.
[0129] In another embodiment, the method determines a first count
for a first microbe and a second count for a second microbe.
[0130] In one further aspect, a method for determining
antimicrobial resistance of a microbe includes adding a sample of
at least one microbe into a well containing at least one
antimicrobial, and measuring an electrical property in the well for
a sample-duration. The sample-duration is at least one hour and not
more than six hours.
[0131] In embodiments, the microbes are selected from Aerobacter,
Bacillus, Bordetella, Brucella, Campylobacter, Chlamydia,
Chromobacterium, Clostridium, Corynebacterium, Enterobacter,
Escherichia, Haemophilus, Klebsiella, Listeria, Mycobacterium,
Mycoplasma, Neisseria, Pneumococcus, Proteus, Pseudomonas,
Providencia, Salmonella, Serratia, Shigella, Staphylococcus,
Streptococcus, Vibrio, Yersinia, Acinetobacter, Bacteroides,
Bifidubacterium, E. kenella corrodens, Gardnerella vaginalis,
Mobiluncus, Proteobacteria, Desulfobacterales, Desulfovibrionales,
Syntrophobacterales, Thermodesulfobacteria, Nitrospirae, gram
positive Peptococcaceae, Archaea, Archaeoglobus, or any
combinations thereof.
[0132] In other embodiments, the antimicrobial agents are selected
from Actinomyces phages, Bacillus phage .PHI.29, bacteriophage
M102, bacteriophage e10, bacteriophage f1, bacteriophage .lamda.,
bacteriophage PI, spherical phage PhiX174, spherical phage G4,
spherical phage S13, bacteriophage T1, bacteriophage T2,
bacteriophage T3, bacteriophage T4, bacteriophage T5, bacteriophage
T6, bacteriophage T7, ssRNA bacteriophages MS2, ssRNA
bacteriophages R17, ssRNA bacteriophages f2, ssRNA bacteriophages Q
beta, S. mutans phages, and any combinations thereof.
[0133] In other embodiments the phage is cultivated and isolated so
that it attacks only the microbe to be identified using methods
well known to those in the field of microbiology. Such phages are
readily available in libraries.
[0134] In embodiments, the electrical property is selected from
conductance, resistance, voltage, amperage, capacitance, impedance,
inductance, and any combinations thereof. In embodiments, any
method is conducted in less than 90 minutes, less than 60 minutes,
less than 45 minutes, less than 30 minutes, less than 25 minutes,
less than 20 minutes, less than 18 minutes, less than 15 minutes,
or less than 12 minutes.
[0135] In embodiments, the sample is taken 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 and any combinations thereof.
[0136] 8. Stackable Units--Sample Preparation Automation
[0137] In yet another aspect, a diagnostic device for detecting at
least one microbe includes a first unit and a second unit; the
first unit is stackable into the second unit. The first unit is a
diagnostic unit comprising at least one well, the at least one well
having electrodes contacting the inside and the outside of the at
least one well. The second unit is a reader unit comprising a
connector section for the electrodes of the diagnostic unit. In
embodiments, the first unit further comprising a sample holder and
filter unit, the sample holder and filter unit being in fluidic
communication.
[0138] In another aspect, a diagnostic device for identifying at
least one bacterium in a sample includes a first unit and a second
unit; the first unit is stackable into the second unit. The first
unit is a diagnostic unit comprising at least one well, the at
least one well having electrodes contacting the inside and the
outside of the at least one well, a fluidic system comprising of
one-way valves and a port for pressurizing the fluidic system. The
second unit is a computational reader unit comprising a connector
section for the electrodes of the diagnostic unit and connection of
at least one micro-pump.
[0139] In another aspect, a diagnostic device for identifying at
least one bacterium in a sample includes a first unit and a second
unit; the first unit is stackable into the second unit. The first
unit is a diagnostic unit comprising at least one well, the at
least one well having electrodes contacting the inside and the
outside of the at least one well. The diagnostic unit can further
comprise at least one bacteriophage. The second unit is a
computational reader unit comprising a connector section for the
electrodes of the diagnostic unit.
[0140] In one further aspect, a diagnostic device for determining a
count of at least one microbe in a sample comprises a diagnostic
unit, which includes at least one well. The at least one well has
electrodes contacting the inside and the outside of the at least
one well. The diagnostic device further includes a reader unit. The
diagnostic unit and the reader unit form a stackable integrated
system. The reader unit includes a memory chip which contains
correlation data. The correlation data provide a count for microbes
taken from data sampled by the reader unit.
[0141] In yet one further aspect, a diagnostic device for
determining antimicrobial resistance of at least one microbial in a
sample includes a first unit and a second unit; the first unit is
stackable into the second unit. The first unit is a diagnostic unit
comprising one or more wells. The wells have electrodes, which
contact the inside and the outside of the at least one well. The
diagnostic unit also includes at least one antimicrobial. The
second unit is a reader unit, which comprises a connector section
for the electrodes of the diagnostic unit.
[0142] In embodiments, the diagnostic devices have electrodes
including a non-oxidizing material. The non-oxidizing materials can
be selected from metals, nonmetals, polymers, composites, resists,
resins, carbon nano-tubes, plastics, or any combinations thereof.
In a particular embodiment, the diagnostic devices have electrodes
that include copper covered with graphene.
[0143] 9. Automated Sample Preparation
[0144] In other embodiments, the diagnostic device further includes
a sample inlet, a sample receptacle, a first compartment connected
to the sample receptacle, the first compartment containing a first
liquid. The diagnostic further includes a filtration chamber
containing a waste compartment and a phage compartment; a second
compartment connected to the filtration chamber, the second
compartment containing a second liquid; and a manifold well unit.
The first or the second liquid can be selected from phosphate
buffer, sodium bicarbonate, dimethlsulfoxide, NaOH, Methanol or
glacial acetic acid, HCL, lactic or hydrochloric acid, aqueous
buffer, saline, de-ionized water, broth, or analyte based on
Clinical and Laboratory Standards Institute's "Performance
Standards for Antimicrobial Susceptibility Testing; Twenty-First
Information Supplement", January 2011, Vol 31 No 1. In embodiments,
the first or second liquid can be used to reconstitute, dissolve,
or prepare agents, such as bacteriophages or antimicrobial
compounds, such as antibiotics, antivirals, antifungals, or
parasiticides, for mixing the agent with a microbe.
[0145] The filtration chamber includes at least one filter
comprising a fluorinated polymer. For example, the fluorinated
polymer is polyvinylidene fluoride (PVDF). In other embodiments,
the filters include a prefilter layer, which can be a cellulose
material. For example, the cellulose material can be a cellulose
ester. The second and third liquid can be selected from de-ionized
water, buffer, saline, broth, analyte, or any combinations thereof.
Another embodiment might include additional liquid chambers to
accommodate the combined antimicrobials different needs for
reconstitution from their dry format. Another embodiment might
include additional filters and one-way valves between the chamber
where the antimicrobial is reconstituted and the chamber containing
the electrodes.
[0146] In other embodiments, the diagnostic device further includes
a sample inlet, a sample receptacle, a first compartment connected
to the sample receptacle, the first compartment containing a first
liquid. The diagnostic further includes a filtration chamber
containing a waste compartment and a phage compartment; a second
compartment connected to the filtration chamber, the second
compartment containing a second liquid; and a manifold well unit.
The first or the second or third liquid can be selected from
phosphate buffer, sodium bicarbonate, dimethlsulfoxide, NaOH, HCL,
lactic hydrochloric acid, aqueous buffer, saline, de-ionized water,
broth, or analyte or other liquid to reconstitute the dry form of
the bacteriophage. The filtration chamber includes at least one
filter comprising a fluorinated polymer. For example, the
fluorinated polymer is polyvinylidene fluoride (PVDF). In other
embodiments, the filters include a prefilter layer, which can be a
cellulose material. For example, the cellulose material can be a
cellulose ester. The second liquid or third can be selected from
de-ionized water, buffer, saline, broth, analyte, or any
combinations thereof. Another embodiment might include additional
liquid chambers to accommodate the combined bacteriophages
different needs for reconstitution from their dry format. Another
embodiment might include additional filters and one-way valves
between the chamber where the bacteriophage is reconstituted and
the chamber containing the electrodes.
[0147] In embodiments, the diagnostic has a reader unit that
further includes one or more analog to digital converter, one or
more memory chip, one or more microprocessor with a computational
unit, a system clock, a display processor, and a display. The
reader unit can further include one or more micro-pumps to
pressurize the diagnostic device and activate the fluidic
system.
[0148] In yet some embodiments, the diagnostic device has a reader
unit that includes a communication device and associated port. In
other embodiments, the reader unit includes a port for submitting
data. In other embodiments, the reader unit includes a port for
receiving data. The port can be a wireless transmitter or a wired
communication device.
[0149] In embodiments, the diagnostic device includes at least one
antimicrobial is selected from aminoglycosides, amphenicols,
ansamycins, beta-lactams, lincosamides, macrolides, polypeptide
antibiotics, tetracyclines, cycloserine, mupirocin, tuberin,
2,4-diaminopyrimidines, nitrofurans, quinolones, sulfonamides,
sulfones, clofoctol, hexedine, methenamine, nitroxoline,
taurolidine, and xibernol.
[0150] In further embodiments, the at least one antimicrobials is
selected from amikacin, azlocillin, carbencillin, cefaclor,
cefemandole, cefonicid, cefotaxime, cefoperazone, cefoxitin,
ceftizoxime, ceftriaxzone, ciprofloxacin, clindamycin,
gatifloxacin, gemifloxacin, gentamicin, kanamycin, linezolid,
mecillinam, meropenem, methicillin, metronidazole, mezlocillin,
minocyclin, moxifloxacin, nafcillin, netilmycin, oxacillin,
penicillin, piperacillin, quinupristin-dalfopristin, sparfloxacin,
sulbactam, tazobactam, teicoplanin, tetracyclines, tobramycin,
trimethoprim, trospectomycin and vancomycin.
[0151] In embodiments, the diagnostic unit has one or more wells
with a holding capacity of at least about 1 .mu.L, at least about
10 .mu.L, at least about 20 .mu.L, at least about 50 .mu.L, at
least about 100 .mu.L, at least about 200 .mu.L, at least about 500
.mu.L, at least about 1 mL, or at least about 1.5 mL or at least
about 2 mL.
[0152] In other embodiments, the diagnostic unit has one or more
wells with a holding capacity of not greater than about 2 mL, not
greater than about 1.5 mL, not greater than about 1 mL, not greater
than about 500 .mu.L, not greater than about 200 .mu.L, not greater
than about 100 .mu.L, not greater than about 50 .mu.L, or not
greater than about 20 .mu.L.
[0153] In yet other embodiments, the diagnostic unit has one or
more wells with a holding capacity between about 1 .mu.L to about 2
mL, between about 10 .mu.L to about 2 mL, between about 100 .mu.L
to about 2 mL, between about 100 .mu.L to about 1.5 mL, between
about 100 .mu.L to about 1 mL, between about 500 .mu.L to about 2
mL, between about 500 .mu.L to about 1.5 mL, or between about 500
.mu.L to about 1 mL.
[0154] The specification and illustrations of the embodiments
described herein are intended to provide a general understanding of
the structure of the various embodiments. The specification and
illustrations are not intended to serve as an exhaustive and
comprehensive description of all of the elements and features of
apparatus and systems that use the structures or methods described
herein. Separate embodiments may also be provided in combination in
a single embodiment, and conversely, various features that are, for
brevity, described in the context of a single embodiment, may also
be provided separately or in any subcombination. Further, reference
to values stated in ranges includes each and every value within
that range. Many other embodiments may be apparent to skilled
artisans only after reading this specification. Other embodiments
may be used and derived from the disclosure, such that a structural
substitution, logical substitution, or another change may be made
without departing from the scope of the disclosure. Accordingly,
the disclosure is to be regarded as illustrative rather than
restrictive.
EXAMPLES
Example 1
Coating of Copper Wire with Graphene
[0155] In the embodiment of a single-cell sensor FIG. 8c, an
aqueous dispersion of graphene has been prepared by catalytic
hydrogenation of humic acid. The humic acid was extracted from
leonardite (Agro-Lig) and then catalytically hydrogenated using
various catalysts in a Parr reactor at 150.degree. C. The solution
is then passed through a strong acid ion exchange column to remove
excess cations. The aqueous dispersion of graphene was applied to
the copper wire contact points in the sensor with a dropper and
allowed to dry. In one embodiment the graphene content might be
0.5% by weight of the aqueous dispersion. In another embodiment the
graphene might be 1% by weight. And yet another embodiment the
graphene might be 2% by weight.
[0156] In further embodiments, alternative techniques are
contemplated of applying graphene to either copper or to other
materials composing the electrodes.
Example 2
Implementation of Bacteria Identity Determination
[0157] Tests were run to show that experiments at room temperature
produced the same function over time as experiments held in a water
bath at 37 degrees Celsius and this data is not shown here.
[0158] In the example implementation, one sensor-well contains the
sample at room temperature; the other sensor-well contained T4
bacteriophage specific to E. coli B and also at room temperature.
In the case of the example implementation the bacteria used were E.
coli B, the phage used was type T4, the analyte was a supportive
culture of LB Broth, but the analyte need not be limited to LB
Broth and will be dependent on the types of bacteria being
targeted. LB Broth is manufactured by Miller, part number BL 729A.
It consists of: Enzymatic Digest of Casein 10 g, Yeast Extract 5 g,
Sodium Chloride 10 g, PH is adjusted to 7.3+/-0.2 at 25.degree. C.
It could be seen that the resistance of the analyte first lowered
during the first part of the phage attack and then returned to
starting point at about 201 seconds as the bacteria reabsorbed the
potassium ions.
Example 3
Implementation of Antimicrobial Determination
[0159] An overnight bacterial culture of E. coli B with a
concentration of around 10.sup.9 cells which had attained room
temperature were diluted using LB Broth which had also attained
room temperature to a final concentration of 10.sup.3 cells. 0.9 ml
of this solution was then placed into one sensor well which was
connected to lead #1 of a reader unit. An additional 0.9 ml of the
bacteria was placed into a second sensor well which was connected
to lead#2 of a reader unit. Data were collected on these solutions
for around 4 minutes before the addition of either antibiotic or
diH.sub.2O. At second 263, 0.1 mL of Sulfamethoxazole stock
solution which had attained room temperature was added to the first
sensor well, and at second 283, room temperature diH.sub.2O was
added to the second sensor well and data collection was continued.
All sensors wells were rinsed with 70% ethanol and the rinsed 10
times with diH.sub.2O before the following test.
[0160] An overnight bacterial culture of E. coli B with a
concentration of around 10.sup.9 cells that had attained room
temperature were diluted to a final concentration of 10.sup.3
cells. 0.9 ml of this solution was then placed into a first sensor
well which was connected to lead #1 of a reader unit. An additional
0.9 ml of the bacteria was placed into a second sensor well which
was connected to lead#2 of a reader unit. Data were collected on
these solutions for around 1 minute before the addition of either
antibiotic or diH.sub.2O. At second 63, 0.1 ml of Trimethoprin
stock solution which had attained room temperature was added to the
first sensor well, and at second 278 room temperature diH.sub.2O
was added to the second sensor well and data collection was
continued for some time. All sensors were rinsed with 70% ethanol
and the rinsed 10 times with diH.sub.2O before the following
test.
[0161] A two hour E. coli B bacteria culture with a concentration
of around 10.sup.7 cells that had attained room temperature were
diluted in LB Broth to a final concentration of 10.sup.3 cells. 0.9
mL of this solution was then placed into sensor well which was
connected to lead #1 of the reader unit. An additional 0.9 mL of
the bacteria was placed into a second sensor well which was
connected to lead #2 of the reader unit. Data was collected on
these solutions which were left at room temperature for around 10
minutes before the addition of either antimicrobial or diH.sub.2O.
At second 679, 0.1 mL of Polymyxin stock solution which had
attained room temperature was added to a sensor well and at second
702, diH.sub.2O was added to another sensor well and data was
collected for some time. All sensors were rinsed with 70% ethanol
and the rinsed 10 times with diH2-O before the following test.
[0162] A E. coli B bacteria culture was grown for around 2 hours
and thirty minutes with a concentration of around 10.sup.7 cells
and which had attained room temperature were further diluted in LB
Broth that had attained room temperature to a final concentration
of 10.sup.3 cells. Then 0.9 mL of this solution was then placed
into a sensor well which was connected to lead #1 of the reader
unit. An additional 0.9 ml of the bacteria was placed into another
sensor well which was connected to lead #2 of the reader unit. Data
was collected on these solutions for around 10 minutes and left to
grow at room temperature before the addition of either antibiotic
or diH.sub.2O. At second 665, 0.1 mL of the Azithromycin which had
attained room temperature was added to the sensor well, and at
second 676, 0.1 mL of diH.sub.2O was added to the other sensor
well. All sensors were rinsed with 70% ethanol and the rinsed 10
times with diH.sub.2O before the following test.
Example 4
Bacteria Viability Determination
[0163] Bacteria viability test was implemented by measuring
resistance of samples containing various concentrations of colony
forming units (CFU) of E. Coli B which had attained room
temperature was suspended in LB Broth which had also attained room
temperature. Samples have the following concentration: 10.sup.2
CFU/mL, 10.sup.4 CFU/mL, 10.sup.5 CFU/mL, 10.sup.6 CFU/mL, 10.sup.7
CFU/mL and 10.sup.9 CFU/mL. The bacteria were left to grow at room
temperature for 111 seconds and the resistance decreased as a
function of time. The test was run at room temperature after the LB
Broth and bacteria had also reached room temperature.
[0164] Bacteria viability test was also implemented in artificial
urine by measuring resistance of samples containing various
concentrations of colony forming units (CFU) of E. Coli B. Samples
have the following concentration: 10.sup.2 CFU/mL, 10.sup.4 CFU/mL,
10.sup.5 CFU/mL, 10.sup.6 CFU/mL, 10.sup.7 CFU/mL and 10.sup.9
CFU/mL. The bacteria were left to grow for 111 seconds at room
temperature and the resistance decreased as a function of time. The
sensors detect the growth of the bacteria indicated by a decrease
in resistance of the analyte. The test was run at room temperature
after the artificial urine and bacteria had also reached room
temperature. Artificial Urine was prepared with ingredients
disclosed in Table 1. Part A and Part B were prepared separately,
ingredients were combined from each part according the amount in
Table 1. The pH was adjusted to 5.8. The solution was sterilized by
filtration. Part B was added aseptically to Part A to give 2 L of
artificial urine. The artificial urine was stored at 4 degree
Celsius and could last for 1-1.5 weeks
TABLE-US-00001 TABLE 1 Artificial Urine Recipe Ingredient Amount
Notes Part A H.sub.2O 1.8 L MgCL.sub.2*6H.sub.2O 1.302 g NaCl 9.2 g
Na.sub.2SO.sub.4 4.6 g Na citrate 1.302 g Na oxalate 0.004 g
KH.sub.2PO.sub.4 (monobasic) 5.6 g KCL 3.2 g TSB (Tryptic Soy
Broth) 20.0 g Part B H.sub.2O 200 mL NH.sub.4CL 2.0 g
CaCl.sub.2*2H.sub.2O 1.302 g Urea 50.0 g Creatinine 2.2 g
Example 5
Implementation of Reader
[0165] FIGS. 8A-8C discloses an implementation of a reader unit
system. A sample well having electrodes is attached to Leads that
are connected to sensors. The reader unit collects data over time
from which signature plots are generated.
[0166] Note that not all of the activities described above in the
general description or the examples are required, that a portion of
a specific activity may not be required, and that one or more
further activities may be performed in addition to those described.
Still further, the order in which activities are listed is not
necessarily the order in which they are performed.
[0167] Benefits, other advantages, and solutions to problems have
been described above with regard to specific embodiments. However,
the benefits, advantages, solutions to problems, and any feature(s)
that may cause any benefit, advantage, or solution to occur or
become more pronounced are not to be construed as a critical,
required, or essential feature of any or all the claims.
* * * * *