U.S. patent application number 17/207616 was filed with the patent office on 2021-12-16 for real-time and label free analyzer for in-vitro and in-vivo detecting the suspicious regions to cancer.
This patent application is currently assigned to Mohammad Abdolahad. The applicant listed for this patent is Fereshteh Abbasvandi, Mohammad Abdolahad, Parisa Aghaee, Mahsa Faramarzpour Darzini, Hadi Ghafari, Zohreh Sadat Miripour, Pooneh Mohaghegh, Naser Namdar Habashi. Invention is credited to Fereshteh Abbasvandi, Mohammad Abdolahad, Parisa Aghaee, Mahsa Faramarzpour Darzini, Hadi Ghafari, Zohreh Sadat Miripour, Pooneh Mohaghegh, Naser Namdar Habashi.
Application Number | 20210386330 17/207616 |
Document ID | / |
Family ID | 1000005798545 |
Filed Date | 2021-12-16 |
United States Patent
Application |
20210386330 |
Kind Code |
A1 |
Abdolahad; Mohammad ; et
al. |
December 16, 2021 |
REAL-TIME AND LABEL FREE ANALYZER FOR IN-VITRO AND IN-VIVO
DETECTING THE SUSPICIOUS REGIONS TO CANCER
Abstract
An apparatus for in-vivo measuring H.sub.2O.sub.2 oxidation
within a living tissue. The apparatus includes an electrochemical
probe and an electrochemical stimulator-analyzer. The
electrochemical probe includes a sensing part and a handle. The
sensing part includes a working electrode, a counter electrode, and
a reference electrode. The working electrode includes a first
biocompatible conductive needle coated with a layer of vertically
aligned multi-walled carbon nanotubes. The counter electrode
includes a second biocompatible conductive needle. The reference
electrode includes a third biocompatible conductive needle. The
electrochemical stimulator-analyzer is configured to generate a set
of electrical currents in a portion of the living tissue.
Inventors: |
Abdolahad; Mohammad;
(Tehran, IR) ; Namdar Habashi; Naser; (Tabriz,
IR) ; Miripour; Zohreh Sadat; (Tehran, IR) ;
Ghafari; Hadi; (Tehran, IR) ; Abbasvandi;
Fereshteh; (Tehran, IR) ; Aghaee; Parisa;
(Tehran, IR) ; Faramarzpour Darzini; Mahsa;
(Tehran, IR) ; Mohaghegh; Pooneh; (Shiraz,
IR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Abdolahad; Mohammad
Namdar Habashi; Naser
Miripour; Zohreh Sadat
Ghafari; Hadi
Abbasvandi; Fereshteh
Aghaee; Parisa
Faramarzpour Darzini; Mahsa
Mohaghegh; Pooneh |
Tehran
Tabriz
Tehran
Tehran
Tehran
Tehran
Tehran
Shiraz |
|
IR
IR
IR
IR
IR
IR
IR
IR |
|
|
Assignee: |
Abdolahad; Mohammad
Tehran
IR
|
Family ID: |
1000005798545 |
Appl. No.: |
17/207616 |
Filed: |
March 20, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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17034067 |
Sep 28, 2020 |
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17207616 |
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16857428 |
Apr 24, 2020 |
10786188 |
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17034067 |
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16010510 |
Jun 17, 2018 |
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16857428 |
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62522115 |
Jun 20, 2017 |
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62563673 |
Sep 27, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61B 5/1473 20130101;
G01N 27/308 20130101; G01N 27/3278 20130101; A61B 5/6848 20130101;
G01N 27/3273 20130101 |
International
Class: |
A61B 5/1473 20060101
A61B005/1473; A61B 5/00 20060101 A61B005/00; G01N 27/327 20060101
G01N027/327; G01N 27/30 20060101 G01N027/30 |
Claims
1-20. (canceled)
21. A system for in-vivo measuring H.sub.2O.sub.2 oxidation within
a living tissue, the system comprising: an electrochemical probe
comprising: a working electrode comprising a first biocompatible
conductive needle; a counter electrode comprising a second
biocompatible conductive needle; and a reference electrode
comprising a third biocompatible conductive needle; wherein each of
the first biocompatible conductive needle, the second biocompatible
conductive needle, and the third biocompatible conductive needle is
coated with a layer of vertically aligned multi-walled carbon
nanotubes (VAMWCNTs) configured to be put in direct contact with
the living tissue; an electrochemical stimulator-analyzer
comprising a potentiostat circuit; a memory having
processor-readable instructions stored therein; and one or more
processors configured to access the memory and execute the
processor-readable instructions, which, when executed by the one or
more processors configures the one or more processors to perform a
method, the method comprising: generating a set of electrical
currents in the living tissue by applying a set of electrical
potentials to the electrochemical probe utilizing the
electrochemical stimulator-analyzer; and recording the set of
electrical currents by measuring an electric current flowing from
the counter electrode to the working electrode utilizing the
electrochemical stimulator-analyzer.
22. The system of claim 21, wherein applying the set of electrical
potentials to the electrochemical probe comprises applying a
sweeping range of electrical potentials between -1 V and 1 V to the
working electrode.
23. The system of claim 21, wherein measuring the electric current
flowing from the counter electrode to the working electrode
comprises measuring the electric current at the working electrode
after applying each electrical potential in the sweeping range.
24. The system of claim 21, wherein recording the set of electrical
currents comprises: generating, utilizing a control amplifier of
the potentiostat circuit, a control voltage at a control output of
the control amplifier, the control output coupled to the counter
electrode, wherein generating the control voltage comprises
amplifying a voltage difference between a first control input of
the control amplifier and a second control input of the control
amplifier, the second control input coupled to the reference
electrode; generating, utilizing a transimpedance amplifier of the
potentiostat circuit, a transimpedance voltage at a transimpedance
output of the transimpedance amplifier by amplifying a voltage of a
transimpedance input of the transimpedance amplifier, the
transimpedance input connected to the working electrode; detecting,
utilizing a peak detector circuit of the potentiostat circuit, a
maximum voltage at a detector input of the peak detector circuit in
a given period of time, the detector input coupled to the
transimpedance output; transmitting, utilizing the peak detector
circuit, the maximum voltage to a detector output of the peak
detector circuit; connecting a feedback network between the
detector input and the transimpedance input; activating, utilizing
a comparator circuit of the potentiostat circuit, a periodic wave
generator of the potentiostat circuit by providing an activation
voltage to an activation input of the periodic wave generator
responsive to a voltage of the detector output being smaller than a
reference voltage; generating, utilizing the periodic wave
generator, a periodic wave at the first control input responsive to
activating the periodic wave generator; deactivating, utilizing the
comparator circuit, the periodic wave generator by providing a
deactivation voltage to the activation input responsive to a
voltage of the detector output being equal to or larger than the
reference voltage; and coupling, utilizing the periodic wave
generator, the first control input to ground responsive to
deactivating the periodic wave generator.
25. The system of claim 24, wherein providing the activation
voltage to the activation input responsive to the voltage of the
detector output being smaller than the reference voltage comprises
providing the activation voltage to a comparator output of the
comparator circuit responsive to a voltage of a first comparator
input of the comparator circuit being smaller than a voltage of a
second comparator input, wherein: the first comparator input is
connected to the detector output; the second comparator input is
coupled to a DC voltage supply comprising a DC voltage; and the
comparator output is connected to the activation input.
26. The system of claim 25, wherein providing the deactivation
voltage to the activation input responsive to the voltage of the
detector output being equal to or larger than the reference voltage
comprises providing the deactivation voltage to the comparator
output responsive to the voltage of the first comparator input
being equal to or larger than the voltage of the second comparator
input.
27. The system of claim 25, wherein the method further comprises:
providing the reference voltage to the second comparator input by
transmitting a fraction of the DC voltage to the second comparator
input utilizing a variable resistor.
28. The system of claim 24, wherein generating the periodic wave
comprises: generating, utilizing a pulse wave generator, a periodic
rectangular wave at an output of the pulse wave generator; coupling
a negative input of an operational amplifier (op-amp) to an output
of the pulse wave generator; coupling a positive input of the
op-amp to ground; coupling an op-amp output of the op-amp to the
first control input; connecting an RC circuit between the op-amp
output and the negative input, the RC circuit comprising a first
resistor and a first capacitor connected in parallel; connecting a
second resistor to the negative input; connecting a third resistor
between the second resistor and the output of the pulse wave
generator; connecting a drain of a field effect transistor (FET)
between the second resistor and the third resistor; connecting a
source of the FET to ground; connecting a gate of the FET to the
activation input; and providing the activation voltage to the
activation input utilizing the comparator circuit.
29. The system of claim 28, wherein coupling the first control
input to ground comprises providing the deactivation voltage to the
activation input utilizing the comparator circuit.
30. The system of claim 28, wherein the first resistor R.sub.1, the
first capacitor C.sub.1, the second resistor R.sub.2, and the third
resistor R.sub.3 satisfy a set of conditions defined by the
following: R.sub.1C.sub.1>100/f.sub.pw
R.sub.2=R.sub.3<0.1R.sub.1 where f.sub.pw is a frequency of the
periodic rectangular wave.
31. The system of claim 24, wherein the method further comprises:
connecting a first unity gain input of a unity gain amplifier of
the potentiostat circuit to the reference electrode; connecting a
unity gain output of the unity gain amplifier to the second control
input; connecting a second unity gain input of the unity gain
amplifier to the unity gain output; and transmitting a voltage at
the reference electrode to the unity gain output by amplifying a
voltage difference between the first unity gain input and the
second unity gain input utilizing the unity gain amplifier.
32. The system of claim 31, wherein utilizing each of the control
amplifier, the transimpedance amplifier, and the unity gain
amplifier comprises utilizing respective operational amplifiers
(op-amps).
33. The system of claim 24, wherein the method further comprises
transferring an electric current flowing through the control output
to the counter electrode utilizing a first current buffer amplifier
(CBA) of the potentiostat circuit, the first CBA connected between
the control amplifier and the counter electrode.
34. The system of claim 33, wherein the method further comprises
transferring an electric current flowing through the transimpedance
output to the detector input utilizing a second current buffer
amplifier (CBA) of the potentiostat circuit, the second CBA
connected between the transimpedance amplifier and the peak
detector circuit.
35. The system of claim 24, wherein the method further comprises
responsive to voltage variations at the first control input,
compensating voltage variations at the counter electrode utilizing
a first analog compensator of the potentiostat circuit, the first
analog compensator connected between the counter electrode and the
first control input.
36. The system of claim 35, wherein the method further comprises
compensating voltage variations at the second control input
utilizing a second analog compensator of the potentiostat circuit,
the second analog compensator connected between the second control
input and ground.
37. The system of claim 36, wherein: utilizing the first analog
compensator comprises utilizing a first compensation capacitor; and
utilizing the second analog compensator comprises utilizing a
second compensation capacitor and a compensation resistor connected
in series.
38. The system of claim 24, wherein connecting the feedback network
between the detector input and the transimpedance input comprises
connecting a feedback resistor between the detector input and the
transimpedance input, a resistance R.sub.f of the feedback resistor
equal to V ref I max , ##EQU00004## where V.sub.ref is the
reference voltage and I.sub.max is an upper limit of an electric
current flowing through the detector input.
39. The system of claim 21, wherein each of the first biocompatible
conductive needle, the second biocompatible conductive needle, and
the third biocompatible conductive needle comprises a respective
sensing tip coated with a layer of VAMWCNTs.
40. The system of claim 21, further comprising a sensing part, the
sensing part comprising: an electrode holder encompassing the
working electrode, the counter electrode, and the reference
electrode; and a releasing button configured to release the sensing
part for replacing the sensing part.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 16/857,428, filed Apr. 24, 2020, and entitled
"REAL-TIME AND LABEL FREE ANALYZER FOR IN-VITRO AND IN-VIVO
DETECTING THE SUSPICIOUS REGIONS TO CANCER," which is a
continuation-in-part of U.S. patent application Ser. No.
16/010,510, filed Jun. 17, 2018, and entitled "REAL-TIME AND LABEL
FREE ANALYZER FOR IN-VITRO AND IN-VIVO DETECTING THE SUSPICIOUS
REGIONS TO CANCER", which takes priority from U.S. Provisional
Patent Application Ser. No. 62/522,115 filed on Jun. 20, 2017, and
entitled "DIAGNOSIS OF CANCER TUMORS IN BIOPSY BREAST TISSUES" and
U.S. Provisional Patent Application Ser. No. 62/563,673 filed on
Sep. 27, 2017, and entitled "CANCER DIAGNOSTIC PROBE", which are
all incorporated herein by reference in their entirety.
TECHNICAL FIELD
[0002] The present disclosure generally relates to cancer
diagnosis, and particularly, to a system, sensor, and method for
diagnosing cancerous regions before and during surgery via a
real-time and label free approach.
BACKGROUND
[0003] Glycolysis is the intracellular biochemical conversion of
one molecule of glucose into two molecules of pyruvate, which can
be used to attain cellular energy. With the assistance of
sufficient oxygen, pyruvate could be converted by pyruvate
dehydrogenase (PDH) into acetylCoA which is crucial in a
metabolizing process to produce ATP in an oxidative way. A
physiological concentration of pyruvate in human normal epithelial
tissue has been reported to 0.7 mmol/g. Also the
lactate-to-pyruvate ratio (L/P ratio) as a reflection of cell's
redox state, illustrates the balance between NAD+ and NADH+H+,
depending on the interconversion of lactate and pyruvate via
lactate dehydrogenase (LDH). The L/P ratio in normal epithelial
tissues is less than 20:1. Markers and assays have been developed
to trace the LADH, P. or L/P in the patients' specimen as
diagnostic or prognostic factors which reveal the interests on
lactate based cancer research. Moreover some methods have been
developed to trace pyruvate by electrochemical methods with the
assistance of chemically labelled working electrodes. However,
there is still no substitutive label free methods and/or devices to
replace expensive, complicated, and late-responsive clinical
methods and devices such as pathology assays.
[0004] Hence, there is a need for cost-effective, label free and
real-time methods and devices, especially sensors and method to use
thereof to detect cancer in suspicious regions especially during
cancer surgery like mastectomy to remove involved regions with
precise margins to reduce resection of normal sites.
SUMMARY
[0005] This summary is intended to provide an overview of the
subject matter of the present disclosure, and is not intended to
identify essential elements or key elements of the subject matter,
nor is it intended to be used to determine the scope of the claimed
implementations. The proper scope of the present disclosure may be
ascertained from the claims set forth below in view of the detailed
description below and the drawings.
[0006] In one general aspect, the present disclosure describes an
exemplary apparatus for in-vivo measuring H.sub.2O.sub.2 oxidation
within a living tissue. An exemplary apparatus may include an
electrochemical probe and an electrochemical stimulator-analyzer.
An exemplary electrochemical probe may include a sensing part and a
handle. An exemplary sensing part may include a working electrode,
a counter electrode, and a reference electrode. An exemplary
working electrode may include a first biocompatible conductive
needle coated with a layer of vertically aligned multi-walled
carbon nanotubes (VAMWCNTs). An exemplary counter electrode may
include a second biocompatible conductive needle. An exemplary
reference electrode may include a third biocompatible conductive
needle.
[0007] An exemplary handle may include an insertion part and a
releasing button. In an exemplary embodiment, the insertion part
may be attached to the sensing part. In an exemplary embodiment,
the insertion part may be configured to be inserted into a portion
of the living tissue. In an exemplary embodiment, the releasing
button may be configured to release the sensing part for replacing
the sensing part.
[0008] In an exemplary embodiment, the electrochemical
stimulator-analyzer may be configured to generate a set of
electrical currents in the portion of the living tissue by applying
a set of electrical potentials to the electrochemical probe and
record the set of electrical currents by measuring an electric
current flowing from the counter electrode to the working
electrode.
[0009] In an exemplary embodiment, the electrochemical
stimulator-analyzer may be further configured to apply the set of
electrical potentials by applying a sweeping range of electrical
potentials between -1 V and 1 V to the working electrode and
measure the electric currents at the working electrode after
applying each electrical potential in the sweeping range.
[0010] An exemplary electrochemical stimulator-analyzer may include
a potentiostat circuit. An exemplary potentiostat circuit may
include a control amplifier, a transimpedance amplifier, a peak
detector circuit, a feedback network, a periodic wave generator,
and a comparator circuit. An exemplary control amplifier may
include a first control input, a second control input coupled to
the reference electrode, and a control output coupled to the
counter electrode. In an exemplary embodiment, the control
amplifier may be configured to generate a control voltage at the
control output by amplifying a voltage difference between the first
control input and the second control input. An exemplary control
amplifier may include an operational amplifier (op-amp).
[0011] An exemplary transimpedance amplifier may include a
transimpedance output and a transimpedance input connected to the
working electrode. In an exemplary embodiment, the transimpedance
amplifier may be configured to generate a transimpedance voltage at
the transimpedance output by amplifying a voltage of the
transimpedance input. An exemplary peak detector circuit may
include a detector output and a detector input coupled to the
transimpedance output. In an exemplary embodiment, the peak
detector circuit may be configured to detect a maximum voltage at
the detector input in a given period of time and transmit the
maximum voltage to the detector output. An exemplary transimpedance
amplifier may include an op-amp.
[0012] In an exemplary embodiment, the periodic wave generator may
be configured to generate a periodic ware at the first control
input responsive to becoming active. In an exemplary embodiment,
the periodic wave generator may be further configured to couple the
first control input to ground responsive to becoming inactive. An
exemplary periodic wave generator may include a pulse wave
generator, an op-amp that may be coupled to the pulse wave
generator, an RC circuit that may be connected between the op-amp
output and the negative input, and a field effect transistor (FET).
An exemplary pulse wave generator may be configured to generate a
periodic rectangular wave. An exemplary op-amp may include a
positive input, a negative input, and an op-amp output. In an
exemplary embodiment, the positive input may be connected to
ground, the negative input may be coupled to an output of the pulse
wave generator, and the op-amp output may be connected to the first
control input. An exemplary RC circuit may include a first resistor
and a first capacitor that may be connected in parallel.
[0013] An exemplary periodic wave generator may further include a
second resistor and a third resistor. In an exemplary embodiment,
the second resistor may be connected to the negative input and the
third resistor may be connected between the second resistor and the
output of the pulse wave generator. An exemplary FET may include a
source, a drain, and a gate. An exemplary source may be connected
to ground. In an exemplary embodiment, the drain may be connected
between the second resistor and the third resistor, and the gate
may be connected to the activation input.
[0014] In an exemplary embodiment, the comparator circuit may be
configured to activate the periodic wave generator by providing an
activation voltage to an activation input of the periodic wave
generator responsive to a voltage of the detector output being
smaller than a reference voltage. In an exemplary embodiment, the
comparator circuit may be further configured to deactivate the
periodic wave generator by providing a deactivation voltage to the
activation input responsive to a voltage of the detector output
being equal to or larger than the reference voltage.
[0015] An exemplary comparator circuit may include a first
comparator input, a second comparator input, and a comparator
output. An exemplary first comparator input may be connected to the
detector output and an exemplary comparator output may be connected
to the activation input. In an exemplary embodiment, the second
comparator input may include the reference voltage. An exemplary
second comparator input may be coupled to a DC voltage supply that
may include a DC voltage. In an exemplary embodiment, the second
comparator input may be coupled to the DC voltage supply via a
variable resistor. An exemplary variable resistor may be configured
to provide the reference voltage to the second comparator input by
transmitting a fraction of the DC voltage to the second comparator
input.
[0016] In an exemplary embodiment, the comparator circuit may be
configured to provide the activation voltage to the comparator
output responsive to a voltage of the first comparator input being
smaller than a voltage of the second comparator input and provide
the deactivation voltage to the comparator output responsive to the
voltage of the first comparator input being equal to or larger than
the voltage of the second comparator input.
[0017] In an exemplary embodiment, the feedback network may be
connected between the detector input and the transimpedance input.
An exemplary feedback network may include a feedback resistor. In
an exemplary embodiment, a resistance R.sub.f of the feedback
resistor may be equal to
V ref I max , ##EQU00001##
where V.sub.ref is the reference voltage and I.sub.max is an upper
limit of an electric current flowing through the detector
input.
[0018] In an exemplary embodiment, the potentiostat circuit may
further include a unity gain amplifier that may be connected
between the reference electrode and the second control input. An
exemplary unity gain amplifier may include a first unity gain
input, a unity gain output, and a second unity gain input. In an
exemplary embodiment, the first unity gain input may be connected
to the reference electrode, the unity gain output may be connected
to the second control input, and the second unity gain input may be
connected to the unity gain output. In an exemplary embodiment, the
unity gain amplifier may be configured to transmit a voltage at the
reference electrode to the unity gain output by amplifying a
voltage difference between the first unity gain input and the
second unity gain input. An exemplary unity gain amplifier may
include an op-amp.
[0019] In an exemplary embodiment, the potentiostat circuit may
further include a first current buffer amplifier (CBA) that may be
connected between the control amplifier and the counter electrode.
An exemplary first CBA may be configured to transfer an electric
current flowing through the control output to the counter
electrode. In an exemplary embodiment, the potentiostat circuit may
further include a second CBA that may be connected between the
transimpedance amplifier and the peak detector circuit. An
exemplary second CBA may be configured to transfer an electric
current flowing through the transimpedance output to the detector
input.
[0020] In an exemplary embodiment, the potentiostat circuit may
further include a first analog compensator that may be connected
between the counter electrode and the first control input. An
exemplary first analog compensator may be configured to compensate
voltage variations at the counter electrode responsive to voltage
variations at the first control input. An exemplary first analog
compensator may include a first compensation capacitor. In an
exemplary embodiment, the potentiostat circuit may further include
a second analog compensator that may be connected between the
second control input and ground. An exemplary second analog
compensator may be configured to compensate voltage variations at
the second control input. An exemplary second analog compensator
may include a second compensation capacitor and a compensation
resistor that may be connected in series.
[0021] In an exemplary embodiment, each of the first biocompatible
conductive needle, the second biocompatible conductive needle, and
the third biocompatible conductive needle may include a respective
sensing tip coated with a layer of VAMWCNTs. In an exemplary
embodiment, each of the working electrode, the counter electrode,
and the reference electrode may include a respective sensing tip
with a diameter between 100 .mu.m and 200 .mu.m, and a length
between 0.1 cm and 1 cm. In an exemplary embodiment, the working
electrode, the counter electrode, and the reference electrode may
be attached to the insertion part at one end of the handle apart
from each other with a distance between 1 mm and 5 mm.
[0022] In an exemplary embodiment, the sensing part may further
include an electrode holder encompassing the working electrode, the
counter electrode, and the reference electrode. In an exemplary
embodiment, the handle may further include a handle head and a
switch located on the handle head. In an exemplary implementation,
the switch may be configured to connect the electrochemical probe
to an electrochemical stimulator-analyzer device, and disconnect
the electrochemical probe from the electrochemical
stimulator-analyzer device.
[0023] Other exemplary systems, methods, features and advantages of
the implementations will be, or will become, apparent to one of
ordinary skill in the art upon examination of the following figures
and detailed description. It is intended that all such additional
systems, methods, features and advantages be included within this
description and this summary, be within the scope of the
implementations, and be protected by the claims herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] The drawing figures depict one or more implementations in
accord with the present teachings, by way of example only, not by
way of limitation. In the figures, like reference numerals refer to
the same or similar elements.
[0025] FIG. 1A illustrates a schematic view of an exemplary
electrochemical system for cancer diagnosis, consistent with one or
more exemplary embodiments of the present disclosure.
[0026] FIG. 1B illustrates a schematic view of an exemplary CNT
based electrochemical chip, consistent with one or more exemplary
embodiments of the present disclosure.
[0027] FIG. 1C illustrates a schematic view of an exemplary sensing
well, consistent with one or more exemplary embodiments of the
present disclosure.
[0028] FIG. 1D illustrates a schematic view of an exemplary
magnified portion of exemplary working electrode within exemplary
sensing well of FIG. 1C, consistent with one or more exemplary
embodiments of the present disclosure.
[0029] FIG. 1E illustrates a schematic view of an exemplary cancer
diagnosis probe (CDP), consistent with one or more exemplary
embodiments of the present disclosure.
[0030] FIG. 1F illustrates a schematic view of an exemplary needle
electrode of exemplary CDP corresponding to the working electrode,
consistent with one or more exemplary embodiments of the present
disclosure.
[0031] FIG. 1G illustrates a schematic view of an exemplary
magnified portion of a tip of exemplary needle electrode of FIG.
1C, consistent with one or more exemplary embodiments of the
present disclosure.
[0032] FIG. 1H illustrates a schematic view of another
implementation of an exemplary cancer diagnosis probe (CDP) for
in-vivo measurement of H.sub.2O.sub.2 oxidation in a living tissue,
consistent with one or more exemplary embodiments of the present
disclosure.
[0033] FIG. 1I illustrates a schematic view of an exemplary
scenario in which an exemplary sensing part has been separated from
an exemplary handle of an exemplary CDP, consistent with one or
more exemplary embodiments of the present disclosure.
[0034] FIG. 1J illustrates a schematic view of an exemplary working
electrode, consistent with one or more exemplary embodiments of the
present disclosure.
[0035] FIG. 1K shows a schematic of a first implementation of a
potentiostat circuit, consistent with one or more exemplary
embodiments of the present disclosure.
[0036] FIG. 1L shows a schematic of a periodic wave generator,
consistent with one or more exemplary embodiments of the present
disclosure.
[0037] FIG. 1M shows a schematic of a second implementation of a
potentiostat circuit, consistent with one or more exemplary
embodiments of the present disclosure.
[0038] FIG. 1N shows a computer system in which an embodiment of
the present disclosure, or portions thereof, may be implemented as
computer-readable code, consistent with exemplary embodiments of
the present disclosure.
[0039] FIG. 2A illustrates an exemplary implementation of a method
for cancer diagnosis, consistent with one or more exemplary
embodiments of the present disclosure.
[0040] FIG. 2B illustrates a schematic implementation of putting
the array of vertically aligned multi-walled carbon nanotubes
(VAMWCNTs) grown on tip of each needle electrode of three needles
electrodes of exemplary CDP in contact with exemplary suspicious
sample, consistent with one or more exemplary embodiments of the
present disclosure.
[0041] FIG. 2C illustrates a schematic view of another exemplary
implementation of putting exemplary electrodes of exemplary CDP in
contact with an exemplary portion of a living tissue, consistent
with one or more exemplary embodiments of the present
disclosure.
[0042] FIG. 2D illustrates an implementation of detecting the
cancerous state in the suspicious sample, consistent with one or
more exemplary embodiments of the present disclosure.
[0043] FIG. 2E illustrates an exemplary implementation of an
exemplary method for in-vivo cancer diagnosis within a living
tissue, consistent with one or more exemplary embodiments of the
present disclosure.
[0044] FIG. 2F illustrates an exemplary implementation of preparing
an exemplary electrochemical probe similar to the exemplary CDP,
consistent with one or more exemplary embodiments of the present
disclosure.
[0045] FIG. 2G illustrates an exemplary implementation of
fabricating three integrated electrodes by coating a layer of
vertically aligned multi-walled carbon nanotubes (VAMWCNTs) on tips
of three electrically conductive biocompatible needles, consistent
with one or more exemplary embodiments of the present
disclosure.
[0046] FIG. 2H illustrates an exemplary implementation of growing
an array of VAMWCNTs on exemplary deposited catalyst layer,
consistent with one or more exemplary embodiments of the present
disclosure.
[0047] FIG. 2I shows an exemplary implementation of detecting the
cancer-involving status of the exemplary portion of the exemplary
living tissue based on the oxidation current peak, consistent with
one or more exemplary embodiments of the present disclosure.
[0048] FIG. 2J shows an exemplary implementation of generating a
set of reference current peak values, consistent with one or more
exemplary embodiments of the present disclosure.
[0049] FIG. 3A illustrates a schematic view of exemplary
electrochemical reactions involved on sensor including exemplary
VAMWCNTs as shown in FIGS. 1D and 1G, consistent with one or more
exemplary embodiments of the present disclosure.
[0050] FIG. 3B illustrates a schematic overview of mitochondrial
electron and proton fluxes in hypoxia, consistent with one or more
exemplary embodiments of the present disclosure.
[0051] FIG. 4 illustrates a field emission scanning electron
microscopy (FESEM) image of the VAMWCNTs array on a portion of an
exemplary fabricated CNT based electrochemical chip, consistent
with one or more exemplary embodiments of the present
disclosure.
[0052] FIG. 5A illustrates a FESEM image of a tip of a needle
electrode of an exemplary fabricated cancer diagnostic probe (CDP)
coated with an array of VAMWCNTs on the tip, consistent with one or
more exemplary embodiments of the present disclosure.
[0053] FIG. 5B illustrates a FESEM image of a first portion of an
exemplary VAMWCNTs array grown on the tip of the needle electrode
of exemplary fabricated CDP, consistent with one or more exemplary
embodiments of the present disclosure.
[0054] FIG. 5C illustrates a FESEM image of a second portion of an
exemplary VAMWCNTs array grown on the tip of the needle electrode
of exemplary fabricated CDP, consistent with one or more exemplary
embodiments of the present disclosure.
[0055] FIG. 5D illustrates a FESEM image of a third portion of an
exemplary VAMWCNTs array grown on the tip of the needle electrode
of exemplary fabricated CDP, consistent with one or more exemplary
embodiments of the present disclosure.
[0056] FIG. 6A illustrates the CV diagrams of L-lactic acid
solution individually recorded by electrochemical sensors
fabricated from platinum (Pt), Gold (Au), amorphous glassy carbon
(GC) and carbon nanotube (CNT) working electrodes (WEs), consistent
with one or more exemplary embodiments of the present
disclosure.
[0057] FIG. 6B illustrates the CV diagrams of solutions with
various concentrations of Hydrogen Peroxide (H.sub.2O.sub.2)
resulted from the lactate turn to H.sub.2O.sub.2 and pyruvate
recorded by electrochemical sensors with CNT arrays working
electrode, consistent with one or more exemplary embodiments of the
present disclosure.
[0058] FIG. 6C illustrates the CV diagrams of H.sub.2O.sub.2
contained lactate solution in comparison with two cell culture
solutions recorded by electrochemical sensors with CNT arrays
working electrode, consistent with one or more exemplary
embodiments of the present disclosure.
[0059] FIG. 7 illustrates the CV diagrams of hypoxic glycolysis in
MCF 10A, MCF-7, MDA-MB-231, and MDA-MB-468 cell tines in comparison
with H.sub.2O.sub.2 contained lactate solution and RPMI measured
and recorded by exemplary CNT based electrochemical chip,
consistent with one or more exemplary embodiments of the present
disclosure.
[0060] FIG. 8A illustrates the CV responses of the solution media
of different normal and cancerous cell lines in various phenotypes
for Colon (COR-L 105, SW-480, HT-29) cell lines in comparison with
Reference diagram for solution H.sub.2O.sub.2 contained solution
with a lactate concentration of about 0.3 mM, consistent with one
or more exemplary embodiments of the present disclosure.
[0061] FIG. 8B illustrates the CV responses of the solution media
of different normal and cancerous cell lines in various phenotypes
for Hematopoietic (1301, LCL-Pl 1) cell lines in comparison with
Reference diagram for H.sub.2O.sub.2 contained solution with a
lactate concentration of about 0.3 mM, consistent with one or more
exemplary embodiments of the present disclosure.
[0062] FIG. 8C illustrates the CV responses of the solution media
of different normal and cancerous cell lines in various phenotypes
for Liver (HEP G2) cell lines in comparison with Reference diagram
for H.sub.2O.sub.2 contained lactate solution with a lactate
concentration of about 0.3 mM, consistent with one or more
exemplary embodiments of the present disclosure.
[0063] FIG. 8D illustrates the CV responses of the solution media
of different normal and cancerous cell lines in various phenotypes
for Lung (QU-DB, MRC-5) cell lines in comparison with Reference
diagram for H.sub.2O.sub.2 contained lactate solution with a
lactate concentration of about 0.3 mM, consistent with one or more
exemplary embodiments of the present disclosure.
[0064] FIG. 8E illustrates the CV responses of the solution media
of different normal and cancerous cell lines in various phenotypes
for Mouth (KB) cell lines in comparison with Reference diagram for
H.sub.2O.sub.2 contained lactate solution with a lactate
concentration of about 0.3 mM, consistent with one or more
exemplary embodiments of the present disclosure.
[0065] FIG. 8F illustrates the CV responses of the solution media
of different normal and cancerous cell lines in various phenotypes
for Neuron (BE(2)-C, LAN-5) cell lines in comparison with Reference
diagram for H.sub.2O.sub.2 contained lactate solution with a
lactate concentration of about 0.3 mM, consistent with one or more
exemplary embodiments of the present disclosure.
[0066] FIG. 8G illustrates the CV responses of the solution media
of different normal and cancerous cell lines in various phenotypes
for Prostate (PC-3, Du-145) cell lines in comparison with Reference
diagram for H.sub.2O.sub.2 contained lactate solution with a
lactate concentration of about 0.3 mM, consistent with one or more
exemplary embodiments of the present disclosure.
[0067] FIGS. 9A-9F illustrate the cytopathological results (top
side) and electrochemical responses (bottom side) of the breast
tissues removed by biopsy or surgery from 6 suspicious patients to
cancer, consistent with one or more exemplary embodiments of the
present disclosure.
[0068] FIG. 10 illustrates a columnar diagram of electrochemical
responses of the breast tissues removed by biopsy or surgery from
11 suspicious patients to cancer, consistent with one or more
exemplary embodiments of the present disclosure.
[0069] FIG. 11A illustrates CV response of exemplary CDP with all
three needles covered by VAMWCNTs immediately after connection to
the tissues, consistent with one or more exemplary embodiments of
the present disclosure.
[0070] FIG. 11B illustrates CV response of exemplary CDP with only
working electrode covered by VAMWCNTs immediately after connection
to the tissues, consistent with one or more exemplary embodiments
of the present disclosure.
[0071] FIG. 11C illustrates CV response of exemplary CDP with
non-CNT covered by needles immediately after connection to the
tissues, consistent with one or more exemplary embodiments of the
present disclosure.
[0072] FIGS. 12A-12E illustrate CV responses recorded by exemplary
CDP (needle based electrochemical sensor) from the resected tissues
from five patients among 50 individual patients suspicious to
breast cancer (bottom side) in comparison with images obtained by
conventional pathological methods (H&E) (top side), consistent
with one or more exemplary embodiments of the present
disclosure.
[0073] FIG. 13 illustrates a summary of categorized regimes of CV
responses recorded by exemplary CDP from the resected tissues from
five patients among 50 individual patients suspicious to breast
cancer representing CV regimes along a spectrum from a completely
non-cancerous state to cancerous state, consistent with one or more
exemplary embodiments of the present disclosure.
[0074] FIG. 14A illustrates a sonography image from a tumor side
taken from an exemplary mouse tumorized by 4T1 breast cancer cell
lines, consistent with one or more exemplary embodiments of the
present disclosure.
[0075] FIG. 14B illustrates H&E image from the tumor side taken
from exemplary tumorized mouse by 4T1 breast cancer cell lines,
consistent with one or more exemplary embodiments of the present
disclosure.
[0076] FIG. 14C illustrates H&E image from a normal/healthy
side taken from exemplary tumorized mouse by 4T1 breast cancer cell
lines, consistent with one or more exemplary embodiments of the
present disclosure.
[0077] FIG. 14D illustrates CV diagrams of normal and tumor
regions/sides of exemplary tumorized mouse by 4T1 breast cancer
cell lines calibrated by a Reference CV diagram from H.sub.2O.sub.2
contained lactate solution with a lactate concentration of about
0.3 mM obtained using exemplary CDP, consistent with one or more
exemplary embodiments of the present disclosure.
[0078] FIG. 15A illustrates a sonography image from a tumor taken
from an exemplary mouse tumorized by 4T1 breast cancer cell lines,
consistent with one or more exemplary embodiments of the present
disclosure.
[0079] FIG. 15B illustrates exemplary six analyzed regions of an
exemplary tumorized mouse among the exemplary five tumorized mice
before surgery, consistent with one or more exemplary embodiments
of the present disclosure.
[0080] FIG. 15C illustrates exemplary six analyzed regions of an
exemplary tumorized mouse among the exemplary five tumorized mice
during surgery, consistent with one or more exemplary embodiments
of the present disclosure.
[0081] FIG. 16 illustrates comparative diagram of CDP responses in
interaction with normal, nonmalignant tumor, and malignant tumor
recorded from individual mice, consistent with one or more
exemplary embodiments of the present disclosure.
[0082] FIG. 17A illustrates CV response diagram obtained by
applying exemplary CDP in detection of suspicious margins during
breast cancer surgery for a known normal region, consistent with
one or more exemplary embodiments of the present disclosure.
[0083] FIG. 17B illustrates CV response diagram obtained by
applying exemplary CDP in detection of suspicious margins during
breast cancer surgery for a suspicious region, consistent with one
or more exemplary embodiments of the present disclosure.
[0084] FIG. 17C illustrates CV response diagram obtained by
applying exemplary CDP in detection of suspicious margins during
breast cancer surgery for another suspicious region, consistent
with one or more exemplary embodiments of the present
disclosure.
[0085] FIG. 17D illustrates an H&E resulted image after the
surgery for a known normal region, consistent with one or more
exemplary embodiments of the present disclosure.
[0086] FIG. 17E illustrates an H&E resulted image after the
surgery for a suspicious region, consistent with one or more
exemplary embodiments of the present disclosure.
[0087] FIG. 17F illustrates an H&E resulted image after the
surgery for another suspicious region, consistent with one or more
exemplary embodiments of the present disclosure.
[0088] FIG. 18 illustrates H&E images from nine exemplary
samples, consistent with one or more exemplary embodiments of the
present disclosure.
[0089] FIG. 19 illustrates classification of current peaks recorded
by exemplary CDP after examining more than 250 samples in
consistence with pathological diagnosis, consistent with one or
more exemplary embodiments of the present disclosure.
[0090] FIG. 20A illustrates an image resulted from frozen H&E
(top-side image), an image resulted from permanent H&E
(middle-side image), and a CV response recorded by exemplary CDP
(bottom-side image) for the anterior IM of a patient (ID 18),
consistent with one or more exemplary embodiments of the present
disclosure.
[0091] FIG. 20B illustrates an image resulted from frozen H&E
(top-side image), an image resulted from permanent H&E
(middle-side image), and a CV response recorded by exemplary CDP
(bottom-side image) for a suspicious margin inside the body of the
patient (anterior margin of patient ID 46), consistent with one or
more exemplary embodiments of the present disclosure.
[0092] FIG. 20C illustrates an image resulted from frozen H&E
(top-side image), an image resulted from permanent H&E
(middle-side image), and a CV response recorded by exemplary CDP
(bottom-side image) for a suspicious margin inside the body of the
patient (posterior IM of patient ID 46), consistent with one or
more exemplary embodiments of the present disclosure.
[0093] FIG. 20D illustrates an image resulted from frozen H&E
(top-side image), an image resulted from permanent H&E
(middle-side image), and a CV response recorded by exemplary CDP
(bottom-side image) for Sentinel Lymph Node (SLN) of patient ID 18,
consistent with one or more exemplary embodiments of the present
disclosure.
[0094] FIG. 21 illustrates a visually summarized comparison between
current peak values of recorded CV responses utilizing the
exemplary CDP via exemplary methods for in-vivo cancer diagnosis
within a living tissue, and CIN pathological classification,
consistent with one or more exemplary embodiments of the present
disclosure.
DETAILED DESCRIPTION
[0095] In the following detailed description, numerous specific
details are set forth by way of examples in order to provide a
thorough understanding of the relevant teachings. However, it
should be apparent that the present teachings may be practiced
without such details. In other instances, well known methods,
procedures, components, and/or circuitry have been described at a
relatively high-level, without detail, in order to avoid
unnecessarily obscuring aspects of the present teachings. The
following detailed description is presented to enable a person
skilled in the art to make and use the methods and devices
disclosed in exemplary embodiments of the present disclosure. For
purposes of explanation, specific nomenclature is set forth to
provide a thorough understanding of the present disclosure.
However, it will be apparent to one skilled in the art that these
specific details are not required to practice the disclosed
exemplary embodiments. Descriptions of specific exemplary
embodiments are provided only as representative examples. Various
modifications to the exemplary implementations will be readily
apparent to one skilled in the art, and the general principles
defined herein may be applied to other implementations and
applications without departing from the scope of the present
disclosure. The present disclosure is not intended to be limited to
the implementations shown, but is to be accorded the widest
possible scope consistent with the principles and features
disclosed herein.
[0096] A number of current methods utilize lactate and/or pyruvate
as cancer markers. However, herein the oxidation of Hydrogen
Peroxide (H.sub.2O.sub.2) molecules measured by carbon nanotubes
(CNTs) based electrodes is utilized to detect cancer and especially
distinguish cancerous regions from healthy regions in a suspicious
tissue. The main consequence of pyruvate formation from lactate is
release of H.sub.2O.sub.2 molecules as the main byproduct of
hypoxia glycolysis. An abnormal redox state appears in cancer cells
based on modulation of hypoxia with increased pyruvate
concentration and lactate-to-pyruvate ratio (L/P ratio) which
results in increasing the concentration of H.sub.2O.sub.2 in
interstitial fluid (stroma). So, determination of H.sub.2O.sub.2
molecules would be an indication for the presence of cancer cells
in a tissue. As H.sub.2O.sub.2 is an active and non-stable molecule
it would turn to O.sub.2, H.sup.+ and release electrons which are
great target charges for electrochemical sensation.
[0097] Herein, an electrochemical approach based on multi-walled
carbon nanotubes (MWCNTs) electrodes is disclosed for fast tracking
of hypoxia glycolysis in the interstitial fluid of biopsied tissues
suspicious to cancer, such as breast tissues. Electrochemical
reduction of H.sub.2O.sub.2 molecules, produced in lactate to
pyruvate transformation, on the electrodes of disclosed system may
present a significant quantitate response signal in correlation
with the presence of cancer cells in a suspicious sample. Here, a
cancer diagnostic probe (CDP) based on vertically aligned
multi-walled carbon nanotubes (VAMWCNTs) arrays as sensing
electrode with direct and selective electron transfer abilities in
interaction with H.sub.2O.sub.2 may be utilized.
[0098] Disclosed herein may include a label free method for
diagnosis of the presence of cancer in suspicious regions based on
determination of the hypoxia glycolysis in a quantitative manner.
The method may be based on measuring the oxidative currents
released during glycolysis from the tissue. A matched diagram
between an electrochemical response measured from a suspicious
sample and cancerous state curves may be utilized for a final
diagnostic result. Over expression of glycolysis assisted mRNAs in
cancerous samples may be observed as an indicator of a presence of
cancer in a sample. Exemplary method may be applied as an
alternative for frozen pathology during the surgery with faster and
more precise efficiency. Furthermore, a label free system including
an electrochemical sensor with integrated three CNT based
electrodes is disclosed for tracking hypoxia glycolysis via
detecting electrochemical reduction of H.sub.2O.sub.2 molecules,
which may be produced in Lactate to pyruvate transformation in
cancer cells. Exemplary simple and label free electrochemical assay
may also be used for measuring the drug resistance of the tumors as
a pre therapeutic prediction (as a new prognostic factor) to
increase the survival rate in future.
[0099] In some implementations, exemplary electrochemical sensor
may include an integrated sensor on the needles, named herein as a
cancer diagnostic probe (CDP). Exemplary CDP may be fabricated and
utilized in real-time on the suspicious regions to cancer before
and during surgery in patients (In vivo). The domain of suspicious
regions with a resolution of about 3 mm may be detected using
exemplary method and CDP. The significant specification of CDP
rather than recently reported real-time diagnostic methods, such as
mass-spec, may allow the CDP to track the cancer involved regions
before surgery by squeezing exemplary CDP to suspicious regions
through the skin with the tracking resolution of 3 mm. In
conventional diagnostic protocols, to precisely remove the cancer
regions during surgery, a frozen sample from each suspicious region
may need to be sent for pathologists. The pathology results may be
available after about 15 minutes with the false negative response
ratio of about 10%. Whereas, a cancer region may be distinguished
in-situ utilizing exemplary CDP in less than about 10 seconds or
even instantaneously before or during surgery and without any need
for resecting and freezing a sample from a patient. The diagnostic
information obtained by exemplary CDP may be used to detect cancer
in marginally suspicious regions with rare distributions of cancer
cells filtrated between normal stroma in less than about 20 seconds
during the surgery or biopsy of live animal or human models without
any requirement of tissue resection and preparation for frozen
pathology. Exemplary CDP may be also utilized to detect an accurate
location of cancer involved regions before surgery in superficial
tumors.
[0100] Moreover, exemplary sensor may include a CNT based
electrochemical chip for in vitro cancer diagnosis in suspicious
samples. Exemplary CNT based electrochemical chip may include an
array of electrodes of VAMWCNTs used in electrochemical assays.
Both liquid and solid suspicious samples may be analyzed using
exemplary CNT based electrochemical chip to detect a cancer
presence within the suspicious samples.
[0101] FIG. 1A shows a schematic view of an electrochemical system
100 for cancer diagnosis, consistent with one or more exemplary
embodiments of the present disclosure. Exemplary electrochemical
system 100 may include an exemplary sensor 102, an electrochemical
stimulator-analyzer 104, and an array of electrically conductive
connectors 106. Exemplary sensor 102 may be configured to put in
contact with a suspicious sample for cancer. Exemplary sensor 102
may include an integrated three-electrodes array, which may include
the working electrode 108, the counter electrode 110, and the
reference electrode 112. Each of the working electrode IN, the
counter electrode 110 and the reference electrode 112 may include
an array of vertically aligned multi-walled carbon nanotubes
(VAMWCNTs). The electrochemical stimulator-analyzer 104 may be
configured to measure electrochemical responses from the working
electrode 108 and sensor 102 may be connected to the
electrochemical stimulator-analyzer 104 via the array of
electrically conductive connectors 166.
[0102] In an exemplary implementation, exemplary electrochemical
system 100 may be configured to detect a cancerous state via
measuring H.sub.2O.sub.2 during hypoxia glycolysis in the
suspicious sample for cancer. Exemplary electrochemical system 100
may be utilized by an exemplary method for cancer diagnosis
described herein below.
[0103] In an exemplary embodiment, electrochemical
stimulator-analyzer 104 may include a device that may be capable of
measuring cyclic voltammetry (CV) based diagrams. In an exemplary
embodiment, electrochemical stimulator-analyzer 104 may include a
potentiostat.
[0104] In an exemplary implementation, electrochemical system 100
may further include a processor 114 that may be utilized for
recording and analyzing electrochemical measurements that may be
measured by electrochemical stimulator-analyzer 104. Processor 114
may also be used for controlling electrochemical stimulations that
may be carried out by electrochemical stimulator-analyzer 184. In
an exemplary embodiment, processor 114 may include an EVIUM readout
system.
[0105] In an exemplary implementation, sensor 102 may include a CNT
based electrochemical chip that may be configured to conduct in
vitro cancer diagnosis assays. FIG. 18 shows a schematic view of
exemplary CNT based electrochemical chip 102, consistent with one
or more exemplary embodiments of the present disclosure. Exemplary
CNT based electrochemical chip 102 may include at least one sensing
well 120 and one array of electrically conductive connectors 106.
FIG. 1C shows a schematic view of exemplary sensing well 120,
consistent with one or more exemplary embodiments of the present
disclosure. Each sensing well 120 may include a substrate 122, a
passivation layer 124 that may be grown on substrate 122, a
catalyst layer 126 that may be coated or deposited and subsequently
patterned on the passivation layer 124, and three arrays of
VAMWCNTs that may be grown on the catalyst layer 126. Three arrays
of VAMWCNTs may include the working electrode 108, the counter
electrode 110, and the reference electrode 112.
[0106] In an exemplary embodiment, substrate 122 may include a
silicon chip or wafer. Passivation layer 124 may include a layer of
SiO.sub.2 with a thickness of less than about 500 nm that may be
grown by wet oxidation furnace on the surface of on substrate 122.
Catalyst layer 126 may include a layer of Nickel (Ni) with a
thickness of less than about 10 nm that may be coated on
passivation layer 124 by an E-beam evaporation system at a
temperature of about 120.degree. C. with depositing rate of about
0.1 Angstroms/s. Three arrays of VAMWCNTs (the working electrode
108, the counter electrode 110, and the reference electrode 112)
may be grown on catalyst layer 126 using a direct current plasma
enhanced chemical vapor deposition (DC-PECVD) system. The growth
process of VAMWCNTs may include three steps of firstly, annealing
at a temperature of about 680.degree. C. in an H.sub.2 environment
with a flow rate of about 35 standard cubic centimeters per minute
(sccm) for about 30 minutes; secondly, graining, including plasma
hydrogenation of surface for about 5 minutes with the intensity of
about 5.5 Wcm.sup.-2 that may result in the catalyst layer 126
graining and formation of Ni nano-sized islands, and finally,
growth of VAMWCNTs by introducing a plasma of C.sub.2H.sub.2 and
H.sub.2 mixture with flow rates of about 5 sccm and about 35 sccm
to the chamber for about 15 minutes. Each of the VAMWCNTs may have
a length between about 0.5 .mu.m and about 5 .mu.m and a diameter
between about 20 nm and about 100 nm. The working electrode 108 may
be grown on an area of about 1 cm.times.1 cm, the counter electrode
110 may be grown on an area of about 1 cmx 1 cm, and the reference
electrode 112 may be grown on an area of about 0.5 cm.times.0.5
cm.
[0107] FIG. 1D shows a schematic view of an exemplary magnified
portion 128 of exemplary working electrode 108 within exemplary
sensing well 120 of FIG. 1C, consistent with one or more exemplary
embodiments of the present disclosure. Exemplary VAMWCNTs 130 of an
army of VAMWCNTs of working electrode 108 may be grown vertically
on catalyst layer 126. Catalyst layer 126 may be coated or
deposited and subsequently patterned on the passivation layer 124,
where passivation layer 124 may be grown on substrate 122.
[0108] In an exemplary implementation, sensor 102 may include a
cancer diagnosis probe (CDP) that may be configured to conduct in
vivo cancer diagnosis assays. FIG. 1E shows a schematic view of
exemplary cancer diagnosis probe (CDP) 102, consistent with one or
more exemplary embodiments of the present disclosure. Exemplary
cancer diagnosis probe (CDP) may include three needle electrodes
132, 134, and 136 as exemplary implementations of the working
electrode 168, the counter electrode 110, and the reference
electrode 112, respectively. Moreover, CDP 102 may include a
holding member 138 that may be configured to hold three needle
electrodes 132, 134, and 136. Three needle electrodes 132, 134, and
136 may be fixed on one end 148 of the holding member 138.
[0109] FIG. 1F shows a schematic view of an exemplary needle
electrode 132 corresponding to the working electrode 108,
consistent with one or more exemplary embodiments of the present
disclosure. Referring to FIG. 1F, each needle electrode of three
needles electrodes 132, 134, and 138 may include a tip 142. Each
needle electrode of the three needles electrodes 132, 134, and 136
may include a catalyst layer 14 that may be deposited on tips 142
of three needles electrodes 132, 134, and 136 and an array of
VAMWCNTs 146 that may be grown on catalyst layer 144 on tip 142 of
each needle electrode of three needles electrodes 132, 134, and
138.
[0110] In an exemplary embodiment, each needle electrode of three
needles electrodes 132, 134, and 138 may include a steel needle
with a diameter between about 100 .mu.m and about 200 .mu.m, and a
length between about 0.1 cm and about 1 cm. Three needle electrodes
132, 134, and 138 may be fixed on the end 140 of the holding member
138 apart from each other with a distance (interspace) between each
other in a range of about 1 mm to about 5 mm.
[0111] In an exemplary embodiment, catalyst layer 144 may include a
layer of Nickel (Ni) with a thickness of less than about 10 nm that
may be coated on tip 142 of each needle electrode by an E-beam
evaporation system at a temperature of about 120.degree. C. with a
depositing rate of about 0.1 Angstroms/s. Three arrays of VAMWCNTs
(the working electrode 108, the counter electrode 116, and the
reference electrode 112) may be grown on catalyst layer 144 using a
direct current plasma enhanced chemical vapor deposition (DC-PECVD)
system as described herein above.
[0112] FIG. 1G shows a schematic view of an exemplary magnified
portion 148 of tip 142 of exemplary needle electrode 132 shown in
FIG. 1F, consistent with one or more exemplary embodiments of the
present disclosure. Exemplary VAMWCNTs 130 of an array of VAMWCNTs
146 may be grown vertically on catalyst layer 144. Catalyst layer
144 may be coated or deposited on a surface of tip 142.
[0113] Exemplary cancer diagnosis probe (CDP) 162 may have various
implementations. Exemplary cancer diagnosis probe (CDP) 102 may
include an electrochemical probe with three integrated
needle-shaped electrodes for in-vivo electrochemical measurements
and diagnosis operations, such as cancer diagnostic techniques or
methods. FIG. 1H shows a schematic view of another implementation
of exemplary cancer diagnosis probe (CDP) 102 for in-vivo
measurement of H.sub.2O.sub.2 oxidation in a living tissue,
consistent with one or more exemplary embodiments of the present
disclosure. Exemplary CDP 102 may be utilized for in-vivo
measurement of H.sub.2O.sub.2 oxidation in a living tissue;
thereby, allowing for detecting a cancerous state within the living
tissue. Exemplary CDP 102 may include a handle 152 and a sensing
part 154, where sensing part 154 may be attached to a first end 156
of handle 152. Exemplary sensing part 154 may include an exemplary
working electrode 158, an exemplary counter electrode 160, and an
exemplary reference electrode 162. Exemplary sensing pert 154 may
further include an electrode holder 164. In an exemplary
embodiment, each of exemplary working electrode 158, counter
electrode 160, and reference electrode 162 may be attached to
electrode holder 164.
[0114] In an exemplary embodiment, handle 152 may include a handle
head 166, an insertion part 168, a switch 176, and a releasing
button 172. Exemplary insertion part 168 may allow for inserting
CDP 102 into a biological sample, for example, an exemplary living
tissue in a patient's body, a tumor in a patient's body, or a
biopsied sample from a patient who may involve with cancer.
Exemplary switch 170 may be located on head 166 and switch 170 may
be configured to connect CDP 102 to an electrochemical
stimulator-analyzer device and/or disconnect CDP 302 from the
electrochemical stimulator-analyzer device. In an exemplary
embodiment, the electrochemical stimulator-analyzer device may
include a potentiostat device or an electrochemical
workstation.
[0115] In an exemplary embodiment, CDP 102 may be connected to an
electrochemical stimulator-analyzer device through an electrical
connector, for example, an electrical wire, that may be connected
to a second end 174 of handle 152. In another exemplary embodiment,
CDP 102 may be connected to the electrochemical stimulator-analyzer
device utilizing a wireless connection between CDP 102 and the
electrochemical stimulator-analyzer device without any needs to
connecting wires. For example, CDP 102 may be connected to the
electrochemical stimulator-analyzer device via Bluetooth devices or
Bluetooth modules that may be embedded in CDP 102 and the
electrochemical stimulator-analyzer device. The wireless connection
may allow for simplifying utilizing CDP 102 in a surgery room by a
surgeon, removing redundant wires that may require to sanitize
iteratively, etc.
[0116] In an exemplary embodiment, sensing part 154 may be
replaceable by releasing from handle 152 using releasing button
172. Although electrochemical measurements that may be carried out
utilizing CDP 102 may be repeatable, sensing part 154 may be
replaced by another sensing part 154 (a fresh/new sensing part 154)
for each insertion into each part of the biological sample, which
may be an obligation regarding medical ethics. Such obligations may
be mandatory to avoid transferring cancer cells from one part of
the biological sample to another part of the biological sample;
i.e., from one part of a patient's body to another part of a
patient's body. In an exemplary embodiment, sensing part 154 may be
attached to insertion part 16 at the first end 156 of handle 152.
Furthermore, releasing button 172 may be located on insertion part
168 in proximity to the first end 156 of handle 152. Exemplary
sensing part 154 may be separated from handle 152 by pressing
releasing button 172. FIG. 11 shows a schematic view of an
exemplary scenario in which sensing part 154 has been separated
from handle 152 of exemplary CDP 102, consistent with one or more
exemplary embodiments of the present disclosure.
[0117] Referring to FIG. 1H, exemplary CDP 102 may include three
exemplary electrodes including working electrode 158, counter
electrode 160, and reference electrode 162. In an exemplary
embodiment, working electrode 158 may comprise of a first needle
coated with a layer of VAMWCNTs (an array of VAMWCNTs). VAMWCNTs
may be great sensitive agents for sensing and measuring
H.sub.2O.sub.2 as well as high electrically conductive agents for
accurate electrochemical measurements. Exemplary counter electrode
160 may comprise of a second needle, and exemplary reference
electrode 162 may comprise of a third needle. In an exemplary
embodiment, the second needle may be coated with a layer of
VAMWCNTs (an array of VAMWCNTs). In another exemplary embodiment,
the third needle may be coated with a layer of VAMWCNTs (an array
of VAMWCNTs).
[0118] In an exemplary embodiment, working electrode 158, counter
electrode 160, and reference electrode 162 may be located apart
from each other with a distance between each two respective
electrodes of between about 1 mm and about 5 mm. In an exemplary
embodiment, the distance between each two respective electrodes may
be more than about 5 mm. It should be noted that the distance
between each two respective electrodes may be selected depending on
size of a sample, in which exemplary CDP 102 may be inserted. The
distance between each two respective electrodes may be selected
less than about 5 mm in order to obtain high-accurate
electrochemical responses (i.e., CV diagrams) from the sample. In
addition, the distance between each two respective electrodes
should not be selected may be selected more than about 1 mm in
order to avoid electrical noise in electrochemical
measurements.
[0119] In an exemplary embodiment, each of the first needle, the
second needle, and the third needle may include a biocompatible
conductive needle with a diameter between about 100 .mu.m and about
200 .mu.m, and a length between about 0.1 cm and about 1 cm. In one
embodiment, each of the first needle, the second needle, and the
third needle may include a biocompatible metallic needle, for
example, a steel needle. In one example, each of the first needle,
the second needle, and the third needle may include an acupuncture
needle.
[0120] In an exemplary embodiment, each of the first needle, the
second needle, and the third needle may comprise a biocompatible
conductive needle with a sensing tip. The sensing tip may have a
diameter between about 100 .mu.m and about 200 .mu.m, and a length
between about 0.1 cm and about 1 cm. In an exemplary embodiment, a
layer (an array) of CNTs, for example, VAMWCNTs may be coated on
each sensing tip of the first needle, the second needle, and the
third needle.
[0121] FIG. 1J shows a schematic view of exemplary working
electrode 158, consistent with one or more exemplary embodiments of
the present disclosure. Each of the exemplary counter electrode 160
and exemplary reference electrode 162 may be similar to exemplary
working electrode 158 shown in FIG. 1J. Exemplary working electrode
158 may include exemplary first needle 180 with sensing tip 182. In
an exemplary embodiment, sensing tip 182 may be coated with an
array of VAMWCNTs 184.
[0122] In an exemplary embodiment, a catalyst layer may be
deposited on sensing tip 182. The catalyst layer may include a
layer of Nickel (Ni) with a thickness of less than about 10 nm that
may be coated on sensing tip 182 by an E-beam evaporation system at
a temperature of about 120.degree. C. with a depositing rate of
about 0.1 Angstroms/s. Exemplary array of VAMWCNTs 134 may be grown
on the catalyst layer using a direct current plasma enhanced
chemical vapor deposition (DC-PECVD) system as described herein
above.
[0123] Referring again to FIG. 1A, in an exemplary embodiment,
electrochemical stimulator-analyzer 104 may include a potentiostat
circuit. An exemplary potentiostat circuit may be configured to
measure electrochemical responses from working electrode 108 by
applying a periodic input signal to reference electrode 112 and
measuring an electric current flowing from counter electrode 110 to
working electrode 108. In an exemplary embodiment, an electric
voltage at working electrode 108 may be amplified by a series of
low noise amplifiers (LNAs) to measure a respective electrochemical
response. Different electrochemical responses may be obtained for
different frequencies by varying a frequency of the input signal.
However, an output signal of the potentiostat circuit may tend to
overshoot at high frequencies, leading to instability of
measurements. In an exemplary embodiment, to prevent an excessive
increase of the output signal, the output signal amplitude may be
compared with a reference value utilizing a comparator circuit, and
an output of the comparator circuit may be utilized to adjust an
input signal amplitude so that the output signal may not exceed the
reference value. In an exemplary embodiment, current buffer
amplifiers (CBAs) and compensators may also utilized in the
potentiostat circuit to further stabilize the output signal by
lowering the impact of input signal variations on the output signal
amplitude.
[0124] FIG. 1K shows a schematic of a first implementation of a
potentiostat circuit, consistent with one or more exemplary
embodiments of the present disclosure. In an exemplary embodiment,
electrochemical stimulator-analyzer 104 may include a potentiostat
circuit 104A. In an exemplary embodiment, potentiostat circuit 104A
may include a control amplifier 10402, a transimpedance amplifier
10404, a peak detector circuit 10406, a feedback network 10408, a
periodic wave generator 10410, and a comparator circuit 10412.
[0125] In an exemplary embodiment, control amplifier 10402 may
include a first control input 10414, a second control input 10416
that may be coupled to reference electrode 112, and a control
output 10415 that may be coupled to counter electrode 110. In an
exemplary embodiment, control amplifier 10402 may be configured to
generate a control voltage at control output 10413 by amplifying a
voltage difference between first control input 10414 and second
control input 10416. In an exemplary embodiment, control amplifier
1402 may include a differential amplifier. An exemplary
differential amplifier may include a first input node that may be
connected to first control input 10414 and a second input node
connected to second control input 10416. Therefore, an exemplary
differential amplifier may obtain the voltage difference between
first control input 10414 and second control input 10416 by
measuring a voltage difference between the first input node and the
second input node. In an exemplary embodiment, the voltage
difference may then multiplied by again of the differential
amplifier to generate the control voltage at control output 10418.
In exemplary embodiment, control amplifier 10402 may include an
operational amplifier (op-amp). As a result, in exemplary
embodiment, an electric potential at first control input 10414 may
be transmitted to reference electrode 112 through second control
input 10416 (since electric potentials at op-amp inputs may be
approximately equal). Therefore, in exemplary embodiment, a voltage
at reference electrode 112 may be adjusted by adjusting a voltage
of first control input 10414. In an exemplary embodiment, periodic
wave generator 10410 may be utilized to adjust the voltage of first
control input 10414, as described below. In exemplary embodiment,
exciting reference electrode 112 with an electric potential may
cause a flow of an electric current from working electrode 108 to
control output 10418 through counter electrode 110.
[0126] In exemplary embodiment, transimpedance amplifier 10404 may
include a transimpedance output 10420 and a transimpedance input
10422. In an exemplary embodiment, transimpedance input 10422 may
be connected to working electrode 108. In an exemplary embodiment,
transimpedance amplifier 10404 may be configured to generate a
transimpedance voltage at transimpedance output 10420 by amplifying
a voltage of transimpedance input 10422. In an exemplary
embodiment, the voltage of transimpedance input 10422 may be
multiplied by a gain of the transimpedance amplifier to generate an
amplified voltage at transimpedance output 10420. In exemplary
embodiment, transimpedance amplifier 10404 may include an op-amp.
In exemplary embodiment, transimpedance input 10422 may be
connected to a negative input of an exemplary op-amp, whereas a
positive input of an exemplary op-amp may be connected to ground
(negative and positive inputs are not shown in FIG. 1K). As a
result, in exemplary embodiment, an electric current flowing
through working electrode 108 may be converted to a respective
voltage at transimpedance output 10420 by passing through feedback
network 10408 (since an amount of electric current that may pass
through transimpedance input 10422 to an exemplary negative op-amp
input may be insignificant). In other words, in exemplary
embodiment, the electric current at working electrode in may be
measured by recording a respective voltage at transimpedance output
1420).
[0127] In an exemplary embodiment, peak detector circuit 10406 may
include a detector output 10424 and a detector input 10426. In an
exemplary embodiment, detector input 10426 may be coupled to
transimpedance output 10420. In an exemplary embodiment, peak
detector circuit 10406 may be configured to detect a maximum
voltage at detector input 10426 in a given period of time and
transmit the maximum voltage to detector output 10420. In an
exemplary embodiment, peak detector circuit 10406 may include a
capacitor which may be charged by through detector input 10426. In
an exemplary embodiment, peak detector circuit 10406 may further
include a diode which may be forward biased when the voltage of
detector input 10426 exceeds a voltage of the capacitor and may be
reverse biased when the voltage of detector input 10426 becomes
lower than the capacitor voltage, causing the capacitor to be
disconnected from detector input 10426. As a result, the maximum
voltage at detector input 10426 may be stored by an exemplary
capacitor as long as the capacitor is not reset. In an exemplary
embodiment, the capacitor may be connected to detector output
10420. Therefore, in an exemplary embodiment, detector output 10420
may hold the maximum voltage until a higher voltage is detected at
detector input 10426 by peak detector circuit 10406. As a result,
in an exemplary embodiment, peak values of electric signals at
working electrode 108 (which may be transmitted to detector input
10426 through transimpedance amplifier 10404) may be detected by
peak detector circuit 10406. In an exemplary embodiment, detected
peak values of signals at working electrode 106 may be utilized to
improve stability of potentiostat circuit 104A. In an exemplary
embodiment, peak detector circuit 10406 may be configured to store
the maximum voltage until the given period of time passes. In an
exemplary embodiment, the given period of time may be determined
based on a duration in which an electrochemical response may be
recorded. For example, the given period of time may be set to an
expected duration in which an exemplary CV diagram is recorded. In
an exemplary embodiment, the maximum voltage may be stored in an
exemplary capacitor for the given period of time. In an exemplary
embodiment, peak detector circuit 10406 may be reset by discharging
the capacitor after the given period of time passes.
[0128] In an exemplary embodiment, periodic wave generator 10410
may be configured to generate a periodic wave at first control
input 10414 responsive to periodic wave generator 10410 becoming
active In an exemplary embodiment, comparator circuit 10412 may be
utilized to activate periodic wave generator 10410, as described
below. When periodic wave generator 10410 is active, an exemplary
periodic wave may be transmitted to counter electrode 110 through
second control input 10416 to reference electrode 112. In an
exemplary embodiment, periodic wave generator 10410 may be
configured to adjust the voltage of first control input 10414 by
generating a periodic sawtooth wave at first control input 10414,
as described below. As a result, in an exemplary embodiment,
counter electrode 110 may be excited with a sawtooth wave, which
may facilitate recording CV diagrams from respective
electrochemical responses due to moderately slow sweep rates of
sawtooth waves. In an exemplary embodiment, periodic wave generator
10410 may be further configured to couple first control input 10414
to ground responsive to periodic wave generator 10410 becoming
inactive. In an exemplary embodiment, comparator circuit 10412 may
be utilized to deactivate periodic wave generator 10410, as
described below.
[0129] FIG. 1L shows a schematic of a periodic wave generator,
consistent with one or more exemplary embodiments of the present
disclosure. In an exemplary embodiment, periodic wave generator
10410 may include a pulse wave generator 10452, an op-amp 10454
that may be coupled to pulse wave generator 10452, an RC circuit
10456, and a field effect transistor (FET) M.sub.1.
[0130] In an exemplary embodiment, pulse wave generator 10452 may
be configured to generate a periodic rectangular wave. In an
exemplary embodiment, either an analog or a digital pulse generator
may be utilized to implement pulse wave generator 10452. In an
exemplary embodiment, a duty cycle of rectangular pulses in the
periodic rectangular wave may be set to 50% to generate square
pulses.
[0131] In an exemplary embodiment, op-amp 10454 may include a
positive input 10458, a negative input 10460, and an op-amp output
10462. In an exemplary embodiment, positive input 10455 may be
connected to ground, negative input 10460 may be coupled to an
output of pulse wave generator 10452, and op-amp output 10462 may
be connected to first control input 10414. In an exemplary
embodiment, RC circuit 10456 may be connected between op-amp output
10462 and negative input 1040, and may include a first resistor
R.sub.1 and a first capacitor C.sub.t that may be connected in
parallel.
[0132] In an exemplary embodiment, periodic wave generator 10410
may further include a second resistor R.sub.2 and a third resistor
R.sub.3. In an exemplary embodiment, second resistor R.sub.2 may be
connected to negative input 10460 and third resistor R.sub.3 may be
connected between second resistor R.sub.2 and the output of pulse
wave generator 10452. As a result, in an exemplary embodiment, an
output current of pulse wave generator 10452 flowing through second
resistor Rb and third resistor R may be transmitted to op-amp
output 10462 through RC circuit 10456 (since an amount of electric
current that may pass into op-amp 10454 through an exemplary
negative input 10460 may be insignificant). Therefore, in an
exemplary embodiment, the transmitted current to op-amp output
10462 may preserve a shape and frequency of the output current of
pulse wave generator 10452. However, a voltage shape at op-amp
output 10462 may be determined based on values of first resistor
R.sub.1 and first capacitor C.sub.1 in RC circuit 10456. In an
exemplary embodiment, different values of first resistor R.sub.1
and first capacitor C.sub.1 may result in different
charge/discharge time for capacitor C.sub.1, resulting in different
voltage shapes at op-amp output 10462. For example, the voltage at
op-amp output 10462 may be shaped as a sawtooth wave if the values
of first resistor R.sub.1 and first capacitor C.sub.1 satisfy a set
of exemplary conditions, as described below.
[0133] In an exemplary embodiment, first resistor R.sub.1, first
capacitor C.sub.1, second resistor R.sub.2, and third resistor
R.sub.3 may satisfy a set of conditions defined by the
following:
R.sub.1C.sub.1>100/f.sub.pw
R.sub.2=R.sub.3<0.1R.sub.1
[0134] where f.sub.pw is a frequency of the periodic rectangular
wave. In an exemplary embodiment, the above set of conditions may
ensure that the time constant of RC circuit 10456 (i.e.,
R.sub.1C.sub.1) may be significantly higher than 10T.sub.pw where
T.sub.pw is a period of the periodic rectangular wave, and each of
second resistor R.sub.2 and third resistor R.sub.3 may be
negligible compared to first resistor R.sub.1. As a result, in an
exemplary embodiment, the periodic rectangular wave may be
converted to a sawtooth wave at op-amp output 10462.
[0135] In an exemplary embodiment, FET M.sub.1 may include a source
10460, a drain 10466, and a gate 10468. In an exemplary embodiment,
source 10460 may be connected to ground. In an exemplary
embodiment, drain 10466 may be connected between second resistor
R.sub.2 and third resistor R.sub.3, and gate 10468 may be connected
to an activation input 10427. In an exemplary embodiment, FET
M.sub.1 may be turned off by applying a deactivation voltage to
gate 10468, for example, by connecting activation input 10427 to
ground. As a result, in an exemplary embodiment, op-amp 10454 may
be coupled to third resistor R.sub.3 through second resistor
R.sub.2 and therefore, an output voltage of pulse wave generator
10452 may be transmitted to op-amp output 10462. On the other hand,
in an exemplary embodiment, FET M.sub.1 may be turned on by
applying an activation voltage (for example, a negative voltage for
an n-channel FET or a negative voltage for a p-channel FET) to
activation input 10427. As a result, in an exemplary embodiment,
second resistor R.sub.2 may be coupled to ground through FET
M.sub.1 and consequently, a voltage at op-amp output 10462 may be
reduced as first capacitor C.sub.1 is being discharged.
[0136] Referring again to FIG. 1K, in an exemplary embodiment,
comparator circuit 10412 may be configured to activate periodic
wave generator 10410 by providing the activation voltage to
activation input 10427 of periodic wave generator 10410 responsive
to a voltage of detector output 10420 being smaller than a
reference voltage 10428. In an exemplary embodiment, comparator
circuit 10412 may be further configured to deactivate periodic wave
generator 10410 by providing the deactivation voltage to activation
input 10427 responsive to a voltage of detector output 10420 being
equal to or larger than reference voltage 10428. As discussed
earlier, since peak values of electric signals at working electrode
108 may be detected by detector circuit 10406, utilizing comparator
circuit 10412 may prevent an excessive increase in amplitudes of
electric signals at working electrode 108 by deactivating periodic
wave generator 10410 which may stop an excitation of reference
electrode 112. As a result, in an exemplary embodiment, a flow
electric current through working electrode IN may be reduced.
[0137] In an exemplary embodiment, comparator circuit 10412 may
include a first comparator input 10446, a second comparator input
10448, and a comparator output 10450. In an exemplary embodiment,
first comparator input 10446 may be connected to detector output
10420 and comparator output 10450 may be connected to activation
input 10427.
[0138] In an exemplary embodiment, second comparator input 10448
may include reference voltage 10428. An exemplary second comparator
input 10448 may be coupled to a DC voltage supply that may include
a DC voltage. In an exemplary embodiment, second comparator input
1448 may be coupled to the DC voltage supply via a variable
resistor. An exemplary variable resistor may be implemented
utilizing a potentiometer or a rheostat. An exemplary variable
resistor may be configured to provide reference voltage 10428 to
second comparator input 10448 by transmitting a fraction of the DC
voltage to second comparator input 10448. In an exemplary
embodiment, the fraction of the DC voltage may be determined by
varying a resistivity of the variable resistor until reference
voltage 10428 is obtained.
[0139] In an exemplary embodiment, comparator circuit 10412 may be
configured to provide the activation voltage to comparator output
10450 responsive to a voltage of first comparator input 10446 being
smaller than a voltage of second comparator input 10448. In an
exemplary embodiment, comparator circuit 10412 may be further
configured to provide the deactivation voltage to comparator output
10450 responsive to the voltage of first comparator input 10446
being equal to or larger than the voltage of second comparator
input 10448. In an exemplary embodiment, an analog comparator may
be utilized to implement comparator circuit 10412. In an exemplary
embodiment, the analog comparator may be configured to generate
each of the activation voltage and the deactivation voltage at an
output node of the analog comparator. An exemplary output node of
the analog comparator may be connected to comparator output 10450
to provide each of the activation voltage and the deactivation
voltage to comparator output 10450.
[0140] In an exemplary embodiment, feedback network 10408 may be
connected between detector input 10426 and transimpedance input
10422. In an exemplary embodiment, feedback network 10408 may
include a feedback resistor. In an exemplary embodiment, a
resistance R.sub.f of the feedback resistor may be equal to
V ref I max , ##EQU00002##
where V.sub.ref is a magnitude of reference voltage 10428 and
I.sub.max is an upper limit of an electric current flowing through
detector input 10426. As a result, in an exemplary embodiment, an
electric voltage at detector input 10426 may not exceed V.sub.ref
as long as the electric current flowing through detector input
10426 remains lower than I.sub.max. In an exemplary embodiment,
upper limit I.sub.max may be determined based on an expected
maximum value of an electric current that flows through working
electrode 108 (which may also flow through feedback network 10406,
as discussed earlier). Therefore, in an exemplary embodiment,
determination of resistance R.sub.f based on I.sub.max may further
stabilize potentiostat circuit 104A by preventing measured electric
potentials from reaching reference voltage 10423.
[0141] In an exemplary embodiment, potentiostat circuit 104A may
further include a unity gain amplifier 10430 that may be connected
between reference electrode 112 and second control input 10416. In
an exemplary embodiment, unity gain amplifier 10430 may include a
first unity gain input 10432, a unity gain output 10434, and a
second unity gain input 10436. In an exemplary embodiment, first
unity gain input 10432 may be connected to reference electrode 112,
unity gain output 10434 may be connected to second control input
10416, and second unity gain input 10436 may be connected to unity
gain output 10434 and therefore, to second control input 10416. In
an exemplary embodiment, unity gain amplifier 10430 may be
configured to transmit a voltage at reference electrode 112 to
unity gain output 10434 by amplifying a voltage difference between
first unity gain input 10432 and second unity gain input 10436. In
exemplary embodiment, unity gain amplifier 10430 may include a
differential amplifier. An exemplary differential amplifier may
include an exemplary first input node that may be connected to
first unity gain input 10432 and an exemplary second input node
connected to second unity gain input 10436. Therefore, an exemplary
differential amplifier may obtain the voltage difference between
first unity gain input 10432 and second unity gain input 10436 by
measuring a voltage difference between the exemplary first input
node and the exemplary second input node. In an exemplary
embodiment, the voltage difference may then be multiplied by a gain
of the differential amplifier to generate an amplified voltage at
unity gain output 10434. In exemplary embodiment, unity gain
amplifier 10430 may include an op-amp. Therefore, in an exemplary
embodiment, the voltage of first control input 10414 (i.e., an
output voltage of periodic wave generator 10410) may be provided to
reference electrode 112 through second unity gain input 10436 that
is connected to second control input 10416, because electric
potentials of first unity gain input 10432 and second unity gain
input 10436 may be almost equal.
[0142] FIG. 1M shows a schematic of a second implementation of a
potentiostat circuit, consistent with one or more exemplary
embodiments of the present disclosure. In an exemplary embodiment,
electrochemical stimulator-analyzer 104 may include a potentiostat
circuit 104B. In an exemplary embodiment, potentiostat circuit 104B
may include potentiostat circuit 104A and some additional elements.
Exemplary additional elements may include current buffer amplifiers
and analog compensators, which may improve stability of
electrochemical stimulator-analyzer 104 at higher signal amplitudes
and frequencies, thereby extending a range of frequency and/or
amplitude in which electrochemical stimulator-analyzer 104 may
operate.
[0143] In an exemplary embodiment, potentiostat circuit 104B may
further include a first current buffer amplifier (CBA) 10438 that
may be connected between control amplifier 10402 and counter
electrode 110. In an exemplary embodiment, first CBA 10438 may be
configured to transfer an electric current flowing through control
output 10413 to counter electrode 110.
[0144] In an exemplary embodiment, potentiostat circuit 104B may
further include a second CBA 10440 that may be connected between
transimpedance amplifier 10404 and peak detector circuit 1406. In
an exemplary embodiment, second CBA 10440 may be configured to
transfer an electric current flowing through transimpedance output
10420 to detector input 10426.
[0145] In an exemplary embodiment, potentiostat circuit 1048 may
further include a first analog compensator 10442 that may be
connected between counter electrode 110 and first control input
10414. In an exemplary embodiment, first analog compensator 10442
may be configured to compensate voltage variations at counter
electrode 110 responsive to voltage variations at first control
input 10414. In an exemplary embodiment, first analog compensator
10442 may include a first compensation capacitor. In an exemplary
embodiment, potentiostat circuit 104B may further include a second
analog compensator 10444 that may be connected to second control
input 10416. In an exemplary embodiment, second analog compensator
10444 may be configured to compensate voltage variations at second
control input 10416. In an exemplary embodiment, second analog
compensator 10444 may include a second compensation capacitor and a
compensation resistor that may be connected in series. In an
exemplary embodiment, values of the first compensation capacitor,
the second compensation capacitor, and the compensation resistor
may be set to about 22 pF, 47 pF, and 4.99 k.OMEGA., respectively.
As a result, in an exemplary embodiment, a scan rate of about 1
V/.mu.s may obtained at a frequency of about f.sub.pw=1 MHz for
recording electrochemical responses utilizing potentiostat circuit
104B, which may be suitable for high speed applications.
[0146] In another aspect of the present disclosure, a method for
cancer diagnosis is disclosed. FIG. 2A shows an exemplary
implementation of method 290 for cancer diagnosis, consistent with
one or more exemplary embodiments of the present disclosure. Method
200 may include putting an array of vertically aligned multi-walled
carbon nanotubes (VAMWCNTs) of a sensor in contact with a
suspicious sample (step 202), recording an electrochemical response
from the suspicious sample, where the electrochemical response may
include an oxidation current peak (step 204), and detecting a
cancerous state in the suspicious sample responsive to a larger
amount of the oxidation current peak than a threshold value (step
206). The sensor may be similar to exemplary sensor 102 described
hereinabove.
[0147] Step 202 may include putting the array of vertically aligned
multi-walled carbon nanotubes (VAMWCNTs) of the sensor in contact
with the suspicious sample. In an exemplary implementation, putting
the array of VAMWCNTs of the sensor in contact with the suspicious
sample may include one of dropping the suspicious sample onto the
sensor, placing the suspicious sample onto the sensor, squeezing
the sensor into the suspicious sample, inserting the sensor into
the suspicious sample, and combinations thereof.
[0148] In an exemplary embodiment, the suspicious sample may
include one of a liquid suspicious sample, a solid suspicious
sample, and combinations thereof. In an exemplary embodiment, the
suspicious sample may include one of a plurality of cell lines, a
biopsied sample from a human or animal body, a removed sample from
a human or animal body by surgery, a portion of a living tissue in
a human or animal body, and a portion of a living tissue in a human
or animal body during surgery.
[0149] In an exemplary implementation, the sensor may be similar to
sensor 162 and may include a substrate, a catalyst layer, and three
arrays of vertically aligned multi-walled carbon nanotubes
(VAMWCNTs) grown on the catalyst layer. Three arrays of VAMWCNTs
may include a working electrode that may include a first array of
VAMWCNTs, a reference electrode that may include a second array of
VAMWCNTs, and a counter electrode that may include a third array of
VAMWCNTs. In an exemplary implementation, the sensor may further
include a passivation layer between the substrate and the catalyst
layer.
[0150] In an exemplary implementation, the sensor may include one
of a CNT based electrochemical chip similar to exemplary CNT based
electrochemical chip 102 shown in FIG. 1B, and a cancer diagnosis
probe (CDP) similar to exemplary CDP 192 shown in FIG. 1E. The
substrate of the cancer CDP may include three needles, where each
needle of the three needles may be coated by an array of VAMWCNTs
of the three arrays of VAMWCNTs. In an exemplary implementation,
the sensor may include exemplary sensor 102 as shown schematically
in FIGS. 1A, 1B, and 1E.
[0151] FIG. 2B shows a schematic implementation of step 202 that
may include putting the army of vertically aligned multi-walled
carbon nanotubes (VAMWCNTs) grown on tip of each needle electrode
of three needles electrodes 132, 134, and 138 of exemplary cancer
diagnosis probe (CDP) 102 in contact with exemplary suspicious
sample 250, consistent with one or more exemplary embodiments of
the present disclosure. Step 102 may include inserting or squeezing
exemplary cancer diagnosis probe (CDP) 102 in exemplary suspicious
sample 250.
[0152] In an exemplary implementation, putting the array of
VAMWCNTs of exemplary sensor 102 in contact with the suspicious
sample may take place temporarily or over a time duration of less
than 1 seconds for a real-time cancer diagnosis case. In an
exemplary embodiment, putting the array of VAMWCNTs of exemplary
sensor 102 in contact with the suspicious sample may take place
temporarily or over a time duration of less than 1 seconds for in
vivo or in vitro cancer diagnosis using exemplary sensor which may
be an exemplary CDP or exemplary CNT based electrochemical chip. In
an exemplary embodiment, putting the array of VAMWCNTs of exemplary
sensor 102 in contact with the suspicious sample may be for a time
duration of about 12 hours or more for in vitro cancer diagnosis
cases with high levels of accuracy utilizing exemplary CNT based
electrochemical chip 102. In an exemplary embodiment, putting the
array of VAMWCNTs of exemplary sensor 102 in contact with the
suspicious sample may be carried out in a time duration of about
0.1 seconds to about 24 hours.
[0153] Step 204 may include recording the electrochemical response
from the suspicious sample, where the electrochemical response may
include an oxidation current peak. In an exemplary embodiment, the
electrochemical response may include a cyclic voltammetry (CV)
diagram of hypoxic glycolysis chemical reaction in biological cells
within the suspicious sample. In an exemplary embodiment, the
electrochemical response may include a cyclic voltammetry (CV)
diagram of H.sub.2O.sub.2 related oxidation/reduction chemical
reaction in biological cells within the suspicious sample. The
concentration of H.sub.2O.sub.2 may be in correlation with the
hypoxia glycolysis occurred in tumor cells. In an exemplary
embodiment, the electrochemical response may include a cyclic
voltammetry (CV) diagram of H.sub.2O.sub.2 oxidation that may be
electrically sensed by VAMWCNTs in biological cells within the
suspicious sample. In an exemplary embodiment, the electrochemical
response may include an oxidation current peak of exemplary CV
diagram of hypoxic glycolysis chemical reaction in biological cells
within a suspicious sample.
[0154] In an exemplary implementation, recording the
electrochemical response from the suspicious sample (step 204) may
include connecting the sensor to an electrochemical
stimulator-analyzer, applying an electrical voltage on the sensor
using the electrochemical stimulator-analyzer, and measuring the
electrochemical response from the suspicious sample using the
electrochemical stimulator-analyzer. In an exemplary embodiment,
the electrochemical stimulator-analyzer may include a
potentiostat.
[0155] Step 206 may include detecting the cancerous state in the
suspicious sample responsive to a larger amount of the oxidation
current peak than a threshold value. In an exemplary embodiment,
the threshold value may include an oxidation current peak of about
700 .mu.A or more when a time duration of putting the array of
vertically aligned multi-walled carbon nanotubes (VAMWCNTs) of the
sensor in contact with the suspicious sample (step 202) may be more
than about 12 hours. In an exemplary embodiment, the threshold
value may include an oxidation current peak of about 80 .mu.A or
more when a time duration of putting the array of vertically
aligned multi-walled carbon nanotubes (VAMWCNTs) of the sensor in
contact with the suspicious sample (step 202) may be about 5
seconds or less.
[0156] FIG. 2C shows an implementation of detecting the cancerous
state in the suspicious sample (step 206), consistent with one or
more exemplary embodiments of the present disclosure. Detecting the
cancerous state in the suspicious sample (step 206) may include
recording a reference electrochemical response from a reference
solution, where the reference electrochemical response may include
a reference oxidation current peak (step 201), comparing the
electrochemical response with the reference electrochemical
response (step 210), and detecting the cancerous state in the
suspicious sample responsive to a larger oxidation current peak of
the electrochemical response in comparison with the reference
oxidation current peak (step 212). In an exemplary embodiment, the
reference solution may include a lactate solution with a lactate
concentration of about 0.05 mM or more.
[0157] Disclosed systems, methods and sensors herein may have
various implementations, all based on measuring H.sub.2O.sub.2
oxidation current peak due to hypoxia glycolysis and reverse
Warburg phenomena in cancer cells in order to for diagnosing
cancerous tumors in real-time and with highly accuracy. Exemplary
CDP 102 may be utilized via exemplary system 100 and/or utilizing
exemplary method 200 for non-invasively diagnosing, in real-time, a
presence of pre-neoplastic/neoplastic cells in either internal or
external margins of a patient during tumor surgery, for example,
breast cancer surgery. The exemplary systems, methods, and sensors
may be capable of instantaneously determining an amount of
H.sub.2O.sub.2 released from cancer or atypical cells, through
reverse Warburg effect and hypoxia assisted glycolysis pathways, in
a quantitative electrochemical manner. Reverse Warburg effect and
hypoxia assisted glycolysis pathways may lead to high levels of
H.sub.2O.sub.2 concentration in cancerous tumors in comparison with
healthy tissues. Due to limited precision of conventional H&E
pathology of biopsy samples and requirement to time-consuming
preparation and consideration of many blocks and slides for
complete evaluation of biopsy samples, exemplary systems, methods,
and sensors of the present disclosure may be applied for cancer
diagnosis, which may be based on live detecting the hypoxia
glycolytic functions of high risk/premalignant cells based on the
H.sub.2O.sub.2 released from cancer or atypical cells (through
reverse Warburg effect 10 and hypoxia assisted glycolysis
pathways). The exemplary systems, methods, and sensors may be
utilized for diagnosing all cancerous tumors, in which hypoxia
glycolysis may be the main differential mechanism between the
phenotypes of healthy, precancerous and cancerous cells.
[0158] In an exemplary implementation, method 200 may further
include fabrication of exemplary sensor 102 (not illustrated), for
example, exemplary CDP 102. In an exemplary implementation, method
200 may be utilized for in-vivo cancer diagnosis within a living
tissue utilizing exemplary CDP 102.
[0159] FIG. 2E shows an exemplary implementation of exemplary
method 220 for in-vivo cancer diagnosis within a living tissue,
consistent with one or more exemplary embodiments of the present
disclosure. Exemplary method 220 may be similar to method 200 shown
in FIG. 2A. Exemplary method 220 may include preparing an
electrochemical probe by fabricating three integrated electrodes
(step 221), putting tips of the three integrated electrodes in
contact with a portion of the living tissue by inserting the tips
of the three integrated electrodes into the portion of the living
tissue (step 222), recording an electrochemical response from the
portion of the living tissue, where the electrochemical response
may include a cyclic voltammetry (CV) diagram with an oxidation
current peak of hypoxic glycolysis chemical reaction in biological
cells within the portion of the living tissue (step 224), and
detecting a cancer-involving status of the portion of the living
tissue based on the oxidation current peak (step 226).
[0160] In detail, step 221 may include preparing an electrochemical
probe. In an exemplary embodiment, the electrochemical probe may be
similar to exemplary CDP 102 that is shown in FIGS. 1E, 1H and 1I.
FIG. 2F shows an exemplary implementation of preparing an exemplary
electrochemical probe similar to CDP 102 (step 221), consistent
with one or more exemplary embodiments of the present disclosure.
Step 221 may include fabricating three integrated electrodes by
coating a layer of vertically aligned multi-walled carbon nanotubes
(VAMWCNTs) on tips of three electrically conductive biocompatible
needles (step 230), and fixing the three integrated electrodes at
one end of a handle (or a holding member)(step 232).
[0161] FIG. 2G shows an exemplary implementation of fabricating
three integrated electrodes by coating a layer of vertically
aligned multi-walled carbon nanotubes (VAMWCNTs) on tips of three
electrically conductive biocompatible needles (step 230),
consistent with one or more exemplary embodiments of the present
disclosure. Step 230 may include depositing a catalyst layer on the
tips of the three electrically conductive biocompatible needles
(step 240), and growing an array of VAMWCNTs on the deposited
catalyst layer (step 242). In an exemplary implementation, step 230
may include fabricating three integrated electrodes by coating
three respective layers of VAMWCNTs on tips of three electrically
conductive biocompatible needles.
[0162] In detail, step 240 may include depositing a catalyst layer
on tips of three electrically conductive biocompatible needles. In
an exemplary implementation, step 240 may include depositing three
catalyst layers on three respective tips of the three electrically
conductive biocompatible needles. In an exemplary implementation,
step 240 may include depositing a respective layer of Nickel (Ni)
with a thickness of less than about 10 nm using an E-beam
evaporation system at a temperature of about 120.degree. C. with a
depositing rate of about 0.1 Angstroms/s on each tip of the tips of
the three electrically conductive biocompatible needles.
[0163] Furthermore, step 242 may include growing an array of
VAMWCNTs on the deposited catalyst layer on each tip of the tips of
the three electrically conductive biocompatible needles. FIG. 2H
shows an exemplary implementation of growing an array of VAMWCNTs
on the deposited catalyst layer (step 242), consistent with one or
more exemplary embodiments of the present disclosure. Step 242 may
include annealing the deposited catalyst layer at a temperature of
about 680.degree. C. in an H.sub.2 environment with a flow rate of
about 20 standard cubic centimeters per minute (sccm) to 35 sccm
for about 30 minutes (step 244), graining the annealed catalyst
layer by plasma hydrogenation of surface of the catalyst layer for
about 5 minutes with an intensity of about 5.5 Wcm.sup.-2 (step
246), and growing VAMWCNTs on the grained catalyst layer in a
chamber by introducing a plasma comprising a mixture of
C.sub.2H.sub.2 with flow rate of about 5 sccm and H.sub.2 with flow
rate of about 35 sccm to the chamber for about 15 minutes (step
248). In an exemplary implementation, graining the annealed
catalyst layer by plasma hydrogenation of surface of the catalyst
layer for about 5 minutes with an intensity of about 5.5 Wcm.sup.-2
(step 246) may result in catalyst graining, and formation of
nano-sized islands of the catalyst.
[0164] Referring to FIG. 2E, step 222 may include putting tips of
the three integrated electrodes in contact with a portion of the
living tissue by inserting the tips of the three integrated
electrodes into the portion of the living tissue. In an exemplary
implementation, step 222 may include putting tips of exemplary
three integrated electrodes of exemplary CDP 102 in contact with a
portion of a living tissue by inserting the tips of the three
integrated electrodes into the portion of the living tissue. As
used herein, "the portion of the living tissue" may refer to an
implementation of "the suspicious sample" described hereinabove,
which may include a biological sample that may be suspicious to be
cancerous. FIG. 2C shows a schematic view of another exemplary
implementation of putting electrodes 158, 160, and 162 of exemplary
CDP 162 in contact with exemplary portion 262 of living tissue 260
(step 222), consistent with one or more exemplary embodiments of
the present disclosure. Putting electrodes 158, 160, and 162 of
exemplary CDP 102 in contact with exemplary portion 262 of living
tissue 260 may include inserting sensing part 154 into exemplary
portion 262 of living tissue 260.
[0165] In an exemplary implementation, putting electrodes 158, 160,
and 162 of exemplary CDP 102 in contact with exemplary portion 262
of living tissue 260 may include at least one of putting electrodes
158, 160, and 162 inside portion 262 of living tissue 260,
inserting electrodes 158, 160, and 162 inside portion 262 of living
tissue 260, interacting a secretion of living tissue 260 with
electrodes 158, 160, and 162, squeezing electrodes 158, 160, and
162 inside portion 262 of living tissue 260, and combinations
thereof. In an exemplary embodiment, inserting electrodes 158, 160,
and 162 inside portion 262 of living tissue 260 may include
penetrating electrodes 158, 160, and 162 into portion 262 of living
tissue 260. In one implementation, putting electrodes 158, 160, and
162 of exemplary CDP 102 in contact with portion 262 of living
tissue 260 (step 222) may be done during at least one of a surgery
operation, a mastectomy operation, a biopsy operation, an
endomicroscopy operation, an optical biopsy operation, a clinical
examination of a patient, and combinations thereof. In one
implementation, putting electrodes 158, 160, and 162 of exemplary
CDP 102 in contact with exemplary portion 262 of living tissue 260
may include inserting electrodes 158, 160, and 162 of exemplary CDP
102 in portion 262 of living tissue 260 with an insertion depth
between about 3 mm and about 5 mm.
[0166] In an exemplary embodiment, portion 262 of living tissue 260
may be in liquid form or solid form. In further detail, in an
exemplary embodiment, portion 262 may comprise at least one of a
liquid sample suspicious to be cancerous, a solid sample suspicious
to be cancerous, and combinations thereof.
[0167] In an exemplary embodiment, portion 262 of living tissue 260
may include at least one of a biopsied sample from a human or
animal body, a sample resected from a human or animal body by
surgery, a portion of living tissue 260 in a human or animal body
near to skin, an exemplary portion 262 of living tissue 260 of a
human or animal body that may be accessible during surgery (tumor
removal surgery) or biopsy operation, a suspicious mass to be
cancerous in a human or animal body, a removed sample from a human
or animal body by surgery, and combinations thereof.
[0168] Moreover, step 224 may include recording an electrochemical
response from the portion of the living tissue, where the
electrochemical response may include a cyclic voltammetry (CV)
diagram with an oxidation current peak of hypoxic glycolysis
chemical reaction in biological cells within the portion of the
living tissue. In an exemplary implementation, step 224 may include
recording a CV diagram with an oxidation current peak of hypoxic
glycolysis chemical reaction from portion 262 of living tissue 260
utilizing exemplary CDP 102. In an exemplary implementation,
recording the electrochemical response from the suspicious sample
(step 224) may include recording the electrochemical response from
exemplary suspicious sample 250 (FIG. 2B) or exemplary portion 262
of living tissue 260 (FIG. 2C). In an exemplary embodiment, the
electrochemical response may be recorded using exemplary CDP 102
that is shown in FIGS. 1E, 1H and 1I. In an exemplary embodiment,
the electrochemical response may include a cyclic voltammetry (CV)
diagram with an oxidation current peak of hypoxic glycolysis
chemical reaction in biological cells within exemplary portion 262
of the living tissue 260.
[0169] In an exemplary implementation, recording the
electrochemical response from exemplary portion 262 of living
tissue 260 may include connecting exemplary CDP 102 to an
electrochemical stimulator-analyzer, applying a set of electrical
potentials to exemplary CDP 102 using the electrochemical
stimulator-analyzer, and recording a set of electrical currents
respective to the applied set of electrical potentials from
exemplary portion 262 of the living tissue 260 using the exemplary
CDP 102 and the electrochemical stimulator-analyzer. In an
exemplary embodiment, each of the measured set of electrical
current may flow from counter electrode 110 to working electrode
108 when a respective electrical potential of the set of electrical
potentials is applied to CDP 102.
[0170] In an exemplary implementation, applying the set of
electrical potentials to exemplary CDP 102 may include applying a
sweeping range of electrical potentials between about -1 V and
about 1 V to exemplary working electrode 158 or exemplary working
electrode 163. In an exemplary implementation, applying the set of
electrical potentials to exemplary CDP 102 may include applying a
sweeping range of electrical potentials between about -0.8 V and
about 0.8 V to exemplary working electrode 158 or exemplary working
electrode 108. In an exemplary embodiment, the electrochemical
stimulator-analyzer may comprise a potentiostat.
[0171] In detail, step 226 may include detecting a cancer-involving
status of the portion of the living tissue based on the oxidation
current peak. In an exemplary implementation, step 226 may include
detecting a cancer-involving status of exemplary suspicious sample
250 (FIG. 2B) or exemplary portion 262 of living tissue 260 (FIG.
2C). In an exemplary implementation, detecting the cancer-involving
status of portion 262 of living tissue 260 based on the oxidation
current peak (step 226) may include detecting a healthy state at
portion 262 of living tissue 260 responsive to a value (an amount)
of the oxidation current peak being smaller than a first threshold
value, detecting a cancerous state at portion 262 of living tissue
260 responsive to the value of the oxidation current peak being
larger than a second threshold value, and detecting a moderately
cancer-involved state at the portion 262 of living tissue 260
responsive to the value of the oxidation current peak being between
the first threshold value and the second threshold value.
[0172] In an exemplary implementation, exemplary method 220
utilizing exemplary CDP 102 may be utilized for in-vivo cancer
diagnosis within all tissues in a human or animal's body. In an
exemplary implementation, exemplary method 220 utilizing exemplary
CDP 102 may be utilized for in-vivo cancer diagnosis of all
cancerous tumors, in which hypoxia glycolysis may be the main
differential mechanism between the phenotypes of healthy,
precancerous and cancerous cells. In an exemplary implementation,
detecting the cancer-involving status of portion 262 of living
tissue 260 based on the oxidation current peak (step 226) may
include detecting presence of human breast cancer in a portion of a
breast tissue responsive to the value of the oxidation current peak
being equal to 203 .mu.A or more, detecting the healthy state at
the portion of the breast tissue responsive to the value of the
oxidation current peak being equal to 137 .mu.A or less, and
detecting a moderately breast cancer-involved state at the portion
of the breast tissue responsive to the value of the oxidation
current peak being between 137 .mu.A and 203 .mu.A.
[0173] In an exemplary implementation, detecting the
cancer-involving status of portion 262 of living tissue 260 based
on the oxidation current peak (step 226) may include detecting
presence of cervical cancer in a portion of a cervical tissue
(cervix) responsive to the value of the oxidation current peak
being equal to 145 .mu.A or more, detecting the healthy state at
the portion of the cervical tissue (cervix) responsive to the value
of the oxidation current peak being equal to 115 .mu.A or less, and
detecting a moderately cervical cancer-involved state (or a
suspicious-involved state) at the portion of the cervical tissue
(cervix) responsive to the value of the oxidation current peak
being between 115 .mu.A and 145 .mu.A.
[0174] FIG. 2I shows an exemplary implementation of detecting the
cancer-involving status of portion 262 of living tissue 260 based
on the oxidation current peak (step 226), consistent with one or
more exemplary embodiments of the present disclosure. In detail,
detecting the cancer-involving status of portion 262 of living
tissue 260 based on the oxidation current peak (step 226) may
include generating a set of reference current peak values (step
270), looking up the oxidation current peak within the generated
set of reference current peak values (step 272), and detecting the
cancer-involving status in portion 262 of living tissue 260 (step
274).
[0175] In detail, step 270 may include generating a set of
reference current peak values. FIG. 2J shows an exemplary
implementation of generating the set of reference current peak
values (step 270), consistent with one or more exemplary
embodiments of the present disclosure. In an exemplary
implementation, generating the set of reference current peak values
(step 270) may include recording a set of CV diagrams from a
plurality of samples of living tissues using the electrochemical
probe (i.e., CDP 102)(step 280), measuring a set of reference
current peaks respective to the recorded set of CV diagrams for
each sample of the plurality of samples of living tissues (step
282), determining status of each sample by applying a pathological
assay to each sample (step 284), and assigning the determined
status of each sample to the respective measured reference current
peak (step 236). In an exemplary implementation, generating the set
of reference current peak values (step 270) may include calibrating
exemplary CDP 102 for an exemplary living tissue similar to living
tissue 260. In such implementation, the plurality of samples of
living tissues may include a plurality of samples of living tissues
from the same organ, for example, breast tissue, of a plurality of
human or animal bodies.
[0176] The determined status may include one of a healthy state, a
cancerous state, and a moderately cancer-involved state, based on
result of the applied pathological assay. In an exemplary
implementation, detecting the cancer-involving status in portion
262 of living tissue 260 (step 274) may include detecting the
healthy state for portion 262 of living tissue 260 responsive to
the oxidation current peak being in a first range of the generated
set of reference current peak values assigned as being of the
healthy state, detecting the cancerous state for portion 262 of
living tissue 260 responsive to the oxidation current peak being in
a second range of the generated set of reference current peak
values assigned as being of the cancerous state, and detecting the
moderately cancer-involved state for portion 262 of living tissue
20 responsive to the oxidation current peak being in a third range
of the generated set of reference current peak values assigned as
being of the moderately cancer-involved state.
[0177] In an exemplary implementation, the whole process of
exemplary method 200 which may include, putting the array of
VAMWCNTs of exemplary sensor 102 in contact with the suspicious
sample (step 202), recording the electrochemical response from the
suspicious sample (step 204), and detecting the cancerous state in
the suspicious sample (step 206) may be carried out in less than
about 30 seconds. Similarly, in an exemplary implementation, steps
222-226 of exemplary method 220 which may include putting
electrodes 153, 160, and 162 of exemplary CDP 102 in contact with
exemplary portion 262 of living tissue 260 (step 222), recording
the electrochemical response from exemplary portion 262 of living
tissue 260 (FIG. 2C) (step 224), and detecting the cancerous state
in 262 of living tissue 260 (step 226) may be carried out in less
than about 30 seconds.
[0178] In an exemplary implementation, conducting cancer diagnosis
process of exemplary method 228 which may further include replacing
a previously used sensing part 154 with a new sensing part 154 may
take place in less than about 40 seconds. In an exemplary
implementation, conducting cancer diagnosis process of exemplary
method 220 which may include replacing a previously used sensing
part 154 with a new sensing part 154, inserting exemplary CDP 102
including the new sensing part 154 into a target tissue by
inserting new sensing part 154 into a target location within the
target tissue, recording a CV response with a current peak from the
target location, and detecting cancerous state of the target tissue
may take place in less than about 40 seconds. For example,
replacing a previously used sensing part 154 with a new sensing
part 154, inserting exemplary CDP 102 including the new sensing
part 154 into portion 262 of living tissue 260 by inserting new
sensing part 154 into portion 262 of living tissue 260, recording a
CV response with a current peak from portion 262 of living tissue
260, and detecting cancerous state of portion 262 of living tissue
266 may take place in less than about 40 seconds.
[0179] In an exemplary implementation, replacing the previously
used sensing part 154 may include removing the previously used
sensing part 154, and connecting the new (fresh) sensing part 154
to handle 152. In an exemplary implementation, replacing the
previously used sensing part 154 may take a time interval of less
than about 20 sec. In an exemplary implementation, recording the CV
response with the current peak from a target location (i.e.,
portion 262 of living tissue 260) may be carried out in about 15
sec or less due to synchronized real-time processing.
[0180] Accordingly, in an exemplary embodiment, methods 200, and
220, provide a quick and efficient approach to instantaneously
detect a tissue's cancerous state, including indicating presence of
cancer. For example, exemplary CDP 102 may be utilized through
exemplary methods 200, and 224 for real-time high-accurate
detecting cancer-involved margins in a patient's body. In such
implementations, exemplary methods 200, and 220 may be applied on
at least one exemplary portion of a living tissue that may be
suspicious to contain a cancerous tumor. In one implementation,
exemplary methods 200, and 220 may be applied before a tumor
removal surgery, during a tumor removal surgery, after a tumor
removal surgery, and combinations thereof. In an exemplary
implementation, exemplary CDP 102 may be utilized through exemplary
methods 200, and 220 at suspicious margins around a dissected tumor
after the tumor removal surgery to determine whether the suspicious
margins are cancerous or not.
[0181] In an exemplary implementation, electrochemical system 100
may be utilized for cancer diagnosis via exemplary method 290. FIG.
3A shows a schematic view of exemplary electrochemical reactions
involved on sensor 102 including exemplary VAMWCNTs 130 as shown in
FIGS. 1D and 1G, consistent with one or more exemplary embodiments
of the present disclosure. Presence of H.sub.2O.sub.2 active
molecule released during hypoxia glycolysis in a suspicious sample
may be the main trigger of the electrochemical reactions. Hence,
the chemical reaction occurring on the working electrode 108
including VAMWCNTs 130 may include:
L - Lactate + O 2 .times. .fwdarw. L - Lactate Oxidase .times.
.times. Pyruvate + H 2 .times. O 2 Eq . .times. 1 H 2 .times. O 2
.fwdarw. O 2 + 2 .times. H + + 2 .times. e - .times. .times.
response .times. .times. reaction .times. .times. of .times.
.times. the .times. .times. CDP Eq . .times. 2 ##EQU00003##
[0182] When the hypoxia glycolysis (Eq. 2) is activated (the
concentration of 02 is less than 5%) in cancer cells, increased
reactive oxygen species (ROS) generated by mitochondria, would
significantly enhance the cathodic peak of an electrochemical
response measured from the suspicious sample which could be sharply
detected by VAMWCNTs 130 electrodes. It may be known that the
lactate released by hypoxic tumor cells during their glycolysis may
not be discharged as a waste product, but may be taken up by
oxygenated tumor cells as energy fuel in which Lactate is converted
to pyruvate and H.sub.2O.sub.2 by LDH-B and then enters the
mitochondria for OXPHOS to generate ATP. Similar to this process,
the lactate released from hypoxic tumor cells may be used herein in
electrochemical assay to trace the concentration of lactate due to
the intensity of the H.sub.2O.sub.2 produced during LADH (Eq. 1)
and released electrons due to the intensity of H.sub.2O.sub.2
oxidation reaction (Eq. 2).
[0183] FIG. 3B shows a schematic overview of mitochondrial electron
and proton fluxes in hypoxia, consistent with one or more exemplary
embodiments of the present disclosure. During normaxia, electrons
released from reduced cofactors (NADH and FADH2), flow through the
redox centers of the respiratory chain (r.c.) to molecular oxygen
(dotted lines), to which a proton flux from the mitochondrial
matrix to the intermembrane space is coupled (grey arrows). Protons
then flow back to the matrix through the FO sector of the ATP
synthase complex, driving ATP synthesis. ATP is carried to the cell
cytosol by the adenine nucleotide translocator (grey arrows). Under
moderate to severe hypoxia, electrons escape the r.c. redox centers
and reduce molecular oxygen to the superoxide anion radical before
reaching the cytochrome c (black arrows). Under these conditions,
to maintain an appropriate .DELTA..sub..psi.m, ATP produced by
cytosolic glycolysis enters the mitochondria where it is hydrolyzed
by the FIFO ATPase with extrusion of protons from the mitochondrial
matrix (black arrows). So, the mechanism of H.sub.2O.sub.2
detection by the VAMWCNTs 130 electrodes in hypoxia glycolysis may
be based on released ion species during reduction of NADH,
generation of ROS and production of superoxide anion radical by
reducing molecular oxygen before reaching to cytochrome c. The
amount of released charged species and increased current
transferred by VAMWCNTs 130 electrodes may be correlated with the
concentration of the lactate and subsequently H.sub.2O.sub.2 which
resulted in ROS generated during hypoxia glycolysis.
[0184] FIG. 1N shows a computer system 114A in which an embodiment
of the present disclosure, or portions thereof, may be implemented
as computer-readable code, consistent with exemplary embodiments of
the present disclosure. For example, steps 204-212 of flowchart
200, steps 224 and 226 of flowchart 220, steps 270-274 of flowchart
226, and steps 280-286 of flowchart 270 may be implemented in
computer system 114A using hardware, software, firmware, tangible
computer readable media having instructions stored thereon, or a
combination thereof and may be implemented in one or more computer
systems or other processing systems. Hardware, software, or any
combination of such may embody any of the modules and components in
FIGS. 1A-1M. In an exemplary embodiment, computer system 114A may
include processor 114.
[0185] If programmable logic is used, such logic may execute on a
commercially available processing platform or a special purpose
device. One ordinary skill in the art may appreciate that an
embodiment of the disclosed subject matter can be practiced with
various computer system configurations, including multi-core
multiprocessor systems, minicomputers, mainframe computers,
computers linked or clustered with distributed functions, as well
as pervasive or miniature computers that may be embedded into
virtually any device.
[0186] For instance, a computing device having at least one
processor device and a memory may be used to implement the
above-described embodiments. A processor device may be a single
processor, a plurality of processors, or combinations thereof.
Processor devices may have one or more processor "cores."
[0187] An embodiment of the invention is described in terms of this
example computer system 500. After reading this description, it
will become apparent to a person skilled in the relevant art how to
implement the invention using other computer systems and/or
computer architectures. Although operations may be described as a
sequential process, some of the operations may in fact be performed
in parallel, concurrently, and/or in a distributed environment, and
with program code stored locally or remotely for access by single
or multi-processor machines. In addition, in some embodiments the
order of operations may be rearranged without departing from the
spirit of the disclosed subject matter.
[0188] Processor device 11404 may be a special purpose or a
general-purpose processor device. As will be appreciated by persons
skilled in the relevant art, processor device 11404 may also be a
single processor in a multi-core/multiprocessor system, such system
operating alone, or in a cluster of computing devices operating in
a cluster or server farm. Processor device 11404 may be connected
to a communication infrastructure 11406, for example, a bus,
message queue, network, or multi-core message-passing scheme.
[0189] In an exemplary embodiment, computer system 114A may include
a display interface 11402, for example a video connector, to
transfer data to a display unit 11430, for example, a monitor.
Computer system 114A may also include a main memory 11406, for
example, random access memory (RAM), and may also include a
secondary memory 11416. Secondary memory 11410 may include, for
example, a hard disk drive 11412, and a removable storage drive
11414. Removable storage drive 11414 may include a floppy disk
drive, a magnetic tape drive, an optical disk drive, a flash
memory, or the like. Removable storage drive 11414 may read from
and/or write to a removable storage unit 11418 in a well-known
manner. Removable storage unit 11415 may include a floppy disk, a
magnetic tape, an optical disk, etc., which may be read by and
written to by removable storage drive 11414. As will be appreciated
by persons skilled in the relevant art, removable storage unit
11418 may include a computer usable storage medium having stored
therein computer software and/or data.
[0190] In alternative implementations, secondary memory 11410 may
include other similar means for allowing computer programs or other
instructions to be loaded into computer system 114A. Such means may
include, for example, a removable storage unit 11422 and an
interface 11420. Examples of such means may include a program
cartridge and cartridge interface (such as that found in video game
devices), a removable memory chip (such as an EPROM, or PROM) and
associated socket, and other removable storage units 11422 and
interfaces 11420 which allow software and data to be transferred
from removable storage unit 11422 to computer system 114A.
[0191] Computer system 114A may also include a communications
interface 11424. Communications interface 524 allows software and
data to be transferred between computer system 114A and external
devices. Communications interface 11424 may include a modem, a
network interface (such as an Ethernet card), a communications
port, a PCMCIA slot and card, or the like. Software and data
transferred via communications interface 11424 may be in the form
of signals, which may be electronic, electromagnetic, optical, or
other signals capable of being received by communications interface
11424. These signals may be provided to communications interface
11424 via a communications path 11426. Communications path 11426
carries signals and may be implemented using wire or cable, fiber
optics, a phone line, a cellular phone link, an RF link or other
communications channels.
[0192] In this document, the terms "computer program medium" and
"computer usable medium" are used to generally refer to media such
as removable storage unit 11418, removable storage unit 11422, and
a hard disk installed in hard disk drive 11412. Computer program
medium and computer usable medium may also refer to memories, such
as main memory 11408 and secondary memory 11410, which may be
memory semiconductors (e.g. DRAMs, etc.).
[0193] Computer programs (also called computer control logic) are
stored in main memory 11408 and/or secondary memory 11410. Computer
programs may also be received via communications interface 11424.
Such computer programs, when executed, enable computer system 114A
to implement different embodiments of the present disclosure as
discussed herein. In particular, the computer programs, when
executed, enable processor device 11404 to implement the processes
of the present disclosure, such as the operations in method 290
illustrated by flowchart 206 of FIGS. 2A and 2D, flowchart 220 of
FIG. 2E, flowchart 226 of FIG. 2I, and flowchart 270 of FIG. 2J
discussed above. Accordingly, such computer programs represent
controllers of computer system 114A. Where an exemplary embodiment
of method 200 is implemented using software, the software may be
stored in a computer program product and loaded into computer
system 114A using removable storage drive 11414, interface 11420,
and hard disk drive 11412, or communications interface 11424.
[0194] Embodiments of the present disclosure also may be directed
to computer program products including software stored on any
computer useable medium. Such software, when executed in one or
more data processing device, causes a data processing device to
operate as described herein. An embodiment of the present
disclosure may employ any computer useable or readable medium.
Examples of computer useable mediums include, but are not limited
to, primary storage devices (e.g., any type of random access
memory), secondary storage devices (e.g., hard drives, floppy
disks, CD ROMS, ZIP disks, tapes, magnetic storage devices, and
optical storage devices, MEMS, nanotechnological storage device,
etc.).
[0195] The embodiments have been described above with the aid of
functional building blocks illustrating the implementation of
specified functions and relationships thereof. The boundaries of
these functional building blocks have been arbitrarily defined
herein for the convenience of the description. Alternate boundaries
can be defined so long as the specified functions and relationships
thereof are appropriately performed.
Example 1: Fabrication of CNT Based Electrochemical Chip for In
Vitro Assays
[0196] In this example, exemplary CNT based electrochemical chips
was fabricated for in vitro assays. First, silicon wafer (p-type
<100>) substrates were cleaned through standard RCA #1 method
(NMOH:H.sub.2O.sub.2:H.sub.2O solution and volume ratio of 1:1:5
respectively). Then, the cleaned substrates were rinsed in
deionized (DI) water and dried by air. A thin layer of SiO.sub.2
with a thickness of about 200 nm was grown by wet oxidation furnace
on the surface of the silicon wafer, as a passivation layer. Nickel
(Ni) catalyst layer for CNT growth with a thickness of about 9 nm
was coated on SiO.sub.2 by E-beam evaporation system at a
temperature of about 120.degree. C. with depositing rate of about
0.1 Angstroms/s. Afterwards, Ni-covered samples were located in a
direct current plasma enhanced chemical vapor deposition (DC-PECVD)
system to grow vertically aligned multi-walled carbon nanotubes
(VAMWCNT). The growth process has three steps, including annealing,
graining and growth. At first, the sample was annealed at a
temperature of about 680.degree. C. in an H.sub.2 environment with
a flow rate of about 35 standard cubic centimeters per minute
(sccm) for about 30 minutes. During the graining, the surface was
plasma hydrogenated for about 5 minutes with the intensity of about
5.5 Wcm.sup.-2 which results in the catalyst graining and formation
of Ni nano-sized islands. In the growth step a plasma of
C.sub.2H.sub.2 and H.sub.2 mixture with flow rates of about 5 sccm
and about 35 sccm were introduced to the chamber for about 15
minutes. Finally, CNT's were characterized with field emission
scanning electron microscopy (FESEM). The length of CNTs ranged
from about 2.5 to about 5 .mu.m and the diameter of CNTs ranged
from about 50 nm to about 70 nm.
[0197] FIG. 4 shows the FESEM image of the VAMWCNTs army on a
portion of an exemplary fabricated CNT based electrochemical chip,
consistent with one or more exemplary embodiments of the present
disclosure. The CNTs were multi-walled carbon nanotubes with high
purity and a presence of nickel on the top side of the CNTs could
be related to the tip-growth mechanism. The CNT has been used as
the work, counter and reference electrodes in exemplary fabricated
CNT based electrochemical chips. The active area of the work,
counter and reference electrodes were about 100 mm.sup.2, 100
mm.sup.2, 50 mm.sup.2, respectively. The CNT based electrochemical
chips were connected to a potentiostat by conductive wires bonded
to the pads of the potentiostat.
Example 2: Fabrication of Cancer Diagnostic Probe (CDP) for In Vivo
Assays
[0198] In this example, the tips of sterile steel needles were
coated by Ni catalyst layers similar to that was described in
EXAMPLE 1 for CNT based electrochemical chips with the assistance
of E-Beam coating system. A fixture was designed and fabricated to
hold the needles both in E-Beam and DC-PECVD systems to limit the
growth of CNTs just in the tips of the needles. Then, the CNT grown
needles were attached to electrical connectors with three pins by a
conductive paste. Just tips of the needle were extended from the
connectors up to about 1 cm. The probe was reinforced with a
homemade holder and connected to a readout system by a noiseless
cable which handled all three electrodes.
[0199] FIGS. 5A-5D show FESEM images of a tip of a needle electrode
of an exemplary fabricated cancer diagnostic probe (CDP) coated
with an array of VAMWCNTs on the tip and exemplary portions 501,
502 and 503 of the tip, consistent with one or more exemplary
embodiments of the present disclosure. FIG. 5A illustrates a FESEM
image of the tip of a needle electrode of an exemplary fabricated
cancer diagnostic probe (CDP) coated with the array of VAMWCNTs on
the tip, consistent with one or more exemplary embodiments of the
present disclosure. FIG. 5B illustrates a FESEM image of portion
501 of the tip, consistent with one or more exemplary embodiments
of the present disclosure. FIG. 5C illustrates a FESEM image of
portion 502 of the tip, consistent with one or more exemplary
embodiments of the present disclosure. FIG. 5D illustrates a FESEM
image of portion 503 of the tip, consistent with one or more
exemplary embodiments of the present disclosure.
Example 3: CV of H.sub.2O.sub.2 Contained Lactate Solution
[0200] In this example, the cyclic voltammetry (CV) diagram of
L-lactic acid solution individually were recorded by exemplary
electrochemical sensors including working electrodes (WEs)
fabricated from platinum (Pt), Gold (Au), amorphous glassy carbon
(GC) and carbon nanotube (CNT).
[0201] FIG. 6A shows the CV diagrams of L-lactic acid solution
individually recorded by electrochemical sensors fabricated from
platinum (Pt) (curve 620), Gold (Au) (curve 630), amorphous glassy
carbon (GC) (curve 640), and carbon nanotube (CNT) working
electrodes (WEs) (curve 650), consistent with one or more exemplary
embodiments of the present disclosure. It may be observed that the
detected cathodic peak by CNT WE was so sharper (about 1500 .mu.A)
in similar concentration of H.sub.2O.sub.2 with respect to other
electrodes (about 717, 5.7 and 0.8 .mu.A in Au, Pt and GC
electrodes, respectively). CNT greatly transfer the released
charges from oxidized H.sub.2O.sub.2 beneath the nanotubes in media
solution. Hence, CNT arrays were used as electrodes of exemplary
sensors in the present disclosure.
[0202] FIG. 6B shows the CV diagrams of solutions with various
concentrations of lactate (and subsequently H.sub.2O.sub.2)
recorded by electrochemical sensors with CNT arrays working
electrode, consistent with one or more exemplary embodiments of the
present disclosure. CV diagrams were recorded for solutions with a
lactate concentration of about 0.025 mM (CV diagram 602), 0.05 mM
(CV diagram 604), 0.1 mM (CV diagram 606), and 0.3 mM (CV diagram
608). CNT working electrode presented a well concentration depended
increased response to the presence of lactate molecules in the
solutions ranged from about 0.025 mM (CV diagram 602) to about 0.3
mM (diagram 608).
[0203] FIG. 6C shows the CV diagrams of H.sub.2O.sub.2 contained
lactate solution with a lactate concentration of about 0.3 mM (CV
diagram 614) in comparison with two cell culture solutions RPMI (CV
diagram 610) and DMEN (CV diagram 612) recorded by electrochemical
sensors with CNT arrays working electrode, consistent with one or
more exemplary embodiments of the present disclosure. It may be
observed that RPMI and DMEN cell culture solutions show less
electrochemical responses in comparison with H.sub.2O.sub.2
contained lactate solution. The RPMI presented no electrochemical
responses in the voltage attributed to the lactate detection. As a
result, RPMI could be applied as cellular and tissue culture media
with a negligible false positive response.
Example 4: Electrochemical Responses of Different Cell Lines
[0204] In this example, electrochemical sensing of H.sub.2O.sub.2
produced during Lactate/Pyruvate hypoxic glycolysis was verified in
four different phenotypes of breast cell lines ranged from normal
to malignant stages, including: MCF10 A, MCF-7, MDA-MB-231, and
MDA-MB-468. Breast cancer cell lines (MCF10A, MCF-7, MDA-MB-231,
MDA-MB-468) were obtained and were maintained at 37.degree. C. (5%
CO.sub.2, 95% air) in RPMI medium supplemented with 5% fetal bovine
serum, and 1% penicillin/streptomycin. The fresh medium was
replaced every other day. All cell lines were tested and found
negative for Mycoplasma contamination. The cells were detached from
the plates by trypsin and counted by neobar laam.
[0205] FIG. 7 shows the CV responses of normal (MCF10A: CV diagram
792) and different grades of cancerous (MCF7: CV diagram 706,
MDA-MB231: CV diagram 705, and MDA-B468: CV diagram 710) breast
cells' solution media cultured for about 48 hours in comparison
with standard H.sub.2O.sub.2 contained lactate solution with a
lactate concentration of about 0.3 mM (CV diagram 712) and RPMI (CV
diagram 704) in individual sensing wells of exemplary fabricated
sensor in EXAMPLE 1 herein above, consistent with one or more
exemplary embodiments of the present disclosure. Lactate production
due to hypoxic glycolysis would be well detectable after about 48
hours of incubation in cancer cell lines. The CV diagrams of FIG. 7
show that the intensity of oxidation peak, located at the position
of H.sub.2O.sub.2 electrochemical response, significantly increased
with the progression in invasive grades of cancer cells in which
hypoxia glycolysis would be enhanced.
[0206] Referring to FIG. 6B and FIG. 7, sharp difference in
electrochemical peaks of H.sub.2O.sub.2 contained lactate solution
was observed from about 0.025 mM to about 0.05 mM which could be
applied to calibrate cancer cells' media from normal ones. Because
the electrochemical responses of cancer cells' media solution was
equal to the response range of H.sub.2O.sub.2 contained lactate
solution with the concentration of more than about 0.05 mM
meanwhile such response in normal cells was equal to the response
range of the H.sub.2O.sub.2 contained lactate solution with the
concentration of less than about 0.025 mM.
[0207] Moreover, similar responses were recorded from the culture
media of colon, prostate, liver, lung, mouth, neural and
hematopoietic cell lines in normal and cancer phenotypes with
invasive and moderate grades by electrochemical sensing of
H.sub.2O.sub.2 produced during Lactate/Pyruvate hypoxic glycolysis
for some other types of colon, neural, prostate, liver, mouth,
hematopoietic and lung cell lines. Colon (COR-L 105, SW-480,
HT-29), Hematopoietic (1301, LCL-PI 1), Liver (HEP G2), Lung
(QU-DB, MRC-5), Mouth (KB), Neuron (BE(2)-C, LAN-5), Prostate
(PC-3, Du-145) cell lines were obtained and were maintained at
37.degree. C. (5% CO.sub.2, 95% air) in RPMI medium supplemented
with 5% fetal bovine serum, and 1% penicillin/streptomycin. The
fresh medium was replaced every other day. All cell lines were
tested and found negative for Mycoplasma contamination. The cells
were detached from the plates by trypsin and counted by neobar
laam.
[0208] FIGS. 8A-8G shows the CV responses of the solution media of
different normal and cancerous cell lines in various phenotypes
including Colon (COR-L 105 802, SW-480 803, HT-29 804) in FIG. 8A,
Hematopoietic (1301 805, LCL-PI 1 806) in FIG. 8B, Liver (HEP G2
807) in FIG. 8C, Lung (QU-DB 808, MRC-5 809) in FIG. 8D, Mouth (KB
810) in FIG. 8E, Neuron (BE(2)-C, LAN-5) in FIG. 8F, and Prostate
(PC-3 813, Du-145 814) in FIG. 8G cell lines in comparison with
Reference diagram $01 for H.sub.2O.sub.2 contained lactate solution
with a lactate concentration of about 0.3 mM, consistent with one
or more exemplary embodiments of the present disclosure. The
current peaks in cancerous samples were observably increased. The
H.sub.2O.sub.2 based oxidative peaks of cancer media solutions were
sharper than that in normal cells. Grade dependent increase was
observed in H.sub.2O.sub.2 peaks of cancer cells with sharp
difference between normal and cancer phenotypes in all of the cell
lines. This reveal the increased hypoxia glycolysis in cancer cells
with respect to that in normal cells. A great correlation was
observed between the cells' phenotypes and their lactate based
H.sub.2O.sub.2 electrochemical responses.
Example 5: In Vitro Diagnosis of Cancer in Samples by
Electrochemical Tracking of Hypoxia Glycolysis in Secretion of the
Samples
[0209] In this example, the electrochemical responses of 6 breast
tissues removed by biopsy (core needle biopsy (CNB)) or surgery
from 6 of suspicious patients to cancer were analyzed using
exemplary CNT based electrochemical chip. The size of the removed
samples was similar (with the non-dehydrated weight of about 25
mg). The electrochemical responses were compared with
cytopathological analysis done by Hematoxylin and Eosin (H & E)
staining of the 6 breast tissues. Each resected sample was
maintained in RPMI for about 24 hours before analyzing by exemplary
CNT based electrochemical chip to be ensured from the lactate
release in hypoxic tumors. Before pathological assaying, each
resected sample was fixed in Formaline. For electrochemical
analysis of hypoxia glycolysis in secretion of the samples, live
spices from CNB or surgically removed samples were cut in similar
specimens and directly transferred through sensing wells of
exemplary CNT based electrochemical chip containing RPMI-1640
without any preprocessing. About 24 hours after maintaining the
samples in incubator, about 200 .mu.l of the culture media was
dropped to individual sensing wells and the cathodic current of
electrochemical responses of H.sub.2O.sub.2 were recorded in CV
profile.
[0210] FIGS. 9A-9F show the cytopathological results (H&E
images) (top side) and electrochemical responses (bottom side) of
the breast tissues removed by biopsy or surgery from 6 suspicious
patients to cancer, consistent with one or more exemplary
embodiments of the present disclosure. The electrochemical
responses were calibrated based on the reference H.sub.2O.sub.2
contained lactate solution with a lactate concentration of about
0.3 mM as used for cell lines in EXAMPLE 4 above. The intensity of
oxidation peak and released electrons strongly correlated to the
lactate produced by hypoxia glycolysis in cancer cells. A well
correlation could be observed between increased H.sub.2O.sub.2
dependent current peak and cancer transformed morphology of the
tissues. A great match observed between the quantified
electrochemical response and pathological result of the samples in
which the normal and hyperplasic tissues expressed low levels of
H.sub.2O.sub.2 related current peak meanwhile the cancerous tissues
exhibited high levels of H.sub.2O.sub.2 related electrochemical
peaks. Accordingly, FIGS. 9A-9C show results obtained from
non-cancerous samples and FIGS. 9D-9F show results obtained from
cancerous samples.
[0211] Similar electrochemical responses of s more samples,
including live spices from CNB or surgically removed samples, were
obtained using exemplary CNT based electrochemical chip. FIG. 10
shows a columnar diagram of electrochemical responses of the breast
tissues removed by biopsy or surgery from 11 suspicious patients to
cancer, consistent with one or more exemplary embodiments of the
present disclosure. Referring to this figure, two regimes 1002 and
1003 of responses were achieved due to the trace of hypoxia
glycolysis based on LADH in comparison with a reference state 1001
of a H.sub.2O.sub.2 contained lactate solution with a lactate
concentration of about 0.3 mM. In regime 1 indicated by 1002, the
oxidation peaks were ranged from about 933.9 .mu.A to about 1068.9
.mu.A, and in regime 2 indicated by 1003, the oxidation peaks were
ranged from about 269.6 .mu.A to about 718 .mu.A. The pathological
results showed a well correlation with this determination. The
samples presented high levels of hypoxia related oxidative peaks
(categorized in regime 1) were verified as cancer in their H&E
assays. Nests of distinguished tumoral cells in H&E images of
those patients could be observed in FIGS. 9D-9F. Such responses
were observed in the H.sub.2O.sub.2 contained lactate solution with
the concentration of more than about 0.05 mM (FIG. 6B). Samples
with low levels of lactate (regime2) were diagnosed as non-cancer
with different types of benign cancer patients such as hyperplasia
(peak: 556.5 .mu.A) in FIG. 9B, lactational changes (peak: 718
.mu.A), and so on. These electrochemical responses were equal to
the peak determined in H.sub.2O.sub.2 contained lactate solution
with the concentration of less than about 0.025 mM (FIG. 6B).
Comparative columnar diagram presented in FIG. 10 would elaborate
the difference in lactate based electrochemical response between
normal and cancer tissues.
Example 6: Standard Colorimetric Lactate Assay Kit
[0212] As the released H.sub.2O.sub.2 concentration have a direct
correlation with lactate concentration, to further investigate the
accuracy of exemplary electrochemical method described above, the
results of both cell lines (described in EXAMPLE 4) and patients'
samples (described in EXAMPLE 5) were compared by standard
colorimetric lactate assay kit. Although this method is so time
consuming and expensive with complicated multi sequential steps, it
was conducted to check the reliability of lactate concentration
based cancer diagnosis measured by exemplary CNT based
electrochemical chip. Comparative responses versus reference
H.sub.2O.sub.2 contained lactate solution for both electrochemical
and Lactate Kit assays are presented in Table 1 and Table 2.
TABLE-US-00001 TABLE 1 Comparative responses of CNT based
electrochemical chip and standard Lactate Kit Assay on 4 different
phenotypes of Breast cell lines. Electrochemical sensor: Lactate
kit: Relative Cell line Relative Current (%) Lactate Concentration
(%) Reference lactate 100 100 solution MCF 10A 31.1 31.1 MCF-7 52.9
56.3 MDA-MB-231 69.5 70.9 MDA-MB-468 91.5 91.5
TABLE-US-00002 TABLE 2 Diagnostic results of 11 patients suspicious
to breast cancer determined by H&E, Lactate kit, and the
cathodic peaks of released H.sub.2O.sub.2 from the cells measured
by CNT based electrochemical chip assays, respectively. Patient
Lactate Kit CNT Electrochemical ID Type of Tissue H & E Result
Result (%) Sensor (%) Reference lactate -- -- 100 100 solution 1
Normal Non Cancer 22.2 22.2 2 Normal Left Hyper Plasy 42.3 42.3 3
Normal Lactational Change 59.1 59.2 4 Normal Adenosis benign
glandular 53.8 53.7 prolifration 5 Normal Hyperplasy and
inflammation 45.9 45.9 6 Suspicious to Cancer Lympho vascular
invasion 88.1 88.2 7 Suspicious to Cancer Cancer 77.2 77.2 8
Suspicious to Cancer Cancer 85.5 85.4 9 Suspicious to Cancer Cancer
80.5 80.5 10 Suspicious to Cancer Cancer 85.1 85.2 11 Suspicious to
Cancer Cancer 62 62.2
[0213] A correlation was observed between the responses of the CNT
based electrochemical chip and kit which revealed the accuracy of
CNT based electrochemical chip in lactate based cancer detection as
shown in Table 1 and Table 2. The raw values recorded by Lactate
kit and electrochemical sensing welts were presented in these
tables. In summary, tracing the hypoxia glycolysis (correlated with
lactate concentration) in the interstitial fluid of biopsy sample
by electrochemical assay with suitable electrode (such as CNT)
exhibited a high correlation with their pathological states and may
be used as a new method in cancer diagnosis.
Example 7: Integrated Assay on the Tip of the Needles of Cancer
Diagnostic Probe (CDP) for Real-Time Cancer Detection Both In Vitro
and In Vivo
[0214] To extend the application of exemplary label free
electrochemical method of the present disclosure in real-time and
precise detection of the tumor tissues during interventional
sonography or surgery, exemplary CDP fabricated by growth of carbon
nanostructures on the tip of the sterile steel needles as described
in EXAMPLE 2 was used herein. Such integrated system contains three
carbonated needles as working electrode (WE), counter electrode
(CE), and reference electrode (RE). The needles were rinsed by PBS,
Ethanol 70% and DI water followed by drying in N.sub.2 ambient and
UV sterile to prevent from any detachment and remaining the
residues of the nanotubes in the tissue. CV responses were taken
immediately after squeeze of exemplary CDP into the breast
cancerous and normal tissues that indicated sharp increase in the
current peak of the CDP with CNT covered electrodes interacted by
cancer tissue. The important point is that the diagnosis was
completed in live time based on monitoring the lactate
concentration of the tissues inner domain.
[0215] FIG. 11A shows CV response of exemplary CDP with all three
needles covered by VAMWCNTs immediately after connection to the
tissues, consistent with one or more exemplary embodiments of the
present disclosure. It may be observed that reversible shapes with
symmetric anodic and catholic peaks were obtained in CV responses.
Distinguishable response between normal and cancer tissues may be
observed.
[0216] FIG. 11B shows CV response of exemplary CDP with only
working electrode covered by VAMWCNTs immediately after connection
to the tissues, consistent with one or more exemplary embodiments
of the present disclosure. Quality of the peaks were degraded when
replacing the RE and CE by steel needle. The intensity and symmetry
of the responses were degraded in the sensor with just CNT covered
WE (CE and RE were steel needles).
[0217] FIG. 11C shows CV response of exemplary CDP with non-CNT
covered by needles immediately after connection to the tissues,
consistent with one or more exemplary embodiments of the present
disclosure. There may be observed a noisy response without any
distinguishable electrochemical peak. When all of the electrodes
were non CNT covered needles, the responses were completely
degraded and not distinguishable between normal and cancer tissues.
This revealed the important role of CNT in selective interaction
and charge transfer from the H.sub.2O.sub.2 released during
transformation of lactate to pyruvate.
[0218] FIGS. 12A-12E show CV responses recorded by exemplary CDP
(needle based electrochemical sensor) from the resected tissues
from five patients among 50 individual patients suspicious to
breast cancer at the bottom side of FIGS. 12A-12E in comparison
with images obtained by conventional pathological methods (H&E)
at the top side of FIGS. 12A-12E, consistent with one or more
exemplary embodiments of the present disclosure. Patients
respective to FIGS. 12A-12C have normal/non-cancerous (FIG. 12A),
hyperplasic (FIG. 12B), and adenosis glandular proliferative
tissues (FIG. 12C). Patients respective to FIGS. 12D and 12E have
cancer tissues. Electrochemical current peaks of cancer tissues are
sharply (more than about 150 .mu.m) higher than benign ones with a
strong correlation by the abundance of distributed cancer cells.
The CV responses recorded from the normal and cancer tissues of
these 50 individual patients by CNT covered needle sensors (CDP)
greatly detected the hypoxic glycolysis just in cancerous samples
due to cathodic peaks of H.sub.2O.sub.2 (FIGS. 12A-12E). Meanwhile,
while the response time of CDP was less than about 1 minute after
the tissue resection, diagnosis by conventional pathological
methods (H&E) requires at least several hours for sample
fixation and staining procedures. The ratio of cathodic peaks of
reference H.sub.2O.sub.2 contained lactate solution vs.
non-cancerous tissues were more than three times (FIGS. 12A-12C)
while such ratio was less than one time in cancerous tissues (FIGS.
12D and 12E).
[0219] Table 3 shows the results recorded by exemplary CDP in
comparison with the results obtained by H&E analysis from live
resected tissues of 50 patients suspicious to breast cancer. They
exhibited great correlations with the pathological results of the
samples assayed by H&E method.
TABLE-US-00003 TABLE 3 Results recorded by exemplary CDP in
comparison with the results obtained by H&E analysis from live
resected tissues of 50 patients suspicious to breast cancer.
Oxidation Patient Current Peaks CDP H&E ID (.mu.A) (Cancer)
(Cancer) 1 0 Negative NO 2 46.6 Negative NO 3 87 Negative NO 4
316.5 Positive YES (Cancer 90%, Normal 10%) 5 287.8 Positive YES
(Cancer 90%, Normal 10%) 6 22 Negative NO 7 142.3 Positive YES
(Cancer 30%, Normal 70%) 8 150 Positive YES (Cancer 30%, Normal
70%) 9 300 Positive YES (Cancer 90%, Normal 10%) 10 13 Negative NO
11 0 Negative NO 12 101.5 Positive YES (Cancer 30%, Normal 70%) 13
180.2 Positive YES (Cancer 60%, Normal 40%) 14 289.4 Positive YES
(Cancer 90%, Normal 10%) 15 302 Positive YES (Cancer 90%, Normal
10%) 16 274.2 Positive YES (Cancer 90%, Normal 10%) 17 0 Negative
NO 18 142.8 Positive YES (Cancer 30%, Normal 70%) 19 80 Negative NO
20 32.5 Negative NO 21 200 Positive YES (Cancer 60%, Normal 40%) 22
188.2 Positive YES (Cancer 60%, Normal 40%) 23 264.5 Positive YES
(Cancer 90%, Normal 10%) 24 23 Negative NO 25 179.5 Positive YES
(Cancer 60%, Normal 40%) 26 55.2 Negative NO 27 52 Negative NO 28
77 Negative NO 29 0 Negative NO 30 201 Positive YES (Cancer 60%,
Normal 40%) 31 75.6 Negative NO 32 155.8 Positive YES (Cancer 30%,
Normal 70%) 33 99.5 Positive YES (Cancer 30%, Normal 70%) 34 305.5
Positive YES (Cancer 90%, Normal 10%) 35 297.7 Positive YES (Cancer
90%, Normal 10%) 36 112 Positive YES (Cancer 30%, Normal 70%) 37
17.8 Negative NO 38 40 Negative NO 39 73 Negative NO 40 290
Positive YES (Cancer 90%, Normal 10%) 41 90 Positive YES (Cancer
30%, Normal 70%) 42 330 Positive YES (Cancer 90%, Normal 10%) 43
197.5 Positive YES (Cancer 30%, Normal 70%) 44 77.5 Negative NO 45
25 Negative NO 46 266.2 Positive YES (Cancer 90%, Normal 10%) 47 0
Negative NO 48 102.5 Positive YES (Cancer 30%, Normal 70%) 49 310
Positive YES (Cancer 90%, Normal 10%) 50 259.3 Positive YES (Cancer
90%, Normal 10%)
[0220] FIG. 13 shows a summary of categorized regimes of CV
responses recorded by exemplary CDP from the resected tissues from
five patients among 50 individual patients suspicious to breast
cancer that were presented in Table 3. It represents CV regimes
along a spectrum from a completely non-cancerous state to cancerous
state, consistent with one or more exemplary embodiments of the
present disclosure. It may be seen that if the H.sub.2O.sub.2
cathodic peak (equal to oxidation current peak) of exemplary CV
response recorded by exemplary CDP from a patient is less than 90
.mu.A, the tissue is in non-cancerous state. On the other hand, if
the CV response recorded by exemplary CDP from the tissue is in a
range more than about 95 .mu.A, there exists a cancerous state
which may be more intensified by increasing the oxidation current
peak. A range of oxidation current peak between 90 .mu.A and 95
.mu.A is the border range.
Example 8: In Vivo Analysis of Observable Tumor with Histologically
Distinct Cancer Margin Before any Mastectomy
[0221] To determine if CDP would in real time identify an
observable tumor in vivo, about 2.3.times.10.sup.6 4T1-derived
cancer cells were implanted into the back of 10 female BALB/C mice,
and the mice were maintained in individual groups with similar size
of formed tumors with sharp histologically distinct patterns. After
about 10 days, individual CDPs were externally squeezed into their
cancerous and normal regions had been specified by sonography. The
space between each assayed regions was about 3 mm. Also the mice
under body were connected to ground potential such as done for any
patient in surgery room.
[0222] FIG. 14A shows a sonography image from a tumor side taken
from an exemplary mouse tumorized by 4T1 breast cancer cell lines,
consistent with one or more exemplary embodiments of the present
disclosure. A tumor with average sizes with a length 1402 of about
2.24 cm and another length 1464 of about 1.60 cm could be observed
in sonography image.
[0223] FIG. 14B shows H&E image from the tumor side taken from
exemplary tumorized mouse by 4T1 breast cancer cell lines,
consistent with one or more exemplary embodiments of the present
disclosure. FIG. 14C shows H&E image from a normal/healthy side
taken from exemplary tumorized mouse by 4T1 breast cancer cell
lines, consistent with one or more exemplary embodiments of the
present disclosure.
[0224] FIG. 14D shows CV diagrams of normal and tumor regions/sides
of exemplary tumorized mouse by 4T1 breast cancer cell lines
calibrated by a Reference CV diagram from H.sub.2O.sub.2 contained
lactate solution with a lactate concentration of about 0.3 mM
obtained using exemplary CDP, consistent with one or more exemplary
embodiments of the present disclosure. It may be observed that the
lactate related peaks were about 3 times higher in cancer region
(CV diagram 1466 and FIGS. 14A and 14B) versus normal ones (CV
diagram 1408 and FIG. 14C). Sharp lactate electrochemical peaks
were observed in tumor locations by about three times higher
current than that recorded from their normal regions
[0225] To more clearly clarify the impact of H.sub.2O.sub.2
monitoring in tumor growth and progression, tumor size effects on
H.sub.2O.sub.2 related electrochemical peaks recorded by CDP were
compared. A distinguishable increasing regime was observed in the
intensity of current peak through increment in the tumor size.
Moreover, Histopathological images taken from the normal and cancer
regions detected by CDP confirmed this result. Hyper chromatic and
irregular nucleus with increased nucleus/cytoplasm ratio may be
observable in H&E images of cancer region.
Example 9: In Vivo Analyses of Suspicious Regions Before and During
the Surgery
[0226] In this example, the ability of the CDP to distinguish
suspicious regions to cancer in mice model was analyzed by the
resolution of about 3 mm which could be translated to human model.
Tissue samples that contained regions of invasive breast cancer
adjacent to normal stroma were experimented.
[0227] Five tumorized mice by 4T1 breast cancer cell lines were
checked by sonography. FIG. 15A shows a sonography image from a
tumor 1501 taken from an exemplary mouse tumorized by 4T1 breast
cancer cell lines, consistent with one or more exemplary
embodiments of the present disclosure. Approximate dimensions of
the tumor could be observed in sonography image of FIG. 15A.
[0228] Exemplary CDP was tested on tumor and suspicious regions of
the five tumorized mice before (by squeezing through skin) and
during the surgery on exemplary six regions. FIGS. 15B and 15C show
exemplary six analyzed regions 1502-1507 of an exemplary tumorized
mouse among the exemplary five tumorized mice before (FIG. 153) and
during surgery (FIG. 15C), consistent with one or more exemplary
embodiments of the present disclosure. Six analyzed regions may
include center 1502 of tumor 1501, left side 1503 of tumor 1501,
right side 1504 of tumor 1501, upside 1505 of tumor 1501, bottom
side 1506 of tumor 1501, and also an exemplary normal side 1507 far
from tumor 1501 all depicted in FIGS. 15B and 15C were analyzed
consecutively before and during surgery using a CDP with an about 3
mm distance between needle electrodes.
[0229] Moreover, frozen H&E assay was used and rechecked by IHC
method to be ensure from the precision of CDP results with respect
to standard protocols. A tissue section of the sample including
exemplary six regions analyzed by exemplary CDP was subjected to
frozen H&E staining processes and evaluated by the pathologist.
Exemplary tumor 1501 containing suspicious regions was removed and
sent for frozen pathology and the H&E images taken from the
center 1502 of tumor 1501 as well as its posterior 155, anterior
1506, right 1504 and left 1503 laterals with the distance of about
3 mm from the histologically distinct region, were demanded by the
CDP results as presented in Table 4. The results of CDP before and
during surgery exhibited a perfect correlation. Ki67 based IHC
assay confirmed the normal state of R5 and cancer involvement in R3
as classifier reference.
TABLE-US-00004 TABLE 4 Results recorded by exemplary CDP before and
during surgery in comparison with the results obtained by H&E
analysis from six suspicious regions of an exemplary tumorized
mouse. CDP Before Surgery CDP During Surgery H&E frozen Region
(Ox Current Peaks (.mu.A)) (Ox Current Peaks (.mu.A)) (Cancer) 1502
(Center) Positive (169.104) Positive (178.621) Yes (Cancer 90%,
Normal 10%) 1503 (Right) Positive (94.773) Positive (96.89) Yes
(Cancer 30%, Normal 70%) 1504 (Left) Positive (122.643) Positive
(122.8) Yes (Cancer 60%, Normal 40%) 1505 (Up) Negative (30.397)
Negative (31.85) No 1506 (Bottom) Negative (0) Negative (0) No 1507
(Normal) Negative (0) Negative (0) No
[0230] As represented in Table 4, it was distinguished from H&E
analyzes that center 1502 was diagnosed by frozen histopathology as
cancer tissue, whereas regions 1505 and 156 were diagnosed as
normal stroma. Region 1504 was in the margin between the cancer and
normal stroma tissue regions, presenting about 40% tumor tissue and
about 60% normal stroma tissue. Region 1503 was a suspicious region
without any tumor margins but the trace of distributed cancer cells
would be observed between stroma. Tumoral cells would be
distinguished due to their hyper chromic nuclei (triangular arrows
in H&E images of regions 1503 and 1504). Tabled result shows
the CDP obtained for regions 1502 and 1504 presented significant
hypoxic lactate peaks meanwhile lower but detectable meaningful
levels of the H.sub.2O.sub.2 was recorded for region 1503. No
detectable trace of any peak was measured for region 156. The CDP
response obtained for 1507, diagnosed as reference normal stroma
tissue, presented no H.sub.2O.sub.2 peak similar to that observed
for 1506.
[0231] The CDP response obtained for regions 1504 and 1506 were
then evaluated by Ki67 IHC as an independent validation set. The
expression of Ki67 has been reported to be correlated with tumor
cell proliferation and growth in routine pathological investigation
and used as a diagnostic marker. Ki67 based IHC classifier
identified no trace of cancer proliferation in region 1506 (as the
normal stroma) and showed different intensities of filtrated cancer
cells in region 1504. Similar results were obtained for 5 other
animal models with suspicious regions in anterior, posterior, right
and left laterals of their tumors.
[0232] In addition, exemplary CDP exhibited a fine distinguishable
response in interaction with another type of cancer tumors (MC4L2)
as cancer cells with lower invasive grades than 4T1 as experimented
on mice models. Tumors formed by the injection of about
5.times.10.sup.5 MC4L2 cells (mice primary breast cancer cell
lines) were analyzed by exemplary CDP on 5 mice.
[0233] FIG. 16 shows comparative diagram of CDP responses in
interaction with normal (curve 1601), non-metastasized tumor (curve
1602) and metastatic tumor (curve 1603) recorded from individual
mice, consistent with one or more exemplary embodiments of the
present disclosure. Tracing the hypoxia glycolysis exhibited a
strong correlation with the invasive state of the tumor. Results
revealed sharply distinguishable responses between cancerous and
normal regions. However the intensity of the response of MC4L2
tumors is lower than that was recorded for malignant tumor, it is
observably higher than the response peak of normal tissue.
[0234] Furthermore, in this example, exemplary CDP was applied in
finding the suspicious margins during tumor resection surgery in
breast cancer patients. Not only the known normal domains were
detected and set as reference point, but also suspicious margins of
cancer and normal domains were precisely diagnosed in real-time and
confirmed by histopathological assays. So, without requirement to
frozen pathological process the surgeon can finish the surgery with
insurance from precise resection of tumor without any additional
mastectomy from the peripheral tissues.
[0235] FIGS. 17A-17C shows CV responses obtained by applying
exemplary CDP in detection of suspicious margins during breast
cancer surgery for a known normal region (FIG. 17A) that was
checked as calibrating data, and two suspicious margins (FIGS. 17B
and 17C) that were precisely diagnosed as cancerous (FIG. 17B) and
normal (FIG. 17C) domains by CDP in real-time, consistent with one
or more exemplary embodiments of the present disclosure. The
results obtained by exemplary CDP were confirmed by H&E
analysis. FIGS. 17D-17F shows H&E results after the surgery for
the known normal region, and two suspicious margins, consistent
with one or more exemplary embodiments of the present
disclosure.
[0236] These results show that the diagnostic information obtained
by exemplary CDP can be used to detect cancer in marginal
suspicious regions with rare distribution of cancer cells filtrated
between normal stroma in less than about 20 seconds during the
surgery or biopsy of live animal as well as human models without
any requirement to tissue resection and preparation for frozen
pathology. Even it may detect the accurate location of cancer
involved regions before surgery in superficial tumors. The
precision of this method is as well as reported for H&E from
the assayed regions.
Example 10: Pathological Classification of Current Peaks Obtained
by the Exemplary Fabricated CDP
[0237] In this example, current peaks of 258 human fresh samples
prepared from 74 breast cancer patients (Biopsied or surgically
removed) were recorded utilizing exemplary CDP 102. 258 human fresh
samples were tested immediately after dissection from the body
(with the non-dehydrated weight of about 15-25 mg and size of up to
about 1 cm.sup.2. All three integrated needle electrodes of
exemplary CDP 102, assembled on the head probe (the exemplary
sensing part 154), were entered into a target tissue of the 258
human fresh samples at the same time. The whole process which may
include replacing a previously used sensing part 154 by removing
the previously used sensing part 154 and connecting a new sensing
part 154 (about 20 sec), entering exemplary CDP 102 to the target
tissue by inserting new sensing part 154 into a target location
within the target tissue, and recording a CV response with a
current peak from the target location (about 15 sec due to
synchronized real-time processing) would take place in less than
about 40 seconds. The permanent pathological diagnostic results of
the samples, which were re-checked by three histological slides
from each sample, were a set of reference data in probable
scalability of the recorded CV responses.
[0238] Meaningful results were observed after comparing an
experimental categorization of samples through their CDP recorded
current peaks with their categorization through their H&E
pathological diagnoses. Table 5 shows CDP current peak results of
258 fresh samples from 74 patients in association with pathological
diagnosis based on pathological classification (DIN, LIN and FEL).
Additionally, FIG. 18 shows H&E images 1802-1818 from nine
exemplary samples associated with nine exemplary categories in
Table 5, consistent with one or more exemplary embodiments of the
present disclosure.
TABLE-US-00005 TABLE 5 CDP current peak results of 258 fresh
samples from 74 patients in association with pathological diagnosis
based on pathological classification (DIN, LIN and FEL).
Pathological scoring Ranges of Number for re-excising recorded CDP
Number of the matched Pathological Diagnosis recommendation peak
current of tested samples in (classification system) (warning
state) (.mu.A) samples CDP ranges Fatty breast tissue (FEL)
Negative 0-40 15 14 Sclerosing adenosis (DIN) Negative 83-115 11 8
FCC with CCC (DIN) Negative 110-117 19 17 Moderate usual ductal
Negative 117-137 12 10 hyperplasia (DIN) Florid ductal hyperplasia
Negative 150-170 10 8 (DIN) (moderate risk) Stroma + one focus
Negative 175-196 5 4 of ADH (DIN) (moderate risk) Stroma + two or
more foci positive 203-260 31 28 of ADH; DIN1b (DIN) Stroma + one
foci of positive 231-290 24 21 DCIS; DIN1c (DIN) IDC >5%
positive 360 and more 19 19
[0239] It was observed that 26 samples among 258 samples were
normal breast stroma, including fatty breast tissues and simple
fibroadenoma, which all showed current peaks in a range of about
0-83 .mu.A as shown in Table 5. Image 1802 of FIG. 13 shows H&E
image from an exemplary sample including fatty breast tissue (FEL)
as a normal breast tissue with a current peak between about 0 .mu.A
and about 40 .mu.A, consistent with one or more exemplary
embodiments of the present disclosure. 18 samples were non
proliferative fibrocystic changes (FCC) which all of them showed
current peaks in a range of about 53-111 .mu.A. 10 samples were
mild usual ductal hyperplasia (UDH) which all of them showed
current peaks in a range of about 83-110 .mu.A. 11 samples were
sclerosis adenosis (SA) which 8 of them showed current peaks in a
range of about 86-115 .mu.A. Image 1834 of FIG. 18 shows H&E
image from an exemplary sample including sclerosis adenosis (SA)
with a current peak between about 83 .mu.A and about 115 .mu.A,
consistent with one or more exemplary embodiments of the present
disclosure. 12 samples were moderate UDH which 10 of them showed
current peaks in a range of about 120-137 .mu.A. 19 samples were
FCC with columnar cell changes (CCC)(some of them also had one foci
suspicious to atypical ductal hyperplasia (ADH)) which 17 of them
showed current peaks in a range of about 110-173 .mu.A. Image 1896
of FIG. 18 shows H&E image from an exemplary sample including
fibrocystic changes (FCC) with columnar cell changes (CCC) with a
current peak between about 110 .mu.A and about 117 .mu.A,
consistent with one or more exemplary embodiments of the present
disclosure. Image 1808 of FIG. 18 shows H&E image from an
exemplary sample including Moderate usual ductal hyperplasia (DIN)
with a current peak between about 117 .mu.A and about 137 .mu.A,
consistent with one or more exemplary embodiments of the present
disclosure. Image 1810 of FIG. 18 shows H&E image from an
exemplary sample including Florid ductal hyperplasia (DIN) with a
current peak between about 150 .mu.A and about 170 .mu.A,
consistent with one or more exemplary embodiments of the present
disclosure. Image 1812 of FIG. 18 shows H&E image from an
exemplary sample including stroma with one focus of atypical ductal
hyperplasia (ADH) with a current peak between about 175 .mu.A and
about 196 .mu.A, consistent with one or more exemplary embodiments
of the present disclosure. In summary, normal breast (e.g. breast
fatty tissue), UDH (e.g. FCC lesions) and DIN1a (e.g. FCC with CCC
and a small foci of ADH) showed response peak ranges from about 0
.mu.A to about 196 .mu.A which were negatively scored by CDP.
[0240] Moreover, 31 samples showed involvement to two or more foci
of ADH which 28 of them showed peak currents in a range of about
203-250 .mu.A. Image 1814 of FIG. 18 shows H&E image from an
exemplary sample including stroma with two or more foci of ADH
(DIN1b) with a current peak between about 203 .mu.A and about 260
.mu.A, consistent with one or more exemplary embodiments of the
present disclosure. 24 samples showed involvement to low grade
ductal carcinoma in-situ (DCIS), from about 10% to about 45% of the
histological pattern, which 21 of those samples showed current
peaks in a range of about 250-360 .mu.A. Image 1816 of FIG. 18
shows H&E image from an exemplary sample including stroma with
one foci of ductal carcinoma in-situ (DCIS) (DIN1c) with a current
peak between about 231 .mu.A and about 290 .mu.A, consistent with
one or more exemplary embodiments of the present disclosure. 12
samples were invasive ductal carcinoma (IDCs) with distributions
between 5-55% of histological pattern, which all of them showed
current peaks in a range of about 610-800 .mu.A. 2 samples were
phylloids tumor which both showed peak currents more than about 260
.mu.A. Just 10 samples were found with lobular based
atypia/neoplasia; 4 of those samples were invasive lobular
carcinoma (ILC) with extensive distribution which all showed
current peaks in a range of about 380-465 .mu.A. 4 samples were
atypical lobular hyperplasia (ALH) which showed current peaks
between in a range of about 257-270 .mu.A. 2 other samples were
lobular carcinoma in-situ (LCIS) which both showed current peaks of
about 290 .mu.A. Image 1818 of FIG. 18 shows H&E image from an
exemplary sample including invasive ductal carcinoma (IDC) with a
current peak more than about 360 .mu.A, consistent with one or more
exemplary embodiments of the present disclosure.
[0241] Considering the above pathological results and current peaks
of the samples together, a classification on CV responses recorded
by exemplary CDP 162 was proposed based on pathological diagnosis.
The lowest cut-off for pathologists on diagnosing a margin as
positive (dissection is mandatory) is presence of at least two foci
of ADH. Therefore, a border line at about 203 .mu.A was proposed as
a cut-off current for positive scoring exemplary CDP 102. Samples
consisting of ADH with more than two foci, DCIS and IDCs most often
showed peak currents higher than about 203 .mu.A while other
samples consisting of one foci of ADH, UDH and FCC lesions showed
peak currents lower than about 196 .mu.A. Hence, the responses of
exemplary CDP 102 were found to be classifiable based on newest
edition of ductal intraepithelial neoplasia (DIN), lobular
intraepithelial neoplasia (LIN) and fibro-epithelial lesion (FEL)
systems reported by world health organization (WHO). FIG. 19 shows
classification of current peaks recorded by exemplary CDP 102 after
examining more than 250 samples in consistence with pathological
diagnosis, consistent with one or more exemplary embodiments of the
present disclosure. It may be observed that samples with positively
scored levels of glycolytic related H.sub.2O.sub.2 peaks were
verified as ADH. DCIS, IDC and metastatic lymph nodes in their
H&E assays showed more than about 203 .mu.A (as the cut-off) in
CDP response peaks. Samples with current peaks lower than about 196
.mu.A were negatively scored by CDP as neither cancerous nor
pre-cancerous tissue with different types of benign states such as
usual ductal hyperplasia, adenosis, fibrosis and non-proliferative
fibrocystic changes. Most of the abnormal samples were found in DIN
classification (as the mostly occurred types of breast diseases
such as IDC, DCIS, ADH and UDH). CDP peak responses were scored
with a defined cut-off between free and involved margins (at 203
.mu.A), respectively assigned as "Negative" and "positive". Such
scoring would consider any pathological involvement to atypical,
pre-invasive and invasive lesions in margin checking. Also, a
warning regime in negative regions was defined in this
classification. These samples with current peaks between about 137
.mu.A and about 196 .mu.A may be classified in this region.
Surgeons need to be aware on these lesions through presence of
complex fibroadenoma, complex SA or single focus of atypia.
Although these lesions are not pathologically high risk
pre-neoplasia, they ought to be reported and considered through
some guidelines. DIN1b (e.g. two or more foci of ADH), DIN1c (e.g.
low grade-DCIS), DIN2 (e.g. intermediate DCIS), DIN3 (e.g. high
grade DCIS) and IDC lesions showed response peaks in the ranges
between 203 to more than 600 .mu.A which were positively scored by
CDP. Based on this example, pathologically validated diagnostic
scores of exemplary CDP 102 were defined for 258 breast tissue
samples with sensitivity of about 95% and specificity of about 92%
respectively. Such results may be achieved by CNT covered sensing
needles of exemplary CDP 102 which may provide selective
interaction with released H.sub.2O.sub.2 from abnormal tissues with
no post recording perturbation on morphology and distributions of
the sterilized CNT-covered needles.
Example 11: Real-Time In-Vivo Scoring of External Margins (EMs) and
Internal Margins (IMs) by CDP During Breast Cancer Surgery in Human
Models
[0242] In this example, exemplary prepared CDP 102 was utilized
through exemplary method 200 as a real-time diagnostic tool to find
involved body side margins (named IMs) during human cancer surgery.
In this regard, exemplary prepared CDP 102 was applied in real-time
finding of suspicious IMs and EMs during lumpectomy and/or
mastectomy of 127 patients with different types of breast tumors in
different steps of treatment. 14 patients were excluded from the
survey and 113 patients (107 female and 6 male) were included. All
of the tests were done under the license of Ethics Committee with
the informed consent of candidate patients. The sensing needles
(i.e., exemplary working electrode 158, counter electrode 160 and
reference electrode 162) were entered into the margins up to a
depth of about 4 mm and a stopping specimen was embedded on
exemplary head 166 to prevent from further entrance of the needles.
A distance between the exemplary three electrodes was about 3 mm.
Hence, more than about 30 mm.sup.3 of exemplary portion of breast
tissue was exposed to exemplary CDP 102 during each test. Depending
on a surgeon's opinion, up to 12 breast margins (6 EMs on dissected
tumor and 6 IMs in cavity side of a patient (including posterior,
anterior, superior, inferior, medial and lateral) were
intraoperatively checked by exemplary CDP 102 for each patient. The
EMs on dissected tumor were diagnosed (scored) by CDP and the
tested lesions were sent for frozen pathology. Subsequently, the
IMs in cavity side (in the body of the patient) were checked by
exemplary CDP 102 and similarly the tested lesions were considered
by standard frozen pathology. Finally, all of EMs and IMs were
diagnosed (scored) by exemplary CDP 162 and tested by frozen
pathology, were individually evaluated by permanent pathology as
reference standard diagnosis based on pathological classifications
of breast tumors. Also, when a permanent histological pattern was
suspicious for pathologist between two different diagnoses (e.g.
UDH and ADH), IHC would be recommended by her/him. Totally, 895
individual EMs and IMs were intra operatively scored by CDP and
diagnosed by pathology.
[0243] FIG. 20A shows an image resulted from frozen H&E
(top-side image 2002), an image resulted from permanent H&E
(middle-side image 2004), and a CV response recorded by exemplary
CDP 102 (bottom-side image 2006) for the anterior IM of a patient
(ID 18), consistent with one or more exemplary embodiments of the
present disclosure. It may be observed that real-time CV response
recorded by exemplary CDP 102 positively scored the margin, and
both frozen and permanent H&E confirmed involvement of invasive
ductal carcinoma for this patient.
[0244] FIG. 20B shows an image resulted from frozen H&E
(top-side image 2006), an image resulted from permanent H&E
(middle-side image 2010), and a CV response recorded by exemplary
CDP 102 (bottom-side image 2012) for a suspicious margin inside the
body of the patient (anterior margin of patient ID 46), consistent
with one or more exemplary embodiments of the present disclosure.
It may be observed that real-time CV response recorded by exemplary
CDP 102 positively scored the margin, and the removed specimen
showed negative result for malignancy in frozen analyses meanwhile
the permanent H&E showed the papillary lesion with Atypia
region, which must be removed by surgeon. This example may show the
significant role of using exemplary CDP 102 for high-accurate
cancer diagnosis.
[0245] FIG. 26C shows an image resulted from frozen H&E
(top-side image 2014), an image resulted from permanent H&E
(middle-side image 2016), and a CV response recorded by exemplary
CDP 102 (bottom-side image 2018) for a suspicious margin inside the
body of the patient (posterior IM of the same patient ID 46),
consistent with one or more exemplary embodiments of the present
disclosure. It may be observed that real-time CV response recorded
by exemplary CDP 102 negatively scored the margin, which was
confirmed by both frozen and permanent H&E as usual
hyperplasia.
[0246] FIG. 20D shows an image resulted from frozen H&E
(top-side image 2020), an image resulted from permanent H&E
(middle-side image 2022), and a CV response recorded by exemplary
CDP 102 (bottom-side image 2024) for Sentinel Lymph Node (SLN) of
patient ID 18, consistent with one or more exemplary embodiments of
the present disclosure. It may be observed that real-time CV
response recorded by exemplary CDP 102 negatively scored the
margin, whereas it was diagnosed as reactive lymphoid hyperplasia
by both H&E assays.
[0247] Regarding EXAMPLEs 10 and 11 described hereinabove, a
matched clinical diagnostic categorization between the pathological
results of the tested tissues and response peaks obtained by
exemplary CDP 102 was proposed based on pathological classification
(ductal intraepithelial neoplasia (DIN), lobular intraepithelial
neoplasia (LIN) and fibro epithelial lesion (FEL)) with the latest
reported modifications. CDP scoring ability in intra-operative
margin detection was verified on more than 890 human in-vivo
clinical breast samples with sensitivity of about 97% and
selectivity of about 94%. The ability of exemplary CDP 102 and
exemplary method 200 in non-invasive and real-time diagnosis of
internal margins with pathological values (from high-risk benign to
pre-invasive and invasive cancer lesions) may make exemplary CDP
102 a distinct intra-operative tool with simple and small handheld
equipment to increase the prognostic factor of the cancer
patients.
Example 12: Real-Time Tracking of Hypoxia Glycolysis in Conization
Sample for Cervical Intraepithelial Neoplasia (CIN) Detection
[0248] In this example, exemplary methods 200 and 220 utilizing
exemplary CDP 102 were applied to precisely diagnose the cervical
intraepithclial neoplasia (CIN) cells in cone biopsy samples in
real-time in order to improve pathological evaluations to find any
missed CIN (I to III) or other high-risk dysplasia in cone biopsy
samples. Electrochemical assays according to method 220 utilizing
CDP 102 were carried out on in-vitro human fresh cervical samples
prepared from 30 patients' candidate for conization through the
history of abnormal cells present in their pap smear results. CV
studies were done using DC voltage, and no AC frequency was
applied. The potential was swept in the range from about -0.8 to
about +0.8 V, using a scan rate of about 100 mV s.sup.-1.
Hematoxylin and Eosin (H&E) staining was used as staining
procedure in histopathology assays. Hypoxia related H.sub.2O.sub.2
ionic currents from the in-vitro human fresh cervical samples
prepared from 30 patients were recorded. Hypoxia glycolysis
metabolism of fresh cervical tissues was monitored immediately
after dissection from the body (with a non-dehydrated size of up to
6 cm.sup.2). In each sample, at least three points on all over the
tissue were recorded and inked the measured locations. Next, the
tissues held in formalin and sent for standard pathological
evaluation (dehydration, block preparation from the tissues,
preparing a thin layer slide from the block, and H&E staining
of the slides). The permanent pathological diagnostic results of
samples were carried out independently from considering the inked
locations. After the declaration of pathological results, the block
of samples was re-molded and re-blocked from the surface in which
the trace of inked points (points tested by exemplary CDP 102)
could be observable. Again, the H&E slide preparation processes
were carried out, and the pathological diagnosis of inked regions
was declared. This second pathological evaluation was assumed as
the reference for responses recorded by exemplary CDP 102.
[0249] FIG. 21 shows a visually summarized comparison between
current peak values of recorded CV responses utilizing exemplary
CDP 102 via exemplary methods 200, and 220 for in-vivo cancer
diagnosis within a living tissue, and CIN pathological
classification, consistent with one or more exemplary embodiments
of the present disclosure. Coherent results were achieved after
categorizing the values of recorded current peaks from the samples
and comparing them with the H&E diagnosis of the inked samples.
As may be observed from FIG. 21, current peak values of the
recorded CV diagrams from the samples were categorized in three
ranges of a first range 2100 assigned as being at healthy state, a
second range 2200 assigned as being at cancerous state, and a third
range 2300 assigned as being at a suspicious state. Among 30
samples, 9 samples were non-CIN tissues (healthy tissues) including
chronic cervicitis and benign flat condyloma tissues, which all
showed current peaks in the first range 2100 as being between about
0 .mu.A and about 115 .mu.A, and their healthy state was confirmed
by H&E results as is exemplary shown in images 2102 (Chronic
cervicitis) and 2104 (flat condyloma). Nineteen samples were low
grade cancerous and high-grade cancerous, including CIN I (image
2202), CIN II (image 2204), and CIN III (image 2206) with
neoplastic mitotic cells, in which all samples showed current peaks
above about 145 .mu.A within the second range 2200. By considering
the pathology reports of tested regions utilizing exemplary CDP
162, a CIN based scoring of CV responses was proposed. The lowest
cut-off for pathologists to declare a positive diagnosis of a cone
biopsied sample may be the presence of low-grade CIN I or
neoplastic mitotic cells in upper layers of basal cells. The
current peak of about 115 .mu.A was the lowest value recorded for
an involved sample. Hence, it was proposed as a cut-off current for
positive scoring of CV recorded responses. Samples with current
peaks in the third range 2300 between about 115 .mu.A and about 145
.mu.A were assigned as suspicious samples to be cancerous being at
a gray zone, for which re-evaluation by pathology is
recommended.
[0250] As described and shown hereinabove, exemplary methods 200,
and 20, and exemplary CDP 102 may be utilized to lively and
selectively determine a value of H.sub.2O.sub.2 released from
cancer or atypical cells, through reverse Warburg effect and
hypoxia assisted glycolysis pathways. The determined value of
released H.sub.2O.sub.2 may be a high-accurate parameter for cancer
detection in any solid or liquid suspicious mass that may be
cancerous. Although the pathology method may be a gold standard of
cancer diagnosis, it needs to prepare a lot of H&E slides to
reach a perfect diagnosis without any false negatives as well as
time consuming pathology procedures. Accordingly, exemplary methods
260, and 226 and exemplary CDP 162 may be applied as a more
accurate and faster diagnostic tool in comparison with pathology
assays, or as a complementary diagnostic tool for pathology assays
in order to reach fast and accurate cancer detection.
[0251] While the foregoing has described what are considered to be
the best mode and/or other examples, it is understood that various
modifications may be made therein and that the subject matter
disclosed herein may be implemented in various forms and examples,
and that the teachings may be applied in numerous applications,
only some of which have been described herein. It is intended by
the following claims to claim any and all applications,
modifications and variations that fall within the true scope of the
present teachings.
[0252] Unless otherwise stated, all measurements, values, ratings,
positions, magnitudes, sizes, and other specifications that are set
forth in this specification, including in the claims that follow,
are approximate, not exact. They are intended to have a reasonable
range that is consistent with the functions to which they relate
and with what is customary in the art to which they pertain.
[0253] The scope of protection is limited solely by the claims that
now follow. That scope is intended and should be interpreted to be
as broad as is consistent with the ordinary meaning of the language
that is used in the claims when interpreted in light of this
specification and the prosecution history that follows and to
encompass all structural and functional equivalents.
Notwithstanding, none of the claims are intended to embrace subject
matter that fails to satisfy the requirement of Sections 101, 102,
or 103 of the Patent Act, nor should they be interpreted in such a
way. Any unintended embracement of such subject matter is hereby
disclaimed.
[0254] Except as stated immediately above, nothing that has been
stated or illustrated is intended or should be interpreted to cause
a dedication of any component, step, feature, object, benefit,
advantage, or equivalent to the public, regardless of whether it is
or is not recited in the claims.
[0255] It will be understood that the terms and expressions used
herein have the ordinary meaning as is accorded to such terms and
expressions with respect to their corresponding respective areas of
inquiry and study except where specific meanings have otherwise
been set forth herein. Relational terms such as first and second
and the like may be used solely to distinguish one entity or action
from another without necessarily requiring or implying any actual
such relationship or order between such entities or actions. The
terms "comprises," "comprising," or any other variation thereof,
are intended to cover a non-exclusive inclusion, such that a
process, method, article, or apparatus that comprises a list of
elements does not include only those elements but may include other
elements not expressly listed or inherent to such process, method,
article, or apparatus. An element proceeded by "a" or "an" does
not, without further constraints, preclude the existence of
additional identical elements in the process, method, article, or
apparatus that comprises the element.
[0256] The Abstract of the Disclosure is provided to allow the
reader to quickly ascertain the nature of the technical disclosure.
It is submitted with the understanding that it will not be used to
interpret or limit the scope or meaning of the claims. In addition,
in the foregoing Detailed Description, it can be seen that various
features are grouped together in various implementations. This is
for purposes of streamlining the disclosure, and is not to be
interpreted as reflecting an intention that the claimed
implementations require more features than are expressly recited in
each claim. Rather, as the following claims reflect, inventive
subject matter lies in less than all features of a single disclosed
implementation. Thus, the following claims are hereby incorporated
into the Detailed Description, with each claim standing on its own
as a separately claimed subject matter.
[0257] While various implementations have been described, the
description is intended to be exemplary, rather than limiting and
it will be apparent to those of ordinary skill in the art that many
more implementations and implementations are possible that are
within the scope of the implementations. Although many possible
combinations of features are shown in the accompanying figures and
discussed in this detailed description, many other combinations of
the disclosed features are possible. Any feature of any
implementation may be used in combination with or substituted for
any other feature or element in any other implementation unless
specifically restricted. Therefore, it will be understood that any
of the features shown and/or discussed in the present disclosure
may be implemented together in any suitable combination.
Accordingly, the implementations are not to be restricted except in
light of the attached claims and their equivalents. Also, various
modifications and changes may be made within the scope of the
attached claims.
* * * * *