U.S. patent application number 15/670579 was filed with the patent office on 2017-11-23 for systems and methods for detecting a substance in bodily fluid.
This patent application is currently assigned to Avails Medical, Inc.. The applicant listed for this patent is Avails Medical, Inc.. Invention is credited to Meike HERGET, Oren S. KNOPFMACHER.
Application Number | 20170336348 15/670579 |
Document ID | / |
Family ID | 56136335 |
Filed Date | 2017-11-23 |
United States Patent
Application |
20170336348 |
Kind Code |
A1 |
HERGET; Meike ; et
al. |
November 23, 2017 |
SYSTEMS AND METHODS FOR DETECTING A SUBSTANCE IN BODILY FLUID
Abstract
Various devices, systems and methods for determining a parameter
of and/or detecting chemical and biological substances in bodily
fluid are described herein. A device or system may include a
substrate. An active sensor having an electrical characteristic
and/or a control sensor may be disposed on the substrate. In
certain variations, a differential between a first signal from the
active sensor, and a second signal from the control sensor may be
used to determine a parameter of the chemical or biological
substance in the sample of bodily fluid.
Inventors: |
HERGET; Meike; (Woodside,
CA) ; KNOPFMACHER; Oren S.; (Palo Alto, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Avails Medical, Inc. |
Menlo Park |
CA |
US |
|
|
Assignee: |
Avails Medical, Inc.
Menlo Park
CA
|
Family ID: |
56136335 |
Appl. No.: |
15/670579 |
Filed: |
August 7, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15081491 |
Mar 25, 2016 |
9766201 |
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15670579 |
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14599190 |
Jan 16, 2015 |
9377456 |
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15081491 |
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14586802 |
Dec 30, 2014 |
9702847 |
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14599190 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/54373 20130101;
G01N 27/60 20130101; G01N 27/4145 20130101; G01N 33/54353 20130101;
G01N 33/54386 20130101 |
International
Class: |
G01N 27/414 20060101
G01N027/414; G01N 27/60 20060101 G01N027/60; G01N 33/543 20060101
G01N033/543 |
Claims
1. A device for determining a parameter of a substance in a sample,
the device comprising: a substrate having an active sensor disposed
above the substrate, wherein the active sensor responds to an
electrostatic or capacitance change; wherein the active sensor
comprises an extended gate and an electrical component having an
electrical characteristic; and a functionalized structure
comprising a plurality of binding receptors, wherein the binding
receptors are secured directly to the extended gate of the active
sensor, wherein the plurality of binding receptors are configured
to bind with the substance to undergo a reaction resulting in the
release of ions such that the release of the ions results in a
change in the electrical characteristic of the electrical component
independent of current flow in solution; and wherein the electrical
component is configured to generate a signal upon the application
of an electrical stimulus, the signal being indicative of the
change in the electrical characteristic of the electrical
component, and wherein the parameter of the substance is determined
by the signal indicative of the change in the electrical
characteristic of the electrical component.
2. The device of claim 1, wherein the binding receptors each have
an engineered protein directly coupled to each of the binding
receptors and the engineered protein secures each of the binding
receptors directly to the extended gate of the active sensor.
3. The device of claim 2, wherein the engineered protein comprises
at least one cysteine group.
4. The device of claim 2, wherein the distance between each of the
binding receptors and the extended gate is 5 nm or less when the
binding receptors are secured directly to the extended gate.
5. The device of claim 1, wherein the extended gate comprises a
metal layer.
6. The device of claim 5, wherein the metal layer is a gold
layer.
7. The device of claim 1, wherein the functionalized structure
includes a permeable membrane, hydrogel, PVC, or other filter, for
allowing only the passage of a target moiety.
8. The device of claim 1, wherein the electrical component
comprises at least one of a transistor, a capacitor, a resistor,
and an inverter.
9. The device of claim 1, wherein the electrical stimulus comprises
altering an electrical parameter selected from the group consisting
of frequency, current, voltage, resistance, impedance, capacitance,
conductivity, induction, threshold voltage, transconductance,
subthreshold swing, piezo-resistivity, magnetic field, and
electrical noise.
10. A device for determining a parameter of a substance in a
sample, the device comprising: a substrate having an active sensor
and a control sensor, wherein the active sensor is disposed above
the substrate and responds to an electrostatic or capacitance
change; wherein the active sensor comprises a first extended gate
and a first electrical component having a first electrical
characteristic and the control sensor comprises a second extend
gate and a second electrical component having a second electrical
characteristic; and a functionalized structure comprising a
plurality of binding receptors such that the binding receptors are
secured directly to the extended gate of the active sensor, wherein
the plurality of binding receptors are configured to bind with the
substance to undergo a reaction resulting in the release of ions
such that the release of the ions results in a change in the first
electrical characteristic of the first electrical component
independent of current flow in solution, wherein the first
electrical component is configured to generate a first signal upon
the application of an electrical stimulus and the second electrical
component is configured to generate a second signal upon the
application of the electrical stimulus, and wherein the parameter
of the substance is determined by a difference between the first
signal and the second signal.
11. The device of claim 10, wherein the binding receptors each have
an engineered protein directly coupled to each of the binding
receptors and the engineered protein secures each of the binding
receptors directly to the extended gate of the active sensor.
12. The device of claim 1, wherein the engineered protein comprises
at least one cysteine group.
13. The device of claim 11, wherein the distance between each of
the binding receptors and the extended gate is 5 nm or less when
the binding receptors are secured directly to the extended
gate.
14. The device of claim 10, wherein the extended gate comprises a
metal layer.
15. The device of claim 14, wherein the metal layer is a gold
layer.
16. The device of claim 10, wherein the functionalized structure
includes a permeable membrane, hydrogel, PVC, or other filter, for
allowing only the passage of a target moiety.
17. The device of claim 10, wherein the first electrical component
and the second electrical component each comprise at least one of a
transistor, a capacitor, a resistor, and an inverter.
18. The device of claim 10, wherein the electrical stimulus
comprises altering an electrical parameter selected from the group
consisting of frequency, current, voltage, resistance, impedance,
capacitance, conductivity, induction, threshold voltage,
transconductance, subthreshold swing, piezo-resistivity, magnetic
field, and electrical noise.
19. A method for determining a parameter of a substance in a
sample, the method comprising: providing a substrate comprising an
active sensor and a control sensor, wherein the active sensor
comprises a first extended gate and a first electrical component
having a first electrical characteristic and a functionalized
structure coupled to the first extended gate, and wherein the
control sensor comprises a second extended gate and a second
electrical component having a second electrical characteristic;
introducing the sample to the substrate, wherein introducing the
sample results in binding of the substance to the functionalized
structure and results in the release of ions such that the release
of ions results in a change in the first electrical characteristic
of the first electrical component; comparing the first electrical
characteristic of the first electrical component with the second
electrical characteristic of the second electrical component; using
the comparison to determine at least one parameter of the substance
in the sample; and producing an output of the at least one
parameter.
20. The method of claim 19, wherein comparing the first electrical
characteristic of the first electrical component with the second
electrical characteristic of the second electrical component
includes comparing at least one of a frequency, current, voltage,
resistance, impedance, capacitance, conductivity, induction,
threshold voltage, transconductance, subthreshold swing,
piezo-resistivity, magnetic field, and electrical noise of the
first electrical component with that of the second electrical
component.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/081,491, filed on Mar. 25, 2016, which is a
continuation of U.S. patent application Ser. No. 14/599,190 (now
U.S. Pat. No. 9,377,456), filed on Jan. 16, 2015, which is a
continuation of U.S. patent application Ser. No. 14/586,802 (now
U.S. Pat. No. 9,702,847), filed on Dec. 30, 2014, the contents of
which are incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present systems and methods relate generally to devices,
systems and methods for detecting various parameters of a chemical
or biological substance in bodily fluid.
BACKGROUND
[0003] Integration of biosensors on a small scale for e.g., in-home
testing is increasingly being favored by healthcare providers,
however it has been a challenge for years. Optical-based biosensors
require bulky detection equipment and access to power supplies.
Vibration sensitive biosensors (AFM, crystal-quartz balance etc.)
cannot be built into a hand-held or portable device since
background vibration will interfere with the signal. Biosensors
made from electrical components have been considered as a good
solution, however existing biosensors face several problems
including limitations caused by electrostatic screening in complex
media. Several methods to circumvent this problem including sample
dilution and pulsed electrical properties have been explored,
resulting in additional sample processing steps and the dilution of
the analyte, or adding complexity to the device design. In
addition, reference and calibration processes prior to use of the
biosensors complicates their use.
[0004] Existing biosensors used to detect substances in bodily
fluid suffer from a number of other limitations as well. For
example, existing biosensors may be utilized for analyte detection;
however, due to the inability to control various environmental
factors surrounding the sample of bodily fluid and the biosensor,
signals associated with this detection are often not accurate, not
reproducible and do not provide a reliable or stable readout.
Examples of existing biosensors can be found in Pedro Estrela et
al., Label-Free Sub-picomolar Protein Detection with Field-Effect
Transistors, Anal. Chem. 2010, 82, 3531-3536 and Eric Salm et al.,
Electrical Detection of Nucleic Acid Amplification Using an On-Chip
Quasi-Reference Electrode and a PVC REFET, Anal. Chem. 2014, 86,
6968-6975, each of which is herein incorporated by reference.
[0005] As a result of the above limitations and restrictions, there
is a need for an improved device, system and method for detecting
chemical and biological substances in bodily fluid that minimizes
or eliminates such limitations and restrictions.
BRIEF SUMMARY
[0006] Various electrical devices, systems and methods for
determining a parameter of and/or detecting a chemical and
biological substances in bodily fluid are described herein.
[0007] In one example, such a device includes a substrate having an
active sensor; wherein the active sensor comprises a first
electrical component having an electrical characteristic; a
functionalized structure comprising a plurality of binding
receptors each having at least one functional group associated
therewith such that the functional group on each of the binding
receptors permits securing each of the binding receptors to a first
layer of the sensor in a uniform manner, wherein the binding
receptor is configured to interact with the substance such that
interaction of the substance with the functionalized structure
results in a change in the electrical characteristic of the first
electrical component; and wherein the first electrical component is
configured to generate a first signal upon the application of an
electrical stimulus, the first signal being indicative of the
changed electrical characteristic of the first electrical component
and where the changed electrical characteristic allows determining
the parameter of the substance.
[0008] A variation of the device can include a configuration where
a distance between a binding site of the functionalized structure
and the active sensor is minimized by the lack of an adhesion
layer.
[0009] In such devices, the electrical stimulus can include
altering an electrical parameter selected from the group
comprising: frequency, current, voltage, resistance, impedance,
capacitance, conductivity, induction, threshold voltage,
transconductance, subthreshold swing, piezo-resistivity, magnetic
field, and electrical noise.
[0010] The binding receptor of the devices described herein can
include a protein selected from the group consisting of an
engineered protein and an engineered scaffold protein.
[0011] In an additional variation, devices under the present
disclosure can further include a second electrical component having
an electrical characteristic, wherein interaction of the substance
with the active sensor or the control sensor does not result in a
change in the electrical characteristic of the second electrical
component.
[0012] The improved binding receptors described herein can include
a functional group that is directly engineered onto the binding
receptor.
[0013] Variations of the device include the functionalized
structure being positioned on the substrate or active sensor or in
a location separate from the substrate, wherein the functionalized
structure is configured to bind the substance, wherein binding of
the substance with the functionalized structure produces or results
in the release of one or more ions which are detected by the first
electrical component or cause a change in the electrical
characteristic of the first electrical component.
[0014] In an additional variation, the functionalized structure can
be positioned on the substrate or active sensor or in a location
separate from the substrate, wherein the functionalized structure
is configured to bind the substance, wherein binding of the
substance with the functionalized structure changes the electrical
characteristic of the first electrical component.
[0015] The substrate can be positioned in a first location and the
functionalized structure can be positioned in a second location
separate from the substrate, wherein the functionalized structure
is configured to bind to the substance, wherein after binding the
substance undergoes a reaction which changes the electrical
characteristic of the first electrical component of the active
sensor.
[0016] The substances screened in the present devices can include a
therapeutic, drug, biological moiety, chemical moiety, protein,
toxin, ion, antibody, peptide, oligonucleotide, pathogen (e.g.,
bacteria, viruses, fungi), cells (tumor cells, blood cells, other
bodily cells), or ligands.
[0017] The high-k dielectric layer described herein can include,
but is not limited to, aluminum oxide, titanium oxide, zirconium
oxide, yttrium oxide, silicon oxide, tantalum oxide, hafnium oxide
and silicon nitride. The immobilization structure can further
include nanoparticles and/or a metal layer for adhering to the
layer.
[0018] In addition, the devices described herein can comprise a
disposable structure, wherein a plurality of active and control
sensors are positioned on the disposable structure.
[0019] In an additional variation, devices for determining a
parameter of a substance in a test substance can include a
substrate; a first electrical component having an electrical
characteristic, the first electrical component comprising an active
sensor having a covering layer; a functionalized structure
comprising a binding receptor free from endogenous functional
groups and having a targeted functional group, where the targeted
functional group immobilizes the binding receptor to the covering
layer such that the binding receptor extends no more than 5 nm from
the covering layer to minimize a screening length of the
functionalized structure, where the binding receptor is configured
to interact with the test substance such that interaction of the
test substance with the functionalized structure alters the
electrical characteristic of the first electrical component; and
wherein application of an electrical stimulus to the first
electrical component generates a first signal from the first
electrical component being indicative of the changed electrical
characteristic of the first electrical component. In additional
variations of the device, the binding receptor can extend beyond 5
nm as needed while still providing an acceptable screening length
for the desired application.
[0020] The present disclosure also includes methods for determining
a parameter of a substance in a test sample. In one variation, such
a method includes providing a substrate having an active sensor
covered in a first layer and having a first electrical component,
wherein the active sensor comprises at least one functionalized
structure in electrical communication with the active sensor and
wherein the control sensor comprises a second electrical component
having an electrical characteristic, where the functionalized
structure includes a binding receptor having a functional group
coupled thereto such that the functional group secures the binding
receptor to the functionalized structure without the need of an
adhesion layer to minimize a distance between a surface of the
functionalized structure and the active sensor; binding the
substance to the functionalized structure, wherein after binding,
the functionalized structure affects the first electrical component
to produce a changed electrical characteristic, where the changed
electrical characteristic varies from the electrical
characteristic; determining a comparison between the electrical
characteristic and the changed electrical characteristic; using the
comparison to determine at least one parameter of substance in the
test sample, and producing an output of the at least one
parameter.
[0021] In an additional variation, the method can include a
functionalized structure that is positioned on the substrate or
active sensor or in a location separate from the substrate, wherein
the functionalized structure binds the substance, wherein binding
of the substance with the functionalized structure produces or
results in the release of one or more ions which are detected by
the first electrical component or cause a change in the electrical
characteristic of the first electrical component.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0022] FIG. 1 illustrates a variation of a system for detecting a
chemical or biological substance in bodily fluid including a
substrate having active and control sensors and an analyzer, and a
reader.
[0023] FIG. 2 illustrates a schematic diagram of the signal
detection and read out of the system according to FIG. 1.
[0024] FIG. 3A illustrates a substrate having an active sensor and
a control sensor where functionalized structures are disposed on
the active sensor.
[0025] FIG. 3B illustrates a side view of the substrate of FIG. 3A,
where a target chemical or biological substance is bound to a
functionalized structure and undergoes a reaction which produces
ions which diffuse to the surface of the active sensor.
[0026] FIG. 4A illustrates a substrate having an active sensor and
a control sensor where functionalized structures are disposed on
the substrate, adjacent to the active sensor.
[0027] FIG. 4B illustrates a side view of the substrate of FIG. 4A,
where a target chemical or biological substance is bound to the
functionalized structure and undergoes a reaction which produces
ions which diffuse to the surface of the active sensor.
[0028] FIG. 5 illustrates a side view of an active sensor having
functionalized structures disposed thereon, where first and second
moieties of a chemical or biological substance undergo a competing
reaction which produces ions which diffuse to the surface of the
active sensor.
[0029] FIG. 6 illustrates various functionalization schemes and
secondary reactions that a bound chemical or biological substance
may undergo.
[0030] FIG. 7 illustrates various reactions that a bound chemical
or biological substance may undergo.
[0031] FIG. 8A provides an illustrative example of the differences
between a binding site of conventional receptor molecule and
binding receptors with a minimized binding site length, which lies
within the or in close proximity to the screening length instead of
outside.
[0032] FIG. 8B provides an example of an endogenous functionalized
group on a conventional receptor molecule and a binding receptors
with an engineered functionalized group that immobilizes the
binding receptor to the sensor.
[0033] FIG. 9 shows one example of a FET sensor used to determine a
parameter of a substance in a test sample.
[0034] FIG. 10A illustrates an example of a device that increases
the reliability of measurement of a substance in the test sample
using a signal comparison between two sensors.
[0035] FIG. 10B illustrates additional variations of functionalized
sensors and a reference sensor.
[0036] FIGS. 11A to 11C provide illustrations of various binding
receptors and secondary reactions that can be measured by the
sensor.
DETAILED DESCRIPTION
[0037] Variations of the devices are best understood from the
detailed description when read in conjunction with the accompanying
drawings. It is emphasized that, according to common practice, the
various features of the drawings may not be to-scale. On the
contrary, the dimensions of the various features may be arbitrarily
expanded or reduced for clarity and not all features may be visible
or labeled in every drawing. The drawings are taken for
illustrative purposes only and are not intended to define or limit
the scope of the claims to that which is shown.
[0038] In certain variations, a device, e.g., an electrical device
or biosensor, for determining a parameter of and/or for detecting a
chemical or biological substance in bodily fluid is provided. The
device includes a substrate. One or more active sensors and one or
more control sensors may be disposed on the substrate.
[0039] The active sensor includes one or more first electrical
components having an electrical characteristic or property. One or
more functionalized structures are disposed on, near or in a
vicinity of the substrate or active sensor. The functionalized
structure is configured to interact with, e.g., couple with or
bind, the chemical or biological substance, e.g., one or more
moieties of the chemical or biological substance. The interaction
of the chemical or biological substance with the functionalized
structure results in a change in the electrical characteristic or
property of the electrical component of the active sensor.
[0040] The control sensor comprises one or more second electrical
components having an electrical characteristic. However, the
control sensor is configured such that interaction of the chemical
or biological substance with the active sensor and/or the control
sensor does not result in a change in the electrical characteristic
of the second electrical components of the control sensor.
[0041] The first electrical component of the active sensor produces
a signal, where the signal is indicative of the change in the
electrical characteristic of the first electrical component caused
by the interaction of the functionalized group with the chemical or
biological substance in the bodily fluid. For example, the signal
may be indicative of a changed current, voltage, capacitance or
other electrical characteristic. The interaction between the
functionalized group and the chemical or biological substance may
take place on the active sensor or off the active sensor or
substrate in a separate location. Simultaneously, the second
electrical component of the control sensor produces a control
signal. The differential between the signal from the first
electrical component of the active sensor, and the signal from the
second electrical component of the control sensor is used to
determine the parameter of the chemical or biological substance in
the sample of bodily fluid. Indeed, the device, via the
simultaneous use and detection of a control sensor, provides a
self-calibration.
[0042] The differential signal may be used to determine a variety
of parameters or characteristics of the chemical or biological
substance, or to detect the presence of the chemical or biological
substance. In certain variations, the differential signal may be
used to determine the concentration of the chemical or active
substance. The differential signal may be used to determine the
concentration of a variety of ions present in the chemical or
biological substance or in the sample of bodily fluid. For example,
the differential signal may be used to determine the pH of the
chemical or biological substance.
[0043] In certain variations, the differential between the signal
produced by the active sensor and the signal produced by the
control sensor is indicative of, corresponds to, or is used to
determine the concentration of the chemical or biological substance
in the sample of bodily fluid. Indeed, the control sensor signal
may correspond to a known concentration, such that the differential
between the control signal and the signal from the active sensor
may be used to deduce or determine the concentration of the
chemical or biological substance in the sample of bodily fluid.
[0044] The comparison between the signal of the active sensor and
the signal of the control sensor may be the differential change
(.DELTA.S) in an electrical characteristic, e.g., current, voltage,
capacitance, resistance or threshold voltage, of the active sensor
(S1) vs the control sensor (S2), where the comparison yields a
differential signal is (.DELTA.S=S1-S2).
[0045] In other variations, the signal produced by the active
sensor, which is indicative of the changed electrical
characteristic or the change in the electrical characteristic of
the first electrical component which change occurs as a result of
the interaction of the functionalized group with the chemical or
biological substance, may be indicative of a changed current,
voltage, capacitance or other electrical characteristic. For
example, the signal or detected current may correspond to a known
concentration, such that the concentration can be deduced from the
detected current, or current change, or the concentration may be
deduced from a detected change in another electrical characteristic
or property which corresponds to a known concentration.
[0046] The functionalized structure is configured to interact with
the chemical or biological substance of the sample of bodily fluid.
As described supra, the interaction of the chemical or biological
substance with the functionalized structure may result in a change
in the electrical characteristic or property of the electrical
component of the active sensor.
[0047] In any of the variations described herein, the electrical
components may include a transistor, a capacitor, a resistor or an
inverter or any other suitable electrical component known to
persons have ordinary skill in the art.
[0048] A variety of interactions between the chemical or biological
substance and the functionalized structure and/or related reactions
are contemplated, where such interactions and/or reactions result
in a change, or cause a change, in the electrical characteristic or
property of the electrical component of the active sensor.
[0049] In one variation, one or more functionalized structures are
disposed on the active sensor and are configured to bind to the
chemical or biological substance. The binding of the chemical or
biological substance by the functionalized structure results in a
change in an electrical characteristic of the electrical component
of the active sensor. For example, the binding of a charged moiety
or ion of a chemical or biological substance by a functionalized
structure may result in an increase or decrease in charge density
on or in a vicinity of the active sensor or a change in
current.
[0050] In another variation, one or more functionalized structures
are disposed on the substrate or the active sensor, and are
configured to bind to the chemical or biological substance. The
binding of the chemical or biological substance by the
functionalized structure produces one or more ions which diffuse to
the surface of the active sensor and cause a change in an
electrical characteristic of the electrical component of the active
sensor. For example, the produced ions may come into contact with,
bind or otherwise interact with the active sensor, e.g., causing an
increase or decrease in charge density of the active sensor or a
change in current. Optionally, the functionalized structures may be
disposed in another location, separate from the substrate. The
produced ions may then flow over the surface of the active sensor,
and interact with the active sensor, causing a change in the
electrical characteristic of the electrical component of the active
sensor.
[0051] In another variation, one or more functionalized structures
are disposed on the substrate or the active sensor, and are
configured to bind to the chemical or biological substance. The
bound chemical or biological substance undergoes a reaction with
one or more reagents, thereby producing one or more ions which
diffuse to the surface of the active sensor and cause a change in
an electrical characteristic of the electrical component of the
active sensor. For example, the produced ions may come into contact
with, bind or otherwise interact with the active sensor, e.g.,
causing an increase or decrease in charge density of the active
sensor or a change in current. Optionally, the functionalized
structures may be disposed in another location, separate from the
substrate. Where the reaction takes place in a location which is
separated from the substrate, the produced ions may flow to and
over the surface of the active sensor, and interact with the active
sensor, causing a change in the electrical characteristic of the
electrical component of the active sensor.
[0052] In another variation, the one or more functionalized
structures may be in the form of a permeable membrane or other
filter, which is disposed on the active sensor. The membrane or
filter is configured to allow for the passage of the target
chemical or biological substance or a produced ion, such that the
substance or ion may interact with the active sensor, while the
membrane or filter blocks or restricts the passage of other
moieties or ions, e.g., based on size or other property. The ions
may be produced as a result of the binding of the chemical or
biological substance by the functionalized structure or as a result
of a reaction between a bound substance and a reagent. The ions may
diffuse or flow, from a local or remote location, over the surface
of the active sensor, after passing through a membrane of filter,
and cause a change in an electrical characteristic of the
electrical component of the active sensor. For example, the
produced ions may come into contact with, bind or otherwise
interact with the active sensor, e.g., causing an increase or
decrease in charge density of the active sensor or a change in
current. Optionally, the membrane, filter or other functionalized
structure may capture the target substance or ions, but allow the
passage of other non-target ions. Optionally, a membrane, filter or
other functionalized structure may block background charge, where a
charge or lack of charge may be detected when a particle flows
through a membrane and past the sensor.
[0053] In certain variations, a device, e.g., an electrical device
or a biosensor, for determining a parameter of and/or for detecting
a chemical or biological substance in bodily fluid is provided. The
device includes a substrate. One or more active sensors may be
disposed on the substrate.
[0054] The active sensor includes one or more first electrical
components having an electrical characteristic or property. One or
more functionalized structures are disposed on, near or in a
vicinity of the substrate or active sensor. The functionalized
structure is configured to interact with, e.g., couple with or
bind, the chemical or biological substance, e.g., one or more
moieties of the chemical or biological substance. The bound
chemical or biological substance undergoes a reaction thereby
producing a product. The product interacts with the first
electrical component which results in a change in the electrical
characteristic of the first electrical component. A signal from the
first electrical component, the signal being indicative of the
changed electrical characteristic or the change in the electrical
characteristic, may be used to determine the parameter of the
chemical or biological substance in the sample of bodily fluid.
[0055] The signal from the first electrical component of the active
sensor may be used to determine a variety of parameters or
characteristics of the chemical or biological substance, or to
detect the presence of the chemical or biological substance. In
certain variations, the signal may be used to determine the
concentration of the chemical or active substance. The signal may
be used to determine the concentration of a variety of ions present
in the chemical or biological substance. For example, the signal
may be used to determine the pH of the chemical or biological
substance.
[0056] In certain variations, the signal produced by the active
sensor is indicative of, corresponds to, or is used to determine
the concentration of the chemical or biological substance in the
sample of bodily fluid. Indeed, the signal may correspond to a
known concentration, such that the signal from the active sensor
may be used to deduce the concentration of the chemical or
biological substance in the sample of bodily fluid.
[0057] In other variations, the signal produced by the active
sensor, which is indicative of the changed electrical
characteristic or the change in the electrical characteristic of
the first electrical component, may be indicative of a changed
current, voltage, capacitance or other electrical characteristic.
For example, the signal or detected current may correspond to a
known concentration, such that the concentration can be deduced
from the detected current.
[0058] In any of the variations described herein, the electrical
components may include a transistor, a capacitor, a resistor or an
inverter or any other suitable electrical component known to
persons have ordinary skill in the art.
[0059] A variety of interactions between the chemical or biological
substance and the functionalized structure and/or related reactions
are contemplated, where such interactions and/or reactions result
in a change, or cause a change, in the electrical characteristic or
property of the electrical component of the active sensor.
[0060] In one variation, one or more functionalized structures are
disposed on the substrate or the active sensor, and are configured
to bind to the chemical or biological substance. The binding of the
chemical or biological substance by the functionalized structure
produces one or more ions which diffuse to the surface of the
active sensor and cause a change in an electrical characteristic of
the electrical component of the active sensor. For example, the
produced ions may come into contact with, bind or otherwise
interact with the active sensor, e.g., causing an increase or
decrease in charge density of the active sensor or a change in
current. Optionally, the functionalized structures may be disposed
in another location, separate from the substrate. The produced ions
may then flow over the surface of the active sensor, and interact
with the active sensor, causing a change in the electrical
characteristic of the electrical component of the active
sensor.
[0061] In another variation, one or more functionalized structures
are disposed on the substrate or the active sensor, and are
configured to bind to the chemical or biological substance. The
bound chemical or biological substance undergoes a reaction with
one or more reagents, thereby producing one or more ions which
diffuse to the surface of the active sensor and cause a change in
an electrical characteristic of the electrical component of the
active sensor. For example, the produced ions may come into contact
with, bind or otherwise interact with the active sensor, e.g.,
causing an increase or decrease in charge density of the active
sensor or a change in current. Optionally, the functionalized
structures may be disposed in another location, separate from the
substrate. Where the reaction takes place in a location which is
separated from the substrate, but the produced ions may flow over
the surface of the active sensor, and interact with the active
sensor, causing a change in the electrical characteristic of the
electrical component of the active sensor.
[0062] In one variation, the bound chemical or biological substance
(on the substrate or in a remote location) may undergo a reaction
which produces or results in the release of one or more ions which
flow over the active sensor and cause a change in pH or other ion
concentration. For example, the change in pH may be detected by the
first electrical component of the active sensor, which may have a
proton sensitive layer disposed thereon. The change in pH or other
ion concentration may cause a change in the electrical
characteristic of the first electrical component.
[0063] In another variation, the one or more functionalized
structures may be in the form of a permeable membrane or other
filter, which is disposed on the active sensor. The membrane or
filter is configured to allow for the passage of the target
chemical or biological substance or a produced ion, such that the
substance or ion may interact with the active sensor, while the
membrane or filter blocks or restricts the passage of other
moieties or ions, e.g., based on size or other property. The ions
may be produced as a result of the binding of the chemical or
biological substance by the functionalized structure or as a result
of a reaction between a bound substance and a reagent. The ions may
diffuse or flow, from a local or remote location, over the surface
of the active sensor, after passing through a membrane of filter,
and cause a change in an electrical characteristic of the
electrical component of the active sensor. For example, the
produced ions may come into contact with, bind or otherwise
interact with the active sensor, e.g., causing an increase or
decrease in charge density of the active sensor or a change in
current. Optionally, the membrane, filter or other functionalized
structure may capture the target substance or ions, but allow the
passage of other non-target ions. Optionally, a membrane, filter or
other functionalized structure may block background charge, where a
charge or lack of charge may be detected when a particle flows
through a membrane and past the sensor.
[0064] The various reactions described herein may allow indirect
detection, where the product of the reaction may diffuse to the
surface of the sensor where it interacts with the sensor. This
helps circumvent electrostatic screening issues that might
otherwise arise.
[0065] In certain variations, the substrate may also include a
control sensor (as described supra). The control sensor includes a
second electrical component having an electrical characteristic.
Binding of the chemical or biological substance to the active
sensor or the control sensor does not result in a change in the
electrical characteristic of the second electrical component of the
control sensor.
[0066] The active and control sensor are used simultaneously, where
both are disposed on the substrate. A differential between a signal
from the first electrical component of the active sensor, the
signal being indicative of the changed electrical characteristic or
the change in the electrical characteristic, and a signal from the
second electrical component of the control sensor may be used to
determine the parameter of the chemical or biological substance in
the sample of bodily fluid, as described supra.
[0067] In certain variations, the devices described herein may be
part of a sensor or detection system. For example, the device may
include, be coupled to, or be in communication with an analyzer.
The analyzer may be configured to analyze the signals received from
the first and/or second electrical components of the device or
biosensor and to determine the differential between the signals.
The system may also include a reader, where the reader includes, is
coupled to or is in communication with the analyzer. The reader is
configured to provide an electrical read-out of the analyzed
signals and/or the determined parameter, based on the differential
signal.
[0068] The analyzer may be used to receive or read the signal from
the active and control sensors and to perform smart operations to
convert measurements to accurate signal readouts and results for
both sensors. The analyzer may include an analog/digital converter
and/or a multiplexer. The analyzer may be used to provide a
comparison, such as a comparison of signals or other differential
readout (See FIG. 2), and/or for amplifying the signals. The
analyzer may include one or more source-meters or other electronics
to apply a voltage or current, to apply a pulsed signal, to
read-out a voltage, to read-out a current, and/or to read out a
resistance and/or capacitance change or other electrical
characteristic change.
[0069] The reader may connect to or be coupled to the device, to
the analyzer, and/or to the device having an analyzer incorporated
therein. The device may be in the form of a strip having a
plurality of sensors as described supra. The reader may receive
input from the analyzer, and may be used to visualize the detected
and analyzed signals or results from the sensors of the device in a
simple and user friendly way. The reader may include one or more
source-meters or other electronics to apply a voltage or current,
to apply a pulsed signal, to read-out a voltage, to read-out a
current, and/or to read out a resistance and/or capacitance change
or other electrical characteristic change.
[0070] In one variation, a device having one or more sensors and an
analyzer, e.g., a device strip having a plurality of sensors as
described herein, may be inserted into the reader to provide a
user-friendly read-out regarding a parameter, (e.g., concentration)
of the chemical or biological substance detected by the sensor. The
reader may apply different voltages or currents or other electronic
properties to the device strip or analyzer and it may receive or
provide an output which may be visualized by a user in a simple and
effective manner. Optionally, the reader may have capabilities or
be configured to communicate via Bluetooth or Wi-Fi or via other
wired or wireless mechanisms or modes of communication to one or
more other device. Optionally, a controller may be provided, where
the controller is coupled to or in communication with the device,
e.g., the sensors, analyzer, and/or a reader, such that the
controller may be used to control or program the functionality of
the device, including the sensors and/or the analyzer. The
controller may be coupled to the analyzer or be integrated in the
reader. In certain variation, a controller may be located in the
substrate (chip), analyzer or the reader.
[0071] In certain variations, methods for determining a parameter
of a chemical or biological substance in a sample of bodily fluid
include one or more of the following steps. Providing a substrate
having an active sensor and a control sensor, wherein the active
sensor comprises a first electrical component having an electrical
characteristic, wherein at least one functionalized structure is
disposed on the substrate or the active sensor or in a location
remote or separated from the substrate and active sensor, and
wherein the control sensor comprises a second electrical component
having an electrical characteristic. Coupling or binding the
chemical or biological substance to a functionalized structure,
wherein the bound chemical or biological substance undergoes a
reaction thereby producing a product. Interacting the product with
the first electrical component which results in a change in the
electrical characteristic of the first electrical component, while
not resulting in a change in the electrical characteristic of the
second electrical component. Comparing a first signal and a second
signal, where the first signal is from the first electrical
component of the active sensor, the first signal being indicative
of the changed electrical characteristic or a change in the
electrical characteristic, and the second signal being from the
second electrical component of the control sensor. Using the
differential to determine the parameter of the chemical or
biological substance in the sample of bodily fluid. Providing an
electronic read-out of the determined parameter, based on the
differential.
[0072] The functionalized structure may be positioned on the
substrate or active sensor or in a location separate from the
substrate, wherein the functionalized structure couples or binds
the chemical or biological substance. Binding or coupling of the
chemical or biological substance with the functionalized structure
produces or results in the release of one or more ions which are
detected by the first electrical component or cause a change in the
electrical characteristic of the first electrical component.
[0073] The bound or coupled chemical or biological substance may
undergo a reaction with one or more reagents which produces or
results in the release of one or more ions which are detected by
the first electrical component or cause a change in the electrical
characteristic of the first electrical component.
[0074] The bound or coupled chemical or biological substance may
undergo a reaction which produces or results in the release of one
or more ions which cause a change in a pH or other ion
concentration, wherein the change in pH other ion concentration is
detected by the first electrical component or causes a change in
the electrical characteristic of the first electrical
component.
[0075] The bound chemical or biological substance may undergo a
reaction in a first location which produces or results in the
release of one or more products or ions which flow to the active
sensor on the substrate positioned in a second, separate location,
where the products or ions are detected by the first electrical
component of the active sensor or cause a change in the electrical
characteristic of the first electrical component of the active
sensor.
[0076] The following documents are incorporated herein by reference
in their entirety: Hammock, M. L. et al. Electronic readout ELISA
with organic field-effect transistors as a prognostic test for
preeclampsia; U.S. Provisional Pat. App. No. 61/907,363; and
Mathias. W. et al. Selective Sodium Sensing with Gold-Coated
Silicon Nanowire Field-Effect Transistors in a Differential Setup.
ACS Nano 7, 5978-5983 (2013).
[0077] The devices, systems or methods for determining a parameter
of and/or for detecting a chemical or biological substance in
bodily fluid described herein may be utilized with various bodily
fluids to detect various parameters of various substances.
[0078] Bodily fluid may include, e.g., blood, urine, saliva, tears,
ejaculate, odor or other body fluids. Detected substances can
include, e.g., hormones, different pathogens, proteins, antibodies,
various drugs or therapeutics or other chemical or biological
substances. Detected or determined parameters may include, e.g., pH
changes, lactose changes, changing concentration, particles per
unit time where a fluid flows over the device for a period of time
to detect particles, e.g., particles that are sparse, and other
parameters.
[0079] The various devices, systems or methods described herein may
include one or more of the following features described below.
[0080] In certain variations, a plurality of conductors may be
coupled to the active sensor and/or a plurality of conductors may
be coupled to the control sensor. The conductors may be adapted to
be electrically coupled to a reader for obtaining an electrical
reading from the electrical components of the active and control
sensors.
[0081] The chemical or biological substance may include, but not be
limited to, a variety of substances, e.g., any substance suitable
for detection or monitoring, such as, a therapeutic, drug,
biological moiety, chemical moiety, protein, ion or antibody.
[0082] A variety of functionalized structures may be utilized, e.g.
proteins, peptides, antibodies, or chemical moieties. Any of these
functionalized structures may be configured or suitable to bind to
a therapeutic, drug, biological moiety, chemical moiety, protein,
antibody, or ion in the sample of bodily fluid. In other
variations, types of functionalized structures include, but are not
limited to, a permeable membrane, hydrogel or other filter, e.g.,
PVC.
[0083] In one variation, a functionalized structure may include a
binding receptor immobilized on the surface of the active sensor,
and the binding receptor (e.g. antibody, protein, peptide) may be
capable of binding to any of the chemical or biological substances
described herein.
[0084] In certain variations, an immobilization structure may be
disposed on or in a vicinity of a substrate or active sensor of a
device, and a functionalized structure may be coupled to the
immobilization structure. For example, the immobilization structure
may include a high-K dielectric layer such as an atomic layer
deposition ("ALD") or any other technique can be used to deposit
the layer (e.g., such as Growing an oxide layer or nitride etc. on
top of the sensor). The high-.kappa. dielectric layer may include,
but not be limited to, aluminum oxide, titanium oxide, zirconium
oxide, yttrium oxide, silicon oxide, tantalum oxide, hafnium oxide
and silicon nitride. Optionally, the immobilization structure may
include at least a portion made up of nanoparticles and/or a metal
layer for adhering to the layer.
[0085] In certain variations, the control sensor may be passivated.
For example, the control sensor may include a passivation structure
such as a self-assembled monolayer (SAM), metal, or polymer layer.
The SAM may include alkane or aromatic thiols, aromatic silanes, or
any chemical entity having a terminal group that is covalently
attached to a surface, a spacer group having a hydrocarbon, or a
head group, e.g., such as, --COOH, --CH3, --SH, --NH2, long chain
alkyl of any length or aromatic and --OH. The SAM layer can include
a silane where the silanes bearing a long, hydrophobic chain of any
length e.g. long alkyl chain of any length, e.g.
Octadecyldimethylmethoxysilane.
[0086] In any of the variations described herein, the device or
substrate may be in the form of a disposable structure. The
disposable structure may include a plurality of active and/or
control or passivated sensors positioned thereon. In certain
variations, the device or strip may include an analyzer and/or a
reader incorporated therein.
[0087] The device or strip may include a plurality of active
sensors and/or control sensors. The signals generated by each
sensor, e.g., signals resulting from a change to an electrical
characteristic of the active sensors, may be read out. The average
of all the determined parameter values based on each sensor, e.g.,
the concentration of a substance, may then be calculated or
deduced.
[0088] In any of the various devices, systems or methods described
herein, various electrical components or sensors may be utilized.
The electrical component or sensor may be any suitable transistor,
e.g. an OFET (organic field effect transistor) or FET (field effect
transistor). For example, a FET may be of any suitable type, and
may include a semiconducting layer doped with a n-type or p-type
material. A source or source electrode and a drain or drain
electrode may be formed in a spaced-apart position on two sides of
the semiconducting layer. The source electrode and dramin electrode
may be each doped having an opposite polarity to the semiconducting
layer. A suitable dielectric layer, such as an oxide layer, may
underlie the semiconducting layer and the source and drain. A gate
electrode underlies the dielectric layer. In other variations, the
gate electrode may be on top of the FET or in its vicinity. A
substrate layer made from any suitable material such as plastic or
glass serves as a support layer and may underlie the gate
electrode. In certain variations, the semiconducting layer may have
a surface that is opposite to the surface to which the dielectric
layer is adhered.
[0089] Any of the readers described herein may include electrical
components for receiving, digitizing and analyzing the analog
electrical signals received from the sensors or for controlling the
sensor. Such electrical components may include a suitable computer
processor or central processing unit, which may be electrically
coupled to the electrical pickups of the reader that electrically
engage the sensors, where such a feature is provided. The reader
may further include suitable storage or memory, electrically
coupled to the processor, for storing computer data. A suitable
display can be included in the reader for displaying desired
information. The display can be a touch screen, for additionally
serving as an input device or terminal. A transmitter or
transceiver can be included in the reader, and electrically coupled
therein with a processor, for wirelessly transmitting or receiving
information between the reader and a suitable remote device.
[0090] The reader, alone or in conjunction with another suitable
computing device, can be calibrated to convert the change in
electrical characteristic of the electrical component into a
concentration level of the targeted drug or other substance. In one
variation, a suitable algorithm can be provided in software and
stored on a memory of the reader or on a remote device in
communication with the reader, or programmed onto a chip provided
on the reader, so as to permit a processor of the reader to
manipulate or process the plurality of measurements provided by the
sensors on the a device or strip and arrive at an immediate
numerical concentration of the targeted substance.
[0091] Exemplary Variations of Systems for Detecting a Biological
or Chemical Substance in Bodily Fluid.
[0092] FIG. 1 illustrates a variation of a system 1 for determining
a parameter of and/or for detecting a chemical or biological
substance in a sample of bodily fluid. The system includes a
substrate 2. One or more active sensors 3 and one or more control
sensors 4 are disposed on the surface of the substrate 2. For
example, FIG. 1 shows three active sensors 3 and three control
sensors 4; however, it is contemplated that any suitable number of
sensors or sensor pairings may be utilized.
[0093] The system 1 also includes an analyzer 10. The analyzer 10
is configured to analyze the signals received from the first
electrical components of the active sensor 3 and the second
electrical component of the control sensor 4. The system also
includes a reader 20. The reader 20 is coupled to or in
communication with the sensors 3, 4 and/or analyzer 10. The reader
20 is configured to receive an analyzed signal from the analyzer
10, and to provide an electronic read-out of the analyzed signal,
e.g., in a visible, user-friendly mode. Optionally, a controller
may be coupled to the analyzer or be integrated in the reader to
provide control and/or programming.
[0094] The active sensor 3 includes one or more first electrical
components having an electrical characteristic or property. One or
more functionalized structures (not shown), may be disposed on,
near or in a vicinity of the substrate 2 or the active sensor 3.
The functionalized structure and functionalized structure
arrangement may include any of the functionalized structures
described herein, e.g., including the functionalized structures
illustrated in FIGS. 3A-6 (discussed below). The functionalized
structure may interact with (e.g., couple with or bind) the
chemical or biological substance. The interaction of the chemical
or biological substance with the functionalized structure, or the
interaction of an ion or product released or produced by a reaction
involving a bound chemical or biological substance, may result in a
change in the electrical characteristic or property of the
electrical component of the active sensor 3.
[0095] The control sensor 4 includes one or more second electrical
components having an electrical characteristic. The control sensor
4 is configured such that interaction of the chemical or biological
substance with the active sensor 3 and/or the control sensor 4 does
not result in a change in the electrical characteristic of the
second electrical components of the control sensor 4. For example,
the control sensor 4 may be passivated such that the target
chemical or biological substance does not bind to or interact with
the control sensor 4.
[0096] As illustrated with reference to the schematic in FIG. 2,
the first electrical component of the active sensor 3 produces an
active signal 30. The active signal 30 is indicative of the changed
electrical characteristic or the change in the electrical
characteristic of the first electrical component caused by the
interaction of the functionalized group or the first electrical
component with the chemical or biological substance in the bodily
fluid, or a product (e.g., an ion) released from a reaction
involving the chemical or biological substance. For example, the
active signal 30 may be indicative of a change in current, voltage,
capacitance or other electrical characteristic. Simultaneously, the
second electrical component of the control sensor 4 produces a
control signal 40. The analyzer 10 receives, as input, the active
signal 30 from the active sensor 3 and the control signal 40 from
the control sensor 4. The analyzer produces a comparison (or a
comparison signal) between the signals from the active sensor and
the control sensor. For example, such a comparison can include a
differential signal 50, being the difference between the active
signal 30 and the control signal 40. The analyzer may convert the
active and control signals from analog to digital. The differential
signal 50 is then transmitted to the reader 20, and used to deduce
a parameter, e.g., concentration of the chemical or biological
substance in the sample of bodily fluid. The reader 20 than
provides a read-out based on the differential signal, in the form
of a value 51 of a parameter of the substance, e.g., the
concentration of the substance, and/or by indicating whether or not
the substance is or is not present 52 in the sample of bodily
fluid.
[0097] The differential signal 50 may be used to determine a
variety of parameters or characteristics of the chemical or
biological substance, or to detect the presence of the chemical or
biological substance. In certain variations, the differential
signal 50 may be used to determine the concentration of various
ions present in a target chemical or biological substance or in the
sample of bodily fluid. For example, the differential signal 50 may
be used to determine the pH of the target chemical or biological
substance.
[0098] FIGS. 3A-3B illustrate one variation of a device or
substrate 61 having one or more functionalized structures 65
disposed thereon. The substrate 61 includes an active sensor 62 and
a control sensor 63. Functionalized structures 65 are disposed on
the surface of the active sensor 62. As shown in FIG. 3B, a target
chemical or biological substance 67 binds to one or more of the
functionalized structure 65 and undergoes a reaction which produces
one or more ions 68, which diffuse to the surface of the active
sensor 62 where they interact with the active sensor 62 and cause a
change in an electrical characteristic of the active sensor 62
and/or are detected by the active sensor 62.
[0099] FIGS. 4A-4B illustrate another variation of a device or
substrate 71 having one or more functionalized structures 75
disposed thereon. The substrate 71 includes an active sensor 72 and
a control sensor 73. Functionalized structures 75 are disposed on
the surface of the substrate 71, adjacent to the active sensor
72.
[0100] As shown in FIG. 4B, a target chemical or biological
substance 77 binds to one or more of the functionalized structure
75 and undergoes a reaction which produces one or more ions 78,
which diffuse to or flow over to the surface of the active sensor
72 where they interact with the active sensor 72 and cause a change
in an electrical characteristic of the active sensor 72 and/or are
detected by the active sensor 72.
[0101] The various devices described herein may utilize or work
with a variety of functionalized structures and functionalized
structure arrangements, as well as reactions between target
chemical or biological substances and a functionalized structure
and/or other reagents, to determine and/or detect various
parameters of chemical or biological substances.
[0102] FIG. 5 illustrates a variation of an active sensor 82.
Functionalized structures 85 are disposed on the surface of the
active sensor 82. In this variation, a first moiety 87 and a second
moiety 88 of a target chemical or biological substance undergo a
competing reaction which produces ions 89, as the two moieties
exchange binding position on the functionalized structure 85. The
ions 89 then diffuse to the surface of the active sensor 82 where
they interact with the active sensor 82 and cause a change in an
electrical characteristic of the active sensor 82 and/or are
detected by the active sensor 82.
[0103] FIG. 6 illustrates various reactions that may be utilized
with the sensor devices and systems described herein, which involve
a chemical or biological substance binding to a functionalized
structure disposed on a substrate, active sensor or in a location
remote or separate from the sensor device or substrate. The
reactions involve various functionalization schemes as described in
more detail below.
[0104] In reaction A, a target moiety 91 binds functionalized
structure 95, where the binding results in the production of a
secondary product 92, which will effect a change in an electrical
characteristic of an active sensor.
[0105] In reaction B, a target moiety 101 binds functionalized
structure 105. The bound target moiety 101 undergoes a reaction
with reagent 103, which results in the production of a secondary
product 102, which will effect a change in an electrical
characteristic of an active sensor.
[0106] In reaction C, a target moiety 111 binds functionalized
structure 115. The bound target moiety 111 binds a secondary
functionalized structure 114. The binding of the secondary
functionalized structure 114 results in the production of a
secondary product 112, which will effect a change in an electrical
characteristic of an active sensor.
[0107] In reaction D, a first target moiety 121 binds
functionalized structure 125. The bound first target moiety 121
binds a secondary functionalized structure 124. The secondary
functionalized structure 124 binds a second target moiety 126. The
bound second target moiety 126 undergoes a reaction with reagent
123, which results in the production of a secondary product 122,
which will effect a change in an electrical characteristic of an
active sensor.
[0108] In another example of a reaction, the reaction may include a
species specific antibody (e.g. anti mouse, anti rabbit, anti goat,
anti guinea pig, anti rat, anti lama), which is immobilized onto
the sensor surface or other location separate from the sensor.
Antigen-specific polyclonal and monoclonal primary antibodies
raised in, e.g. mouse, rabbit, goat, guinea pig, rat or lama may be
added and recognized by the secondary antibody immobilized to the
sensor surface or other surface. For a stable interaction, chemical
bifunctional cross linkers will be used to irreversibly connect
both antibodies.
[0109] In other variations, peptides, oligos, ligands or other
structures or molecules may be utilized to provide
functionalization to a sensor or other surface. The functionalized
structures may be involved or take part in various reactions, which
can be detected or produce products that can be detected by the
sensor.
[0110] In certain variations, the devices, systems and methods
described herein may provide point-of-care, portable and real-time
diagnostic tools. They may provide an electronic readout of an
enzyme linked immunosorbant assay (ELISA) or other assays to detect
various chemical or biological substances. The electronic
components may be configured to transduce or convert a biochemical
binding event or reaction into an electrical signal, which may be
read out. Indirect detection of a freely diffusing, electronically
active species produced at the site of a bound chemical or
biological substance may be performed utilizing the described
biosensor devices. Electronic readout ELISA schemes where an enzyme
capable of producing an electronically active species may be
utilized.
[0111] In one variation, indirect detection may be utilized in a
device or system described herein where a surface is functionalized
with binding receptors, such as capture antibodies or engineered
proteins, in order to provide specific binding site. In one
example, fins-like tyrosine kinase (sFltl) may be detected. After
sFltl is introduced to the device, it binds to the previously
immobilized capture Abs. A secondary, biotin-labeled detection Ab
is then introduced, which binds to a different epitope of sFltl.
Streptavidin (SA) conjugated GOx (SA-GOx) tagged enzyme is
introduced to bind specifically to the detection Ab. Finally,
glucose is introduced and the enzyme-mediated conversion of glucose
to gluconic acid elicits a pH change that can be measured by the
sensor.
[0112] FIG. 7 shows examples of reactions which cause secondary
cascade reactions, which may be utilized with the devices described
herein. The reactions listed in FIG. 7 are merely examples and not
meant to be limiting, as other reactions my also cause secondary
cascade reactions.
[0113] In certain variations, a functionalization area close to the
sensor system or on the sensor surface where a functionalization
and reaction take place is provided. The functionalization area may
include an oxide surface, nanoparticles, a metal, polymer or any
other kind of material. The functionalization can be a protein,
antibody or a chemical moiety immobilized using a linker, which may
consist of a chemical surface modification, immobilization linkers
(such as ProLinker.TM.) or anything else which allows to bind the
functionalization moiety to the desired surface.
[0114] In certain variations, the functionalization can be in the
form of an assay, e.g. sandwich assay. A reagent may be introduced
to the sensor starting a cascade reaction creating the release of a
moiety, e.g. ions. Secondary reagents may freely diffuse to the
sensor surface or may be pushed to the surface using a force (e.g.
pumps, capillary forces, etc).
[0115] In certain variations, a competing reaction may take place
exchanging a previously captured moiety with another one, where the
exchange of the moiety releases secondary ions.
[0116] In other variations, indirect detection of a freely
diffusing, electronically active species produced at the site of a
binding receptor-immobilized analyte can be performed. The reaction
can create a change in the concentration of the released secondary
ions. It may cause a change in pH (acid or base). Ions that can be
released can be but are not limited to H+, Na+, K+, Cl-, COOH.
[0117] Functionalized Sensors Using Engineered Proteins.
[0118] Electrostatic- and charge-sensitive devices are often used
in liquids as biochemical sensors. These sensors can directly
transduce a biological binding event or biological reaction into an
electronic signal in a label-free manner and are advantageous for
sensor technologies demanding digital readout. Bio-detection in
solution is inherently difficult due to the need of operating in
buffered solutions with typically high ionic strength. Many times
the ionic strength of a buffer has a strong influence on the
sensitivity of the sensor as a result of its inverse square root
relationship with the charge screening distance more commonly known
as the Debye length: the higher the ionic concentration the lower
is the screening length, which is available to detect the
target.
[0119] Engineered proteins can provide a variety of advantageous
features over other receptor molecules currently use for
biomolecule detection. For example, Affimers.TM.: a) small in size
(.about.3 nm), b) highly stable (temperature, pH, proteolysis), c)
highly selective, d) can be engineered in vitro to most target
molecules including small molecules, and d) do not contain
endogenous cysteines or functional groups apart from engineered
ones that can be used for targeted surface immobilization.
[0120] When using electrostatic and charge-sensitive biochemical
sensors a number of factors require consideration: i) specificity,
ii) selectivity, iii) the distance of the binding receptor to the
surface of the sensor (or target surface), and iv) uniform surface
immobilization--meaning the binding receptor be placed uniformly. A
number of binding groups and/or functional groups e.g., e.g.
antibodies, aptamers, DNA, etc. can sufficiently address
specificity and selectivity. However, the distance of the binding
receptor the target surface and uniform surface immobilization are
not well addressed by many binding groups. Such considerations
render many FET based sensors using conventional binding receptors
molecules with an inability to measure detection events in high
ionic strength buffers.
[0121] For example, the distance of the binding receptor's binding
site to the target surface is limited by the actual size of the
binding receptor. Antibodies for instance are .about.15 nm in size
and require buffered solutions at high ion concentrations of
.about.150 mM for proper performance. However, operating the sensor
at these parameters, the charge screening length is reduced to a
few nm such that the sensor will not sense most of the binding
reactions and signals. Moreover, for most binding receptors,
including antibodies, an adhesion layer is required for surface
immobilization thereby increasing the distance to the target
surface even more.
[0122] The use of small receptors, such as engineered proteins
(e.g., 3 nm in size), can overcome the inability to measure
detection events in high ionic strength buffers. This permits
improved performance since biomolecules and their biochemical
reactions can occur at ionic strengths that mimic physiological
environments. The use of a binding receptor that is closer to the
sensor interface dramatically helps overcome many conventional
screening limitations. In addition a more stable binding receptor
allows use of the FET sensor in different surrounding conditions,
such as temperature, lower ionic or variable pH solutions and
thereby facilitates sample preparation and increases the storage
capacity and shelf life of the functionalized sensor.
[0123] FIG. 8A illustrates an example of a conventional receptor
molecule 130 currently use for biomolecule detection. As shown, the
screening distance or target binding site 132 of a conventional
receptor molecule 130 can be 15 nm from the immobilization surface
of the sensor 150 and thus outside the screening distance. Reducing
the distance of the binding site, for example, using the engineered
protein 134 reduces the binding distance of the target binding site
136 to approximately around 3 nm or less. Typically, the screening
length is depended on the background ion concentration where
reducing the ion concentration can increase the screening length.
In any case, the greater the distance of the binding site from the
surface, the lower the probability of detection of a binding event.
Therefore, reducing the distance of the binding site increases the
probability that a binding event will even be sensed/detected by
the sensor and is especially useful where solutions of high ion
concentrations are desired. As noted above, many conventional
receptor molecules also require an adhesion layer to immobilize the
receptor molecule onto the surface of the sensor. Such an
additional layer further increases the binding distance between the
binding site and the surface of the sensor 150. In contrast, the
use of an engineered protein 134 eliminates the need for such an
adhesion layer.
[0124] Moreover, use of a binding receptor that is free of
endogenous functional groups improves uniform surface
immobilization--meaning the binding receptor can be placed
uniformly. In many cases, use of a binding receptor that has one or
more targeted surface functional groups engineered directly onto
the binding receptor further decreases the distance to the sensor
interface and also allows for a highly uniform surface.
[0125] For example, FIG. 8B illustrates a conventional receptor
molecule 130 as having one or more endogenous functional groups 138
(e.g., cysteines) throughout the molecule 130. The presence of
these endogenous functional groups 138 introduces variability in
the location of the adhesion site relative to the surface of the
sensor. In certain cases, an additional adhesion layer is required
to improve uniformity. In contrast, the engineered protein 134
depicted in FIG. 8B can include a targeted functionalization group
135 to functionalize (or immobilize) the engineered protein 134
onto the desired surface of the sensor in a consistent or desired
manner. Although the targeted functional group 135 of the
engineered protein 134 is depicted to be located on an end of the
structure 134, one or more functionalization groups can be
positioned as desired on any respective part of the protein
structure 134.
[0126] In one example electrostatic- and charge-sensitive devices
used in a liquid as biochemical sensor can be covered with a thin
metal layer, such as gold or other metal that is resistant to
corrosion and oxidation in moist air (e.g., a noble metal). In
another variation, the same sensor can be covered with
nanoparticles, including but not limited to metal nanoparticles,
semiconductor layers, an insulator material, or a magnetic
material. Further any other shape of a material can be used to
cover the sensor. The improved binding receptor (e.g., the
engineered protein) is then directly attached to the metal layer or
nanoparticle using any conventional process (e.g. thiol chemistry
to bind to gold or other metal). In additional variations the
sensor does not require any metal layer. Instead, the sensor can be
functionalized on any number of surfaces on the sensor or next to
the sensor (e.g. oxide surface or any other type of surface).
[0127] FIG. 9 shows one example of a FET sensor 150 used to
determine a parameter of a substance in a test sample 140
(typically a liquid). The charge sensitive or electrostatic device
152 can be covered with a layer such as nanoparticles as described
above, or a metal, to form an extended gate 154. The extended gate
(e.g., the metal or nanoparticle layer) 154 is then covered with an
encapsulation layer 156 that expose a portion of the extended gate
154. The encapsulation layer 156 serves to protect the device from
being exposed to environment factors that would damage or degrade
the sensor (e.g, such as excess fluids or leakage currents). In
certain variations, the encapsulation layer 156 covers the whole
sensor chip/strip with an only opening for the extended gate area
or other geometry opening over the sensor area.
[0128] The encapsulation layer can be any material that is
unaffected or less affected by the environment of the test sample
or by general conditions prior to use of the FET sensor 150. The
improved binding receptor (such as the engineered protein) 160 is
then directly attached to the extended gate layer 154 using any
known technique (e.g., in cases of a gold gate layer, thiol
chemistry can bind the binding receptor 160 to the extended gate
154). The sensor can also include a non-conductive material 155,
including but not limited to a polymer, dielectric, oxide, nitride,
etc. As shown, the exposed gate 154 is in contact with the test
sample 140 that is functionalized with the engineered protein 160
as described above.
[0129] FIG. 10A illustrates another example that increases the
reliability of measurement of a substance in the test sample 140
using an assessment and/or comparison of signals between two
sensors. For example, the two sensors 150, 151 can both be covered
with an encapsulation layer such the described metal or
nanoparticle layer 154 where only one sensor 150 is functionalized
with the engineered protein 160 while the other sensor 151 is
either passivated (with a passivation layer) or is simply not
functionalized with any binding receptor 160. In the illustrated
example, the second reference sensor 151 can optionally include a
second passivation layer 162 that makes the sensor 151 just
inherent or prevents a reaction with the target moiety. In the
illustrated examples, the sensor 150 includes an extended gate 154
having a surface that is functionalized with the binding receptor
160, where the functionalized surface interacts with the sample
140.
[0130] As described above, the functionalized sensor 150 is
functionalized with binding receptors (e.g., the engineered
protein) that have at least one functional group associated
therewith such that the functional group on each of the binding
receptors permits securing each of the binding receptors to a first
layer of the sensor (in this case an extended gate 154) in a
desired manner (e.g., the functional groups can secure the binding
receptors in a uniform manner). When placed in contact with the
test sample, the binding receptor interacts with the substance in
the test sample such that interaction of the substance with the
functionalized sensor results in a change in the electrical
characteristic of the sensor (in this example the electrical
characteristic is affected by the change of the extended gate
across the sensor channel 158 or in its vicinity). This change
causes the sensor 152 to produce a changed electrical
characteristic that can be detected upon the application of an
electrical stimulus. Such a stimulus can include, but is not
limited to application of a voltage, current, frequency or other
signal allowing detection of the changed electrical characteristic
from the native electrical characteristic of the sensor. Comparison
of the changed electrical characteristic against the native
electrical characteristic or the control sensor allows determining
the parameter of the substance within the test sample.
[0131] FIG. 10B illustrates additional variations of structures for
determining a parameter of a substance in a test substance, the
device comprising two functionalized sensors 150, 153 and a control
sensor. 151. The illustrations are intended to show various
configurations of the sensors. As illustrated, the sensors can be
placed on substrate layer 180 that can comprise any material such
as an oxide layer or a polymer layer. In one example, the substrate
180 comprises silicon or a similar material that allows a voltage
to be applied through the substrate layer 180 as opposed to the
liquid sample 140. Next a dielectric layer 182 (such as an oxide
material) is applied to permit electrical isolation of the various
sensors. The sensor contacts 152 are located on the dielectric
layer 182 and are bridged with a sensor channel 158. The sensor
channel 158 can comprise any electrically conductive material,
structure, layer or coating that allows for electrical
communication between the sensor contacts 152. Next, a high-k
dielectric material 184 (including but not limited aluminum oxide,
titanium oxide, zirconium oxide, yttrium oxide, silicon oxide,
tantalum oxide, hafnium oxide and silicon nitride) is deposited
over the gate and/or sensor contacts to permit immobilization of
the functionalization layer or binding receptors and to reduce
potential leakage currents (as shown in sensor 150).
[0132] As noted above, variations of the device (e.g., sensor 153)
can include a high-k dielectric layer 184 with an additional metal
and/or nanoparticle layer 186 disposed on the high-k dielectric
layer 184 and a functionalization layer 160 immobilized on the
metal/nanoparticle layer 186. Alternatively, the layer 182 can be
completely removed.
[0133] Reference sensor 151 of FIG. 9B illustrates a passivation
layer 162 located on a high-k dielectric layer 184. However, in
additional variations, the reference sensor can include a
metal/nanoparticle layer 186 with or without a passivation layer
162. In either case, the reference sensor will not include a
functionalization layer.
[0134] As noted above, the test sample can comprise a bodily fluid,
or any other fluid that contains a substance that can be detected
upon binding to the binding receptor.
[0135] For example, engineered proteins can be used in conjunction
with the active or functionalized sensor 150 to detect biological
and chemical molecules from human or other animal bodily fluids,
including but not limited to urine, blood, saliva, tears,
ejaculate.
[0136] Engineered proteins can be engineered with targeted terminal
or internal functional groups such as cysteines and immobilized
onto a surface (e.g., gold or other noble metal) or suitable nano
particles of the active sensor (or an extension of the active
sensor such as an extended gate) using any known process for
immobilization.
[0137] In another variation, the engineered proteins can be
engineered with N- or C-terminal or internal cysteines and
immobilized onto the active sensor surface/nanoparticles modified
by self-assembled monolayers (SAMs) using thiol (--SH)
chemistry.
[0138] Engineered proteins can be immobilized onto the active
sensor surface/nanoparticles coated with carboxylic acid-SAM and
using amide coupling. Alternatively, any other type of SAM layer
can be used.
[0139] The functionalized sensors can use engineered proteins
engineered with terminal streptavidin and immobilized onto (solid
supports and) the active sensor surface/nanoparticles coated with
self biotin-SAMs.
[0140] Engineered proteins can be engineered with terminal
histidine tags and immobilized on (solid supports and) the active
sensor gold surface/gold nanoparticles coated with a Ni2+-NTA
(nitrilotriacetic acid) chelating moiety. In another application
the proteins can be engineered as biotin fusion proteins and
immobilized onto streptavidin functionalized surfaces (or vice
versa). Any number of tags can be engineered depending upon the
desired application.
[0141] Immobilization of the engineered proteins can occur via an
NH2-functionality onto the SiO2 surface of the active sensor by
silane chemistry. In another application, modification of the
charge of SiO2 can be made by application of short amphiphylic
synthetic peptides.
[0142] In an additional variation, engineered proteins are
generated or used as specific ligands for bacterial endo- and
exotoxins. In another application, endo- and exotoxin binding
(analyte binding) to the engineered protein happens directly on the
sensor surface biofunctionalized and activated with the engineered
capture proteins.
[0143] For example FIGS. 11A to 11C provide illustrations of a
binding receptor, such as the engineered protein, functionalized on
the sensor 152 (or on the extended gate, not shown).
[0144] FIG. 11A shows an analyte 170 (e.g., a substance contained
in the test sample) binding to a binding receptor 160 that is
immobilized on a surface of an active sensor 153 to produce a
change in the electrical characteristics electrical component of
the sensor.
[0145] FIG. 11B illustrates a variation of a functionalized sensor
150 where the binding receptor 160 is immobilized on a layer of the
sensor 153 (e.g., the gate, metal layer, and/or high-k dielectric
as described above) that allows binding of the analyte 170.
However, in this variation, a second binding receptor 172 (such as
a glucose oxidase tethered to the engineered protein) is introduced
to the sensor and binds to a different analyte 170 binding site.
Next, a substance can be added to enzymatically cause the second
binding receptor 172 to generate a reaction that can be measured by
the active sensor 153. In the present example, the addition of
glucose can cause a reaction that generates gluconic acid and
elicits a pH change that can be measured by the active sensor.
[0146] FIG. 11C shows another variation or application where endo-
and exotoxin binding to the binding receptor 160 occurs indirectly
on a sensor biofunctionalized with the binding receptor separate
from the active sensor 153. As shown, binding receptors 160
immobilized on the sensor 153 to bind their target analyte 160.
Next, a detection antibody 174 is introduced that binds to a
different site of the analyte molecule 170. Then, a secondary,
biotin labeled antibody 176 can be added that recognizes and binds
to the primary detection antibody 174. Next, a streptavidin
(SA)-bound glucose oxidase is introduced to bind biotin tethered to
the secondary antibody 176. This converts glucose to gluconic acid,
eliciting a pH change that can be measured by the active sensor in
a second location.
[0147] In another variation, an actual target molecule (i.e., the
substance to be detected by sensor) can be immobilized on the
sensor or substrate surface and the binding receptor (e.g. an
engineered protein or engineered scaffold protein, an antibody,
peptide etc.) is applied to bind the target molecule. When the
actual substance in a test sample is applied to the sensor, the
substance in the test sample competes for the bound engineered
protein, thereby releasing the bound receptor and producing a
change in the electric signal of the sensor as noted above.
[0148] The binding receptors can be generated or used as specific
ligand for a number of applications, including but not limited to
disease causing microorganisms including bacteria, yeast, fungi,
viruses, parasites; for yeast biomarker, fungal biomarker, viral
proteins; a specific ligand for tumor cells or other cells; as
specific ligands for disease-related and drug-related biomarkers
(proteins, antibodies, peptides, polysaccharides, lipids,
hormones); as specific ligand for small molecules (drugs,
therapeutics) or drugs subject to abuse; as specific ligand for
nucleic acids, as specific ligand for heavy metal ions; and as
specific ligand for multi-drug resistance proteins causing
resistance to distinct antimicrobials, antibiotics, antifungal
drugs, antiviral medications, antiparasitic drugs, chemicals of a
wide variety of structure and function targeted at eradicating the
organism.
[0149] Any of the functionalization schemes and reactions described
herein may take place in a first location remote from or separated
from the sensor or device located in a second location. The
reaction products or ions may then flow to and/or over the sensor
or device where detection takes place.
[0150] Each of the individual variations described and illustrated
herein has discrete components and features which may be readily
separated from or combined with the features of any of the other
variations. Modifications may be made to adapt a particular
situation, material, composition of matter, process, process act(s)
or step(s) to the objective(s), spirit or scope of the present
invention.
[0151] Methods recited herein may be carried out in any order of
the recited events which is logically possible, as well as the
recited order of events. Furthermore, where a range of values is
provided, every intervening value between the upper and lower limit
of that range and any other stated or intervening value in that
stated range is encompassed within the invention. Also, any
optional feature of the inventive variations described may be set
forth and claimed independently, or in combination with any one or
more of the features described herein.
[0152] All existing subject matter mentioned herein (e.g.,
publications, patents, patent applications and hardware) is
incorporated by reference herein in its entirety except insofar as
the subject matter may conflict with that of the present invention
(in which case what is present herein shall prevail). The
referenced items are provided solely for their disclosure prior to
the filing date of the present application. Nothing herein is to be
construed as an admission that the present invention is not
entitled to antedate such material by virtue of prior
invention.
[0153] Reference to a singular item, includes the possibility that
there are plural of the same items present. More specifically, as
used herein and in the appended claims, the singular forms "a,"
"an," "said" and "the" include plural referents unless the context
clearly dictates otherwise. It is further noted that the claims may
be drafted to exclude any optional element. As such, this statement
is intended to serve as antecedent basis for use of such exclusive
terminology as "solely," "only" and the like in connection with the
recitation of claim elements, or use of a "negative" limitation.
Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
[0154] This disclosure is not intended to be limited to the scope
of the particular forms set forth, but is intended to cover
alternatives, modifications, and equivalents of the variations
described herein. Further, the scope of the disclosure fully
encompasses other variations that may become obvious to those
skilled in the art in view of this disclosure. The scope of the
present invention is limited only by the appended claims.
* * * * *