U.S. patent application number 16/674770 was filed with the patent office on 2020-04-16 for system and method for detecting neural injury.
The applicant listed for this patent is CASE WESTERN RESERVE UNIVERSITY. Invention is credited to Chung-Chiun Liu.
Application Number | 20200115734 16/674770 |
Document ID | / |
Family ID | 54699690 |
Filed Date | 2020-04-16 |
United States Patent
Application |
20200115734 |
Kind Code |
A1 |
Liu; Chung-Chiun |
April 16, 2020 |
SYSTEM AND METHOD FOR DETECTING NEURAL INJURY
Abstract
A detection system for determining pyruvate dehydrogenase (PDH)
levels in a bodily sample includes at least one reaction solution
for generating NAD.sup.+ upon combination with PDH in the bodily
sample, the reaction solution including pyruvate and NADH and a
biosensor for determining the level of generated NAD.sup.+.
Inventors: |
Liu; Chung-Chiun;
(Cleveland, OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CASE WESTERN RESERVE UNIVERSITY |
Cleveland |
OH |
US |
|
|
Family ID: |
54699690 |
Appl. No.: |
16/674770 |
Filed: |
November 5, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15314393 |
Nov 28, 2016 |
10465229 |
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PCT/US15/32609 |
May 27, 2015 |
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16674770 |
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62003221 |
May 27, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/32 20130101; G01N
2800/28 20130101; G01N 27/301 20130101; G01N 27/327 20130101; G01N
2333/90203 20130101; G01N 27/3277 20130101 |
International
Class: |
C12Q 1/32 20060101
C12Q001/32; G01N 27/327 20060101 G01N027/327; G01N 27/30 20060101
G01N027/30 |
Claims
1. A detection system for determining pyruvate dehydrogenase (PDH)
levels in a bodily sample, comprising: at least one reaction
solution for generating NAD.sup.+ upon combination with PDH in the
bodily sample, the reaction solution including pyruvate and NADH;
and a biosensor for determining the level of generated
NAD.sup.+.
2. The detection system of claim 1, the bodily sample comprising a
bodily fluid selected from the group consisting of saliva, breath,
blood, plasma, sera, and urine.
3. The detection system of claim 1, being suitable for detection of
neural injury in a subject.
4. The detection system of claim 1, the neural injury comprising
traumatic brain injury.
5. The detection system of claim 1, the biosensor including a
working electrode formed on a surface of a substrate, a counter
electrode formed on the surface of the substrate, and a measuring
device for applying voltage potentials to the working electrode and
counter electrode and measuring the current flow between the
working electrode and counter electrode.
6. The detection system of claim 5, wherein the working electrode
and the counter electrode comprise metalized films.
7. The detection system of claim 5, wherein the working electrode
and counter electrode independently comprise gold, platinum,
palladium, silver, carbon, alloys thereof, and composites
thereof.
8. The detection system of claim 5, the metalized films are
provided on the surface of the substrate by sputtering or coating
the films on the surface and wherein the working electrode and the
counter electrode are formed using laser ablation
9. An assay for detecting neural injury in a subject comprising: at
least one reaction solution for generating NAD.sup.+ upon
combination with pyruvate dehydrogenase PDH in a bodily sample
obtained from the subject, the reaction solution including pyruvate
and NADH; and a biosensor for determining the level of generated
NAD.sup.+.
10. The assay of claim 9, the bodily sample comprising a bodily
fluid selected from the group consisting of saliva, breath, blood,
plasma, sera, and urine.
11. The assay of claim 9, the neural injury comprising traumatic
brain injury.
12. The assay of claim 9, the biosensor including a working
electrode formed on a surface of a substrate, a counter electrode
formed on the surface of the substrate, and a measuring device for
applying voltage potentials to the working electrode and counter
electrode and measuring the current flow between the working
electrode and counter electrode.
13. The assay of claim 12, wherein the working electrode and the
counter electrode comprise metalized films.
14. The assay of claim 12, wherein the working electrode and
counter electrode independently comprise gold, platinum, palladium,
silver, carbon, alloys thereof, and composites thereof.
15. The assay of claim 15, the metalized films are provided on the
surface of the substrate by sputtering or coating the films on the
surface and wherein the working electrode and the counter electrode
are formed using laser ablation.
Description
RELATED APPLICATION
[0001] This application claims priority from U.S. Provisional
Application No. 62/003,221, filed May 27, 2014, the subject matter
of which is incorporated herein by reference in its entirety.
BACKGROUND
[0002] Traumatic brain injury (TBI) and related head concussion are
a health issue of the civilian population due to car accidents, of
people participating in contact sports, such as football, soccer,
and hockey as well as a major health concern associated with the
United States military. 1.7 million people suffer from TBIs
annually in the United States alone. The cost of dealing with head
injury, including the athletes involved in football, is both
economically and emotionally expensive. TBI is the leading cause of
death and disability in children and young adults. In the U.S.,
nearly 1-2% of the population lives with a degree of TBI-related
disability.
[0003] Currently, the diagnosis of TBI-related symptoms is
generally accomplished by neurological examinations and
neuro-imaging tests, such as magnetic resonance imaging (MRI) and
computerized tomography (CT) scanning. These tests are expensive,
time-consuming, and require highly sophisticated equipment and
skillful operators. Furthermore, the results are not available in a
real time fashion, and the testing results are often inconclusive.
Furthermore, non-contrasted CT scan may be insensitive to mild
injury and can be obscured by extra-cranial injuries or the
necessity for sedation and airway protection. Timely management
decisions are critical in optimizing outcomes of TBI. Consequently,
the value of real time diagnostic assessment given by point-of care
testing of TBI is desirable to expedite appropriate treatment.
SUMMARY
[0004] Embodiments described herein relate to a detection system
and in vitro assay for detecting, identifying, quantifying, and/or
determining in a bodily sample the biomarkers of neural injuries,
such as traumatic brain injury, as well as to a detection system
and in vitro assay for diagnosing, identifying, staging, and/or
monitoring neural injuries, such as traumatic brain injury, in a
subject having or suspected of having a neural injury and/or
neuronal disorder, such as traumatic brain injury.
[0005] In some embodiments, the detection system includes at least
one reaction solution for generating nicotinamide adenine
dinucleotide (NAD.sup.+) upon combination with pyruvate
dehydrogenase (PDH) in a bodily sample and a biosensor for
determining the level of the generated NAD.sup.+. In some
embodiments, the at least one reaction solution can include
pyruvate and NADH in quantities effective to provide substrates for
reaction with PDH and the formation of lactate and NAD.sup.+.
[0006] In some embodiments, the bodily sample can include a bodily
fluid, such as saliva, blood, plasma, sera, breath, or urine, which
can potentially include PDH.
[0007] In other embodiments, the biosensor can include a substrate,
a working electrode formed on a surface of the substrate, a counter
electrode formed on the surface of the substrate and a dielectric
layer, which covers a portion of the working electrode and counter
electrode and defines an aperture exposing other portions of the
working electrode and counter electrode.
[0008] In still other embodiments, the working electrode and the
counter electrode can include metalized films. For example, the
working electrode and counter electrode can independently comprise
gold, platinum, palladium, silver, carbon, alloys thereof, and
composites thereof. The metalized films can be provided on the
surface of the substrate by sputtering or coating the films on the
surface and then laser ablating the films to form the working
electrode and counter electrode.
[0009] In other embodiments, the sensor can include a reference
electrode on the surface of the substrate. The dielectric can cover
a portion of the reference electrode. The sensor can also include a
measuring device for applying voltage potentials to the working
electrode and counter electrode and measuring the current flow
between the working electrode and counter electrode to determine
the level of NAD.sup.+ in a sample, such as a bodily sample.
[0010] In still other embodiments, the detection system can include
other biosensors that are used to detect other biomarkers, besides
PDH, which are indicative of neural injury, such as traumatic brain
injury, in bodily samples from the subject. These other biomarkers
can include, for example, S100B, neuron-specific enolase (NSE), and
sectretagogin (e.g., SCGN, SEGN, CALBL, or setgin). The biosensors
can include a substrate, a working electrode formed on a surface of
the substrate, a counter electrode formed on the surface of the
substrate, and a dielectric layer covering a portion of the working
electrode and counter electrode and defining an aperture exposing
other portions of the working electrode and counter electrode. The
working electrode can be functionalized or chemically
functionalized to include a receptor(s) for at least one of the
biomarkers of interest. The receptor can bind selectively to one or
more of the biomarkers of interest in the bodily sample.
[0011] The detection system can also include a measuring device for
applying a voltage potential to the working electrode, counter
electrode, and/or reference electrode and measuring the current
flow between the working electrode and counter electrode. The
interaction of the biomarker and the receptor, e.g., the bound
biomarker, can be detected using electrochemical analytical
techniques, such as cyclic voltammetry (CV), differential pulse
voltammetry (DPV), to determine the presence of the biomarker in
the bodily sample and whether the subject has a neural injury or
the extent of the neural injury of the subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 is a schematic illustration of a biosensor in
accordance with an aspect of the application.
[0013] FIG. 2 is a top plan view of an array of biosensors in a row
manufactured by a screen-printing process.
[0014] FIG. 3 illustrates plots showing cyclic voltammetric
measurements of pyruvate dehydrogenase over the concentration of
0-7.5 .mu.M. Both pyruvate and NADH are 375 .mu.M.
[0015] FIG. 4 illustrates a calibration curve of PDH over the
concentration of 0-7.5.+0.65 V is used as the potential for the
current of the cyclic voltammogram.
DETAILED DESCRIPTION
[0016] Unless specifically addressed herein, all terms used have
the same meaning as would be understood by those of skilled in the
art of the subject matter of the application. The following
definitions will provide clarity with respect to the terms used in
the specification and claims.
[0017] As used herein, the term "monitoring" refers to the use of
results generated from datasets to provide useful information about
an individual or an individual's health or disease status.
"Monitoring" can include, for example, determination of prognosis,
risk-stratification, selection of drug therapy, assessment of
ongoing drug therapy, determination of effectiveness of treatment,
prediction of outcomes, determination of response to therapy,
diagnosis of a disease or disease complication, following of
progression of a disease or providing any information relating to a
patient's health status over time, selecting patients most likely
to benefit from experimental therapies with known molecular
mechanisms of action, selecting patients most likely to benefit
from approved drugs with known molecular mechanisms where that
mechanism may be important in a small subset of a disease for which
the medication may not have a label, screening a patient population
to help decide on a more invasive/expensive test, for example, a
cascade of tests from a non-invasive blood test to a more invasive
option such as biopsy, or testing to assess side effects of drugs
used to treat another indication.
[0018] As used herein, the term "quantitative data" or
"quantitative level" or "quantitative amount" refers to data,
levels, or amounts associated with any dataset components (e.g.,
markers, clinical indicia,) that can be assigned a numerical
value.
[0019] As used herein, the term "subject" refers to a human or
another mammal, which can be afflicted with a neural injury, such
as a traumatic brain injury, but may or may not have such an
injury. Typically, the terms "subject" and "patient" are used
herein interchangeably in reference to a human individual.
[0020] As used herein, the term "injury or neural injury" is
intended to include a damage which directly or indirectly affects
the normal functioning of the central nervous system (CNS). For
example, the injury can be damage to retinal ganglion cells; a
traumatic brain injury (TBI); a stroke related injury; a cerebral
aneurism related injury; a spinal cord injury, including
monoplegia, diplegia, paraplegia, hemiplegia and quadriplegia; a
neuroproliferative disorder or neuropathic pain syndrome. Examples
of CNS injuries or disease include TBI, stroke, concussion
(including post-concussion syndrome), cerebral ischemia,
neurodegenerative diseases of the brain such as Parkinson's
disease, Dementia Pugilistica, Huntington's disease and Alzheimer's
disease, Creutzfeldt-Jakob disease, brain injuries secondary to
seizures which are induced by radiation, exposure to ionizing or
iron plasma, nerve agents, cyanide, toxic concentrations of oxygen,
neurotoxicity due to CNS malaria or treatment with anti-malaria
agents, trypanosomes, malarial pathogens, and other CNS
traumas.
[0021] As used herein, the term "stroke" is art recognized and is
intended to include sudden diminution or loss of consciousness,
sensation, and voluntary motion caused by rapture or obstruction
(e.g., by a blood clot) of an artery of the brain.
[0022] As used herein, the term "traumatic brain injury" is art
recognized and is intended to include the condition in which, a
traumatic blow to the head causes damage to the brain, often
without penetrating the skull. Usually, the initial trauma can
result in expanding hematoma, subarachnoid hemorrhage, cerebral
edema, raised intracranial pressure (ICP), and cerebral hypoxia,
which can, in turn, lead to severe secondary events due to low
cerebral blood flow (CBF).
[0023] As used herein, the term "subject suspected of having neural
injury" refers to a subject that presents one or more symptoms
indicative of neural injury, such as TBI, or that is being screened
for neural injury, such as TBI. A subject suspected of having
neural injury, such as TBI, may also have one or more risk factors.
The term encompasses individuals who have not been tested for
neural injury, such as TBI, and individuals who have received an
initial diagnosis but for whom the extent of the neural injury is
not known.
[0024] As used herein, the term "providing a prognosis" refers to
providing information regarding the impact of the presence of or
extent of neural injury, such as TBI, (e.g., as determined by the
methods described herein).
[0025] As used herein, the term "bodily sample" refers to a sample
that may be obtained from a subject (e.g., a human) or from
components (e.g., tissues) of a subject. The sample may be of any
biological tissue or fluid with which biomarkers described herein
may be assayed. Frequently, the sample will be a "clinical sample",
i.e., a sample derived from a patient. Such samples include, but
are not limited to, bodily fluids, e.g., saliva, breath, urine,
blood, plasma, or sera; and archival samples with known diagnosis,
treatment and/or outcome history. The term biological sample also
encompasses any material derived by processing the bodily sample.
Processing of the bodily sample may involve one or more of,
filtration, distillation, extraction, concentration, inactivation
of interfering components, addition of reagents, and the like.
[0026] As used herein, the terms "normal" and "healthy" are used
interchangeably. They refer to an individual or group of
individuals who have not shown any symptoms of neural injuries, and
have not been diagnosed with neural injuries. Preferably, the
normal individual (or group of individuals) is not on medication
affecting neural injuries. In certain embodiments, normal
individuals have similar sex, age, body mass index as compared with
the individual from which the sample to be tested was obtained. The
term "normal" is also used herein to qualify a sample isolated from
a healthy individual.
[0027] As used herein, the terms "control" or "control sample"
refer to one or more biological samples isolated from an individual
or group of individuals that are normal (i.e., healthy). The term
"control", "control value" or "control sample" can also refer to
the compilation of data derived from samples of one or more
individuals classified as normal, and/or one or more individuals
diagnosed with a neural injury.
[0028] As used herein, the term "indicative of neural injury", when
applied to an amount of at least one PDH in a bodily sample, refers
to a level or an amount, which is diagnostic of neural injury such
that the level or amount is found significantly more often in
subjects with the injury than in subjects without the injury (as
determined using routine statistical methods setting confidence
levels at a minimum of 95%). Preferably, a level or amount, which
is indicative of a neural injury, is found in at least about 60% of
subjects who have the neural injury and is found in less than about
10% of subjects who do not have the neural injury. More preferably,
a level or amount, which is indicative of neural injury, is found
in at least about 70%, at least about 75%, at least about 80%, at
least about 85%, at least about 90%, at least about 95% or more in
subjects who have the neural injury and is found in less than about
10%, less than about 8%, less than about 5%, less than about 2.5%,
or less than about 1% of subjects who do not have the neural
injury.
[0029] Embodiments described herein relate to a detection system
and in vitro assay for detecting, identifying, quantifying, and/or
determining in a bodily sample biomarkers of neural injuries, such
as traumatic brain injury, as well as to a detection system and in
vitro assay for diagnosing, identifying, staging, and/or monitoring
neural injuries, such as traumatic brain injury, in a subject
having, suspected of having a neural injury, such as traumatic
brain injury.
[0030] The detection systems and methods described herein provide a
single use, disposable, and cost-effective means for simple
point-of-care, real time assessment of neural injuries, such as
TBI, using bodily samples, such as bodily fluids, obtained by
non-invasive or minimally invasive means, which minimizes
complicated clinical procedures for detecting and monitoring TBI
and related brain concussion. Detection of the chemical biomarkers
of TBI has been attempted using a dipstick based immunoassay test.
Unfortunately, this test still required expensive and non-portable
spectrophotometric equipment. Also, the assessment is more
qualitative than quantitative and inconclusive.
[0031] In some embodiments, the detection systems and assays or
methods described herein include at least one reaction solution
that can be used to generate a detectable and/or quantifiable
analyte, which is indicative of the amount, concentration, or level
of pyruvate dehydrogenase in a bodily sample of a subject suspected
of having a neural injury, and a biosensor for detecting the
amount, level, or concentration of the analyte in the reaction
solution.
[0032] The detection of pyruvate dehydrogenase (PDH) can employ the
following reaction mechanism:
##STR00001##
[0033] In the above reaction, pyruvate and NADH are provided in a
reaction solution at quantities effective to act as substrates for
reaction with PDH in a bodily sample mixed with the reaction
solution, and for the production of NAD.sup.+. NAD+ is an
electro-chemically active species, which can be reduced at a given
Gibbs free energy, or in turn, an electrochemical potential. The
reduction current can be used to quantify the PDH involved in the
reaction at a fixed pyruvate and NADH concentration. The amount,
concentration, or level of NAD.sup.+ generated by biochemical
reaction of the reaction solution and PDH in the bodily sample
obtained from the subject suspected of having a neural injury can
be measured using a biosensor to determine the amount,
concentration, or level of PDH in the bodily fluid and hence
whether the subject has a neural injury.
[0034] The quantity of PDH in the bodily sample obtained from a
subject suspected of having neural injury can directly affect the
production of NAD.sup.+. Thus, the quantified level of NAD.sup.+
generated can be compared to a control or predetermined value to
determine the level of PDH in the bodily sample, and if or whether
the subject has a neural injury, such as TBI. For example, an
increase in the detected level of NAD.sup.+ in a bodily sample
mixed with the reaction solution compared to a control value is
indicative of the subject having a neural injury, such as TBI.
[0035] The reaction solution can be prepared, for example, by
mixing quantities of pyruvate and NADH with phosphate buffered
saline solution (PBS), so that the molar ratio between pyruvate and
NADH is about 1:1. PBS solution with a pH of 6.5 can be prepared by
mixing monobasic and dibasic sodium phosphates with deionized
water, and 200 mM of potassium chloride can be added as a
supporting electrolyte to improve conductivity of the buffer.
Advantageously, the reaction solution does not include any reagents
or byproducts that would potentially contribute to background
oxidation current of the biosensor and impair detection and
quantification of the NAD.sup.+ generated.
[0036] The reaction solution so formed can be mixed with a bodily
sample, such as a bodily fluid (e.g., saliva, blood, sera, plasma,
or urine), obtained from the subject. In some aspects, the bodily
fluid can be saliva that is obtained from a subject having or
suspected of having a neural injury. The amount of saliva obtained
from the subject can be about 0.1 ml or more. The obtained saliva
can then be added to the reaction solution. For example, the amount
of saliva added to about 6 .mu.l of the reaction solution can be
about 1 .mu.l or less.
[0037] The bodily samples can be obtained from the subject using
sampling devices, such as syringes, swabs or other sampling
devices, used to obtain liquid and/or solid bodily samples either
invasively (i.e., directly from the subject) or non-invasively.
These samples can then be stored in storage containers. The storage
containers used to contain the collected sample can include a
non-surface reactive material, such as polypropylene. The storage
containers are generally not made from untreated glass or other
sample reactive material to prevent the sample from becoming
absorbed or adsorbed by surfaces of the glass container.
[0038] Collected samples stored in the container may be stored
under refrigeration temperature. For longer storage times, the
collected sample can be frozen to retard decomposition and
facilitate storage. For example, samples obtained from the subject
can be stored in a falcon tube and cooled to a temperature of about
-80.degree..
[0039] The NAD.sup.+, which is generated by mixing of the bodily
sample containing PDH with the reaction solution, is an
electrochemically active species that can be oxidized or reduced
under appropriate conditions and detected using an NAD.sup.+
biosensor to quantify the level of PDH in the bodily sample and
determine whether the subject has a neural injury. In some
embodiments, the biosensor can include a two or three electrode
electrochemical biosensor.
[0040] FIG. 1 illustrates a biosensor 10 in accordance with an
embodiment of the application. The sensor 10 is a three-electrode
sensor including a counter electrode 12, a working electrode 14,
and a reference electrode 16 that are formed on a surface of a
substrate. A dielectric layer 40 covers a portion of the working
electrode 12, counter electrode 14 and reference electrode 16. The
dielectric layer 40 includes an aperture 20, which defines a
detection region of the working electrode 12, counter electrode 14,
and reference electrode 16 that is exposed to samples in which the
levels of PDH are detected.
[0041] A voltage source 22 is connected to the working and
reference electrodes 14, 16. A current measuring device 24 is
connected to the working and counter electrodes 14, 12 to measure
the current generated by the redox reaction of NAD.sup.+ when the
mixture of the reaction solution and bodily sample contacts the
detection region 20 of the sensor 10.
[0042] The working electrode 14 is the site of the redox reaction
of NAD.sup.+, and where the charge transfer occurs. The function of
the counter electrode 12 is to complete the circuit, allowing
charge to flow through the sensor 10. The working electrode 14 and
the counter electrode 12 are preferably formed of the same
material, although this is not a requirement. Examples of materials
that can be used for the working electrode 14 and counter electrode
12 include, but are not limited to, gold, platinum, palladium,
silver, carbon, alloys thereof, and composites thereof.
[0043] Examples of materials that can be used to form the reference
electrode 16 are silver-silver chloride and mercury-mercuric
chloride (Calomel). Silver-silver chloride is preferred. The silver
can be applied to a substrate in the form of a silver ink, which is
commercially available, or can be made using finely dispersed metal
particles, solvent, and a binder. Respective silver contact pads
30, 32, and 34 are connected with each of the electrodes 12, 14,
and 16. This reference electrode can be thick film printed on the
same substrate of the working and counter electrode and also can be
used externally.
[0044] In some embodiments, the working and counter electrodes 14,
12 can include a layer of particles, such as micro-, meso- or
nano-sized particles of active carbon or porous carbon. The active
carbon nanoparticles may be combined with metallic catalyst
particles that increase the rate of electrochemical
oxidation-reduction reaction with NAD.sup.+ and provide the
detection of NAD.sup.+ at a lower oxidation potential than without
the presence of the catalyst particles. In terms of the practical
applications, the metallic catalyst particles can shorten the
reaction time and lower the applied electrochemical potential for
detection of NAD.sup.+ in the mixture of the reaction solution and
biological sample. Lowering the applied potential often leads to
the minimization of electrochemical oxidation or reduction of other
species presented, resulting in a minimization of interference
caused by the unwanted reaction of the confounding species. As a
result, a highly specific biosensor can be obtained and
produced.
[0045] The metallic catalyst particles can include nano-, meso-, or
micro-scale particles of a unary metal (M), a binary metal (M-X), a
unary metal oxide (MOy), a binary metal oxide (MOy-XOy), a
metal-metal oxide composite material (M-MOy) or a combination of
which, wherein y is less than 3, and M and X are independently
selected from a group consisting of Li, Na, Mg, Al, K, Ca, Cr, Mn,
Fe, Co, Ni, Cu, Zn, Ga, Sr, Y, Zr, Nb, Mo, Ru, Rh, Pd, Ag, Cd, In,
Sn, Ba, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Lu, Ta, W,
Os, Ir, Pt, Au, and Pb. In one embodiment, for example, the
metallic catalyst particles may be composed of a unary metal, unary
metal oxide binary metal, or binary metal oxide, such as iridium,
iridium oxide, platinum, ruthenium, platinum-ruthenium,
platinum-nickel, and platinum-gold.
[0046] The voltage source can apply a voltage potential to the
working electrode 14 and reference and/or counter electrode 16, 12,
depending on the design of the sensor 10. The current between the
working electrode 14 and counter electrode 16 can be measured with
a measuring device or meter. Such current is due to the reduction
occurring at the working electrode 12 of NAD.sup.+ in the mixture
of the reaction solution and bodily sample and provided at the
detection region.
[0047] The amount or level of current measured is proportional to
the level or amount of NAD.sup.+ in the mixture of the reaction
solution and bodily sample. In some embodiments, where the sample
is a bodily sample obtained from a subject that has or is suspected
of having a neural injury, once the current level generated by the
reaction solution tested with the sensor is determined, the level
can be compared to a predetermined value or control value to
provide information for diagnosing or monitoring of the neural
injury in a subject. For example, the current level can be compared
to a predetermined value or control value to determine if a subject
has a TBI. An increased current level compared to a predetermined
value or control value can be indicative of the subject having TBI;
whereas similar or decreased current level compared to a
predetermined value or control value can be indicative of the
absence of TBI of the subject
[0048] The current level generated by the mixture of the reaction
solution and bodily sample obtained from the subject can be
compared to a current level of a mixture of the reaction solution
and bodily sample previously obtained from the subject, such as
prior to administration of a therapeutic. Accordingly, the methods
described herein can be used to measure the efficacy of a
therapeutic regimen for the treatment of a condition, pathology, or
disorder associated with neural injury in a subject by comparing
the current level obtained before and after a therapeutic regimen.
Additionally, the methods described herein can be used to measure
the progression of the neural injury in a subject by comparing the
current level in a bodily sample obtained over a given time period,
such as days, weeks, months, or years.
[0049] The current level generated by the mixture of the reaction
solution and bodily sample of the subject may also be compared to a
predetermined value or control value to provide information for
determining the severity of neural injury in the subject. A
predetermined value or control value can be based upon the current
level in comparable samples obtained from a healthy or normal
subject or the general population or from a select population of
control subjects.
[0050] The predetermined value can take a variety of forms. The
predetermined value can be a single cut-off value, such as a median
or mean. The predetermined value can be established based upon
comparative groups such as where the current level in one defined
group is double the current level in another defined group. The
predetermined value can be a range, for example, where the general
subject population is divided equally (or unequally) into groups,
or into quadrants, the lowest quadrant being subjects with the
lowest current level, the highest quadrant being individuals with
the highest current level. In an exemplary embodiment, two cutoff
values are selected to minimize the rate of false positive and
negative results.
[0051] The biosensor illustrated in FIGS. 1 and 2 can be fabricated
on a substrate 100 formed from polyester or other electrically
non-conductive material, such as other polymeric materials, alumina
(Al.sub.2O.sub.3), ceramic based materials, glass or a
semi-conductive substrate, such as silicon, silicon oxide and other
covered substrates. Multiple sensor devices 102 can thus be formed
on a common substrate 100 (FIG. 2). As will be appreciated,
variations in the geometry and size of the electrodes are
contemplated.
[0052] The biosensor can be made using a thin film, thick film,
and/or ink-jet printing technique, especially for the deposition of
multiple electrodes on a substrate. The thin film process can
include physical or chemical vapor deposition. Electrochemical
sensors and thick film techniques for their fabrication are
discussed in U.S. Pat. No. 4,571,292 to C. C. Liu et al., U.S. Pat.
No. 4,655,880 to C. C. Liu, and co-pending application U.S. Ser.
No. 09/466,865, which are incorporated by reference in their
entirety. By way of example, in the case of the carbon electrodes,
active carbon is mixed with a binder, deposited like an ink on the
substrate, and allowed to dry.
[0053] In some embodiments, the working electrode, counter
electrode, and reference electrode may be formed using laser
ablation, a process which can produce elements with features that
are less than one-thousandth of an inch. Laser ablation enables the
precise definition of the working electrode, counter electrode, and
reference electrode as well as electrical connecting leads and
other features, which is required to reduce coefficient of
variation and provide accurate measurements. Metalized films, such
as Au, Pd, and Pt or any metal having similar electrochemical
properties, that can be sputtered or coated on plastic substrates,
such as PET or polycarbonate, or other dielectric material, can be
irradiated using laser ablation to provide these features.
[0054] In one example, a gold film with a thickness of about 300 to
about 2000 A can be deposited by a sputtering technique resulting
in very uniform layer that can be laser ablated to form the working
and counter electrodes. The counter electrode can use other
materials. However, for the simplicity of fabrication, using
identical material for both working and counter electrodes will
simplify the fabrication process providing the feasibility of
producing both electrodes in a single processing step. An Ag/AgCl
reference electrode, the insulation layer, and the electrical
connecting parts can then be printed using thick-film screen
printing technique.
[0055] FIGS. 3 and 4 show that the detection system can be used to
detect PDH in a solution. In preliminary testing, 3 .mu.M of
pyruvate solution (concentration of 375 .mu.M) and 3 .mu.l of NADH
(concentration of 375 .mu.M) were used with a PDH volume of 2 .mu.l
at a concentration varying from 0 to 7.5 .mu.M. The results shown
in FIGS. 3 and 4 employed a phosphate buffer solution (PBS).
Similar to the preliminary test in PBS, initial investigation with
substrate material of 3 .mu.L of pyruvate solution (concentration
375 .mu.M) and 3 .mu.L of NADH (concentration 375 .mu.M) and a PDH
volume of 2 .mu.L with a concentration varying from 0-7.5 .mu.M
performed in artificial saliva instead of PBS can be used to show
saliva is a biological fluid that can be easily used to detect TBI
in real time.
[0056] In other embodiments, the detection system can include other
biosensors that can detect other biomarkers, besides PDH, which are
indicative of neural injury, such as traumatic brain injury, in
bodily samples from a subject. These other biomarkers can include,
for example, S100B, neuron-specific enolase (NSE), and
sectretagogin (e.g., SCGN, SEGN, CALBL, or setgin). These
biosensors can include a substrate, a working electrode formed on a
surface of the substrate, a counter electrode formed on the surface
of the substrate, a dielectric layer covering a portion of the
working electrode and counter electrode and defining an aperture
exposing other portions of the working electrode and counter
electrode. The working electrode can be functionalized or
chemically functionalized to include a receptor(s) for at least one
of the biomarkers of interest. The receptor can bind selectively to
one or more of the biomarkers of interest, which are indicative of
neural injury, in the bodily sample from the subject.
[0057] The detection system can also include a measuring device for
applying a voltage potential to the working electrode, counter
electrode, and/or reference electrode and measuring the current
flow between the working electrode and counter electrode. The
interaction of the biomarker and the receptor can be detected using
electrochemical analytical techniques, such as cyclic voltammetry
(CV), differential pulse voltammetry (DPV), to determine the
presence of the biomarker in the bodily sample and whether the
subject has a neural injury or the extent of the neural injury of
the subject.
[0058] In some embodiments, a receptor that binds selectively to a
biomarker, which is indicative of neural injury, is a molecule that
binds preferentially to that biomarker (i.e., its binding affinity
for that biomarker is greater than its binding affinity for any
other biomarker). Its binding affinity for the biomarker of
interest may be 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,
8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold,
40-fold, 50-fold, 100-fold or more than its binding affinity for
any other biomarker. In addition to its relative binding affinity,
the receptor must also have an absolute binding affinity that is
sufficiently high to efficiently bind the biomarker of interest
(i.e., it must have a sufficient sensitivity). Receptors having
binding affinities in the picomolar to micromolar range are
suitable. Such interaction can be reversible.
[0059] The receptor may be of any nature (e.g., chemical, nucleic
acid, peptide, lipid, combinations thereof and the like). The
biomarker, which is indicative of neural injury, too may be of any
nature provided there exists a receptor that binds to it
selectively and in some instances specifically.
[0060] The term "functionalized" or "chemically functionalized," as
used herein, means addition of functional groups onto the surface
of a material by chemical reaction(s). As will be readily
appreciated by a person skilled in the art, functionalization can
be employed for surface modification of materials in order to
achieve desired surface properties, such as biocompatibility,
wettability, and so on. Similarly, the term "biofunctionalization,"
"biofunctionalized," or the like, as used herein, means
modification of the surface of a material so that it has desired
biological function, which will he readily appreciated by a person
of skill in the related art, such as bioengineering.
[0061] The receptors may be functionalized to the working electrode
covalently or non-covalently. Covalent attachment of a receptor to
working electrode may be direct or indirect (e.g., through a
linker). Receptors may be immobilized on the working electrode
using a linker. The linker can be a linker that can be used to link
a variety of entities.
[0062] In some embodiments, the linker may be a homo-bifunctional
linker or a hetero-bifunctional linker, depending upon the nature
of the molecules to be conjugated. Homo-bifunctional linkers have
two identical reactive groups. Hetero-bifunctional linkers are have
two different reactive groups. Various types of commercially
available linkers are reactive with one or more of the following
groups: primary amines, secondary amines, sulphydryls, carboxyls,
carbonyls and carbohydrates. Examples of amine-specific linkers are
bis(sulfosuccinimidyl) suberate,
bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone, disuccinimidyl
suberate, disuccinimidyl tartarate, dimethyl adipimate 2HCl,
dimethyl pimelimidate 2HCl, dimethyl suberimidate HCl, ethylene
glycolbis-[succinimidyl-[succinate]], dithiolbis(succinimidyl
propionate), and 3,3'-dithiobis(sulfosuccinimidylpropionate).
Linkers reactive with sulfhydryl groups include bismaleimidohexane,
1,4-di-[3'-(2'-pyridyldithio)-propionamidol]butane,
1-[p-azidosalicylamido]-4-[doacetamido]butane, and
N-[4-(p-azidosalicylamido)butyl]-3'-[2'-pyridyldithio]propionamide
Linkers preferentially reactive with carbohydrates include
azidobenzoyl hydrazine. Linkers preferentially reactive with
carboxyl groups include 4-[p-azidosalicylamido]butylamine.
[0063] Heterobifunctional linkers that react with amines and
sulfhydryls include N-succinimidyl-3-[2-pyridyldithio]propionate,
succinimidyl[4-iodoacetyl]aminobenzoate, succinimidyl
4-[N-maleimidomethyl]cyclohexane-1-carboxylate,
m-maleimidobenzoyl-N-hydroxysuccinimide ester, sulfosuccinimidyl
6-[3-[2-pyridyldithio]propionamido]hexanoate, and sulfosuccinimidyl
4-[N-maleimidomethyl]cyclohexane-1-carboxylate. Heterobifunctional
linkers that react with carboxyl and amine groups include
1-ethyl-3-[3-dimethylaminopropyl]-carbodiimide hydrochloride.
Heterobifunctional linkers that react with carbohydrates and
sulfhydryls include
4-[N-maleimidomethyl]-cyclohexane-1-carboxylhydrazide HCl,
4-(4-N-maleimidophenyl)-butyric acid hydrazide.2HCl, and
3-[2-pyridyldithio]propionyl hydrazide.
[0064] Alternatively, receptors may be non-covalently coated onto
the working electrode. Non-covalent deposition of the receptor to
the working electrode may involve the use of a polymer matrix. The
polymer may be naturally occurring or non-naturally occurring and
may be of any type including but not limited to nucleic acid (e.g.,
DNA, RNA, PNA, LNA, and the like, or mimics, derivatives, or
combinations thereof), amino acid (e.g., peptides, proteins (native
or denatured), and the like, or mimics, derivatives, or
combinations thereof, lipids, polysaccharides, and functionalized
block copolymers. The receptor may be adsorbed onto and/or
entrapped within the polymer matrix.
[0065] Alternatively, the receptor may be covalently conjugated or
crosslinked to the polymer (e.g., it may be "grafted" onto a
functionalized polymer).
[0066] An example of a suitable peptide polymer is poly-lysine
(e.g., poly-L-lysine). Examples of other polymers include block
copolymers that comprise polyethylene glycol (PEG), polyamides,
polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene
oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl
ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone,
polyglycolides, polysiloxanes, polyurethanes, alkyl cellulose,
hydroxyalkyl celluloses, cellulose ethers, cellulose esters,
nitrocelluloses, polymers of acrylic and methacrylic esters, methyl
cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl
methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate,
cellulose propionate, cellulose acetate butyrate, cellulose acetate
phthalate, carboxylethyl cellulose, cellulose triacetate, cellulose
sulphate sodium salt, poly(methyl methacrylate), poly(ethyl
methacrylate), poly(butylmethacrylate), poly(isobutyl
methacrylate), poly(hexylmethacrylate), poly(isodecyl
methacrylate), poly(lauryl methacrylate), poly(phenyl
methacrylate), poly(methyl acrylate), poly(isopropyl acrylate),
poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene,
polypropylene, poly(ethylene glycol), poly(ethylene oxide),
poly(ethylene terephthalate), poly(vinyl alcohols), polyvinyl
acetate, polyvinyl chloride, polystyrene, polyhyaluronic acids,
casein, gelatin, glutin, polyanhydrides, polyacrylic acid,
alginate, chitosan, poly(methyl methacrylates), poly(ethyl
methacrylates), poly(butylmethacrylate), poly(isobutyl
methacrylate), poly(hexylmethacrylate), poly(isodecyl
methacrylate), poly(lauryl methacrylate), poly(phenyl
methacrylate), poly(methyl acrylate), poly(isopropyl acrylate),
poly(isobutyl acrylate), and poly(octadecyl acrylate),
poly(lactide-glycolide), copolyoxalates, polycaprolactones,
polyesteramides, polyorthoesters, polyhydroxybutyric acid,
polyanhydrides, poly(styrene-b-isobutylene-b-styrene) (SIBS) block
copolymer, ethylene vinyl acetate, poly(meth)acrylic acid, polymers
of lactic acid and glycolic acid, polyanhydrides,
poly(ortho)esters, polyurethanes, poly(butic acid), poly(valeric
acid), and poly(lactide-cocaprolactone), and natural polymers such
as alginate and other polysaccharides including dextran and
cellulose, collagen, albumin and other hydrophilic proteins, zein
and other prolamines and hydrophobic proteins, copolymers and
mixtures thereof, and chemical derivatives thereof including
substitutions and/or additions of chemical groups, for example,
alkyl, alkylene, hydroxylations, oxidations, and other
modifications routinely made by those skilled in the art.
[0067] In one particular embodiment, the working electrode can
comprise working electrdode that is crosslinked or biotinylated
chemically in order to allow attachment of an antibody or biotin
containing molecule. The gold working electrode can be cross-linked
for example with dithiolbis(succinimidyl propionate) (DSP), which
contains amine reactive N-hydroxysuccinimide (NHS) ester that can
react with amine groups of proteins and antibodies.
[0068] It will be appreciated, the flexibility of the chemical
functionalization makes the biosensor useful for attaching
essentially any ligand having an affinity for a biomarker. Examples
of biomarkers for which ligands having affinity therefore may be
attached to the working electrode include, but are not limited to
DNA, oligo-nucleotides, proteins, biotin, and streptavidin. Protein
ligands include monoclonal antibodies (mABs); however enzyme
substrates may also be used as ligands having affinity for a
corresponding enzyme.
[0069] In some embodiments, the working electrode is functionalized
with monoclonal antibodies or antigen binding fragments thereof by
means of a reactive amino group on the mAB. Because most antibodies
have lysine groups, they can be attached to the device at the
lysine amino group.
[0070] The chemical functionalization method also enables the
bioconjugation of DNA aptamers having an amino group. These
aptamers could potentionally bind small molecules and proteins.
Once bound, the change in the charge on the surface of the working
electrode would enable the device to detect the target biomolecule
or small molecule.
[0071] Similar to the biosensor shown in FIG. 1 for the detection
of PDH, the biosensors for the detection of other biomarkers,
besides PDH, which are indicative of neural injury can be made
using a thin film, thick film, and/or ink-jet printing technique,
especially for the deposition of multiple electrodes on a
substrate. The thin film process can include physical or chemical
vapor deposition.
[0072] In some embodiments, the working electrode, counter
electrode, and reference electrode of these sensors may be formed
using laser ablation, a process which can produce elements with
features that are less than one-thousandth of an inch. Metalized
films, such as Au, Pd, and Pt or any metal having similar
electrochemical properties, that can be sputtered or coated on
plastic substrates, such as PET or polycarbonate, or other
dielectric material, can be irradiated using laser ablation to
provide these features.
[0073] In other embodiments, the detection system can include a
plurality of biosensors that can be provided in an array on a
surface of a substrate. The biosensor array can be configured to
detect NAD+ concentration changes as well as the concentrations of
other analytes indicative neural injury in a bodily sample of a
subject. The biosensor array can include a plurality biosensors
arranged in a plurality of rows and a plurality of columns Each
biosensor comprises on a working electrode and a counter electrode.
The working electrode can be functionalized or chemically
functionalized to include a receptor(s) for at least one of the
analytes of interest. The receptors can be the same or different
for each biosensor of the array and can bind selectively to one or
more of the analytes of interest. The biosensors of the array can
be configured to provide at least one output signal representing
the presence and/or concentration of an analyte in the bodily
sample. For each column of the plurality of columns or for each row
of the plurality of rows, the array further comprises column or row
circuitry configured to provide voltage potentials to respective
biosensors in the column or row. Each biosensor in the row or
column can potentially detect a different analyte and/or biased to
detect different analytes.
[0074] From the above description of the invention, those skilled
in the art will perceive improvements, changes and modifications.
Such improvements, changes and modifications within the skill of
the art are intended to be covered by the appended claims. All
references, publications, and patents cited in the present
application are herein incorporated by reference in their
entirety.
* * * * *