biosensors

Article
A Single-Use, In Vitro Biosensor for the Detection of
T-Tau Protein, A Biomarker of Neuro-Degenerative
Disorders, in PBS and Human Serum Using
Differential Pulse Voltammetry (DPV)
Yifan Dai 1 , Alireza Molazemhosseini 2 and Chung Chiun Liu 1, *
1 Department of Chemical & Biomolecular Engineering and Electronics Design Center,
Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106, USA; yxd176@case.edu
2 Dip. Chimica Materiali e Ing. Chimica “Giulio Natta”, Politecnico di Milano, Via Mancinelli 7, 20131, Italy;
axm1058@case.edu
* Correspondence: cxl9@case.edu; Tel.: +1-216-368-2935

Academic Editor: Jeff D. Newman

Received: 7 November 2016; Accepted: 14 February 2017; Published: 19 February 2017

Abstract: A single-use, in vitro biosensor for the detection of T-Tau protein in phosphate-buffer
saline (PBS) and undiluted human serum was designed, manufactured, and tested. Differential
pulse voltammetry (DPV) served as the transduction mechanism. This biosensor consisted of three
electrodes: working, counter, and reference electrodes fabricated on a PET sheet. Both working and
counter electrodes were thin gold film, 10 nm in thickness. Laser ablation technique was used to define
the size and structure of the biosensor. The biosensor was produced using cost-effective roll-to-roll
process. Self-assembled monolayers (SAM) of 3-mercaptopropionic acid (MPA) were employed to
covalently immobilize the anti-T-Tau (T-Tau antibody) on the gold working electrode. A carbodiimide
conjugation approach using N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC)
and N–hydroxysuccinimide (NHS) cross-linked anti-T-Tau to the carboxylic groups on one end
of the MPA. A T-Tau protein ladder with six isoforms was used in this study. The anti-T-Tau
concentration used was 500,000 pg/mL. The T-Tau protein concentration ranged from 1000 pg/mL to
100,000 pg/mL. DPV measurements showed excellent responses, with a good calibration curve. Thus,
a practical tool for simple detection of T-Tau protein, a biomarker of neuro-degenerative disorders,
has been successfully developed. This tool could also be extended to detect other biomarkers for
neuro-degenerative disorders, such as P-Tau protein and β-amyloid 42.

Keywords: T-Tau protein detection; differential pulse voltammetry; [Fe(CN)6 ]3−/4− redox probe;
3-mercaptopropionic acid (MPA)

1. Introduction
Neurological degenerative disorders are a frightening health burden. These disorders include
Alzheimer’s disease (AD), traumatic brain injury (TBI), different forms of dementia including
Parkinson’s disease, and Creutzfeldt–Jacob disease, among others. Both the physical and
socio-economic burden of neuro-degenerative disorders are immense. For example, there were
currently 5.2 million Americans living with Alzheimer’s disease in 2016, and this number will increase
to 23.8 million in 2050. The cost of caring for Alzheimer’s patients in the U.S. is estimated to be
$236 billion in 2016 [1]. Furthermore, over 40% of family caretakers report that the emotional stress of
their role is high to very high. TBI is a major cause of long-term disability, affecting 100,000 individuals
in the U.S. annually. There are no officially established diagnostic methods for TBI. The annual cost

Biosensors 2017, 7, 10; doi:10.3390/bios7010010 www.mdpi.com/journal/biosensors

Biosensors 2017, 7, 10 2 of 11

of TBI in U.S. is estimated to be $48.3 billion, including $31.7 billion spent on hospital costs, while
$16.6 billion goes toward costs associated with fatalities [2].
Researchers have scientifically assessed amyloidopathy and tauopathy and their effects on
neuro-degenerative disorders. In amyloidopathy, the formation and characterization of β-amyloid
42 biomolecules are of significance [3–6]. In tauopathy, the microtubule associate protein, Tau, which
binds to and stabilizes microtubules axons, is the protein of focus. The levels of total Tau (T-Tau) and
the phosphorylated Tau (P-Tau) are considered useful biomarkers for assessing neuro-degenerative
disorders [7–11]. It is generally agreed that any additional tools for the detection of these biomolecules,
β-amyloid 42, T-Tau, and P-Tau, will be very useful in evaluating neuro-degenerative disorders.
Tau protein is expressed in the neurons of central nervous system and it is critically important in axonal
maintenance and axonal transport [12]. T-Tau and P-Tau levels in brain, blood, and cerebrospinal fluid
related to neuro-degenerative disorders are well recognized [12,13].
Cerebrospinal fluid (CSF) is commonly chosen as the physiological fluid test medium for the
assessment of β-amyloid 42, T-Tau, P-Tau, and others. CSF provides good information for neurological
related phenomena [5], but the collection of CSF is an elaborate and complicated process. On the
other hand, the collection of a small blood sample (20–30 µL) is a minimally invasive procedure and
can be administered relatively simply. Measurements of Tau level in blood as a reliable biomarker
for Alzheimer’s disease have been postulated and reported [13–15]. The technical challenge will be
providing additional tools for the detection of these biomarkers in a simple and accurate manner.
Diagnostic analysis of T-Tau protein in blood samples on brain injury in concussed ice hockey
players by the Simoa technique has been reported [16]. This detection was very time consuming, and
the blood samples were collected at 1, 12, 36, and 48 h and collected in an ethlenediaminetetracetic
acid (EDTA) tube, and an immuno-assay was then employed in the blood sample analysis. The results
of this analysis can be very informative, and it is very elaborate, expensive, and time-consuming.
Thus, the development of a cost-effective, single-use, disposable in vitro biosensor system which can
measure T-Tau in blood accurately can be a first step in providing a practical and useful tool in the
assessment of neuro-degenerative disorders, and it is the objective of this study. The bio-recognition
mechanism of this biosensor system is based on the antibody and antigen interaction and the effect of
a [Fe(CN)6 ]3−/4− redox probe by this interaction. The transduction mechanism is the electrochemical
differential pulse voltammetry (DPV) technique. The biosensor was manufactured using a cost-effective
roll-to-roll process. Both the working and the counter electrodes were thin gold films, and the reference
electrode was a thick-film-printed Ag/AgCl electrode. The Thiol based chemical functionalization step
was used to link the antibody to the gold electrode surface. Activation of the carboxylic group on one
end of the MPA molecule for the immobilization of anti-T-Tau (antibody of T-Tau) was accomplished by
the carbodiimide conjugation technique and showed excellent MPA surface coverage of the biosensor
by X-ray photoelectron spectroscopy (XPS) characterization. DPV measurements of T-Tau antigen in
both 0.1 M PBS and undiluted human serum showed excellent results. Therefore, the results of this
study suggested that a useful tool for the single-use, disposable in vitro measurement of T-Tau to assess
neuro-degenerative disorders including Alzheimer’s disease, TBI, and other dementia symptoms in
blood serum was feasible.
Diagnosis of T-Tau in one of the neuro-degenerative disorders, Creutzfeldt–Jacob disease, in
cerebrospinal fluid used a 1400 pg/mL as the cutoff level of T-Tau in CSF based on Swedish Mortality
Registry [17]. Thus, the level of 1400 pg/mL of T-Tau in a physiological fluid is used as the guide for
identifying neuro-degenerative disorders.

2. Materials and Methods

2.1. Apparatus and Reagents

Tau protein, 6 isoforms (Cat. No. T7951), and anti-T-Tau (Cat. No. SAB 5500182) of
rabbit monoclonal antibody were both obtained from Sigma Aldrich (St. Louis, MO, USA).
Biosensors 2017, 7, 10 3 of 11

Biosensors 2017, 7, 10 3 of 11

Phosphate-buffer saline (PBS) 1.0 M (pH 7.4), human serum, 3-mercaptopropionic acid (MPA),
dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC), and N–hydroxysuccinimide
N-(3 dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC), and N–hydroxysuccinimide
(NHS) were also purchased from Sigma-Aldrich (St. Louis, MO, USA). Potassium hydroxide pellets,
(NHS) were also purchased from Sigma-Aldrich (St. Louis, MO, USA). Potassium hydroxide pellets,
concentrated H2SO4 (95.0 to 98.0 w/w %), and concentrated HNO3 (70% w/w %) were obtained from
concentrated H2 SO4 (95.0 to 98.0 w/w %), and concentrated HNO3 (70% w/w %) were obtained from
Fisher Scientific (Pittsburgh, PA.). Recombined human beta-amyloid 42 was used in the interference
Fisher Scientific (Pittsburgh, PA.). Recombined human beta-amyloid 42 was used in the interference
study (Cat. No. ab82795) ABCAM, (Cambridge, MA, USA). All chemicals were used without further
study (Cat. No. ab82795) ABCAM, (Cambridge, MA, USA). All chemicals were used without further
purification. A CHI 660C (CH Instrument, Inc., Austin, TX, USA) Electrochemical Workstation was
purification. A CHI 660C (CH Instrument, Inc., Austin, TX, USA) Electrochemical Workstation
used for DPV and EIS investigations. All experiments were conducted at room temperature. X-ray
was used for DPV and EIS investigations. All experiments were conducted at room temperature.
photoelectron spectroscopy (XPS) was performed by a PHI Versaprobe 5000 Scanning X-Ray
X-ray photoelectron spectroscopy (XPS) was performed by a PHI Versaprobe 5000 Scanning X-Ray
Photoelectron Spectrometer.
Photoelectron Spectrometer.

2.2.
2.2. Design
Design of
of the
the Biosensor
Biosensor
This
This biosensor
biosensor prototype
prototype forfor the
the detection
detectionof ofT-Tau
T-Tau was
was based
based onon aa platform
platform biosensor
biosensor design.
design.
It
It has
has aa three-electrode
three-electrode configuration.
configuration. Both
Both the
the working
working andand counter
counter electrodes
electrodes were
were thin
thin gold
gold films
films
10 nm in thickness. The gold film was deposited by sputtering physical vapor deposition
10 nm in thickness. The gold film was deposited by sputtering physical vapor deposition at an atomic at an atomic
level.
level. No
No binder
binder was
was employed
employed in in any
anythick-film-printed
thick-film-printed biosensor
biosensor including
including commercially
commercially available
available
three-electrode based biosensors. Therefore, the surface of the gold film working
three-electrode based biosensors. Therefore, the surface of the gold film working electrode was uniform electrode was
uniform and reproducible. The reference electrode was a thick-film-printed
and reproducible. The reference electrode was a thick-film-printed Ag/AgCl electrode. The whole Ag/AgCl electrode. The
whole biosensor
biosensor was formed
was formed on a polyethylene
on a polyethylene terephalate
terephalate (PET)(PET) substrate.
substrate. LaserLaser ablation
ablation technique
technique was
was used to define the size and dimensions of the biosensor and its electrode
used to define the size and dimensions of the biosensor and its electrode elements. The insulator elements. The insulator
was
was aa thick-film-printed
thick-film-printed silicon-free
silicon-free dielectric
dielectric layer
layer made
made ofof Nazdar
Nazdar APLAPL 34 34ink
ink(Shawnee,
(Shawnee,KS, KS,USA).
USA).
This
This was
was aa cost-effective
cost-effectiveroll-to-roll
roll-to-rollfabrication
fabricationprocess.
process.InInthisthisstudy,
study,100100individual
individualbiosensors
biosensors inin
4
rows were produced on each PET substrate (355 × 280 mm 2).
2 Figure 1 shows the
4 rows were produced on each PET substrate (355 × 280 mm ). Figure 1 shows the structure and actual structure and actual
dimensions
dimensions of of this
this biosensor.
biosensor. The overall dimensions
The overall dimensions of of an
an individual
individual biosensor
biosensor werewere 33.0
33.0 ×× 8.0
8.0 mm
mm2. .
2

The
The working
working electrode
electrode area
area was
was 1.54
1.54 mm
mm2 accommodating
2
accommodating 20–25 20–25 µL µL ofof liquid
liquid test
test sample
sample required
required in in
this study. A more detailed description of the design and fabrication process of
this study. A more detailed description of the design and fabrication process of this biosensor has been this biosensor has
been presented
presented elsewhere
elsewhere [18]. [18].

Figure 1. Structure and dimensions of the thin film gold-based T-Tau biosensor prototype [19].
Figure 1. Structure and dimensions of the thin film gold-based T-Tau biosensor prototype [19].

2.3. Pretreatment
2.3. Pretreatment of
of the
the Biosensor
Biosensor
Result reproducibility
Result reproducibilityof ofthe
thebiosensor
biosensorprototype
prototypeisisimportant
important forfor any
any accurate
accurate measurement
measurement of
of the analyte (Tau antigen in this study). Thus, a pretreatment experimental
the analyte (Tau antigen in this study). Thus, a pretreatment experimental protocol intendedprotocol intended toto
enhance the reproducibility of this thin gold film-based biosensor was established based
enhance the reproducibility of this thin gold film-based biosensor was established based on previous on previous
reports [20–21].
reports [20,21]. Since
Since the
the gold
gold film
film electrodes
electrodes used in this
used in this study
study were
were relatively
relatively thin,
thin, and
and in
in order
order to
to
maintain the integrity of the biosensor prototype, modifications of the pretreatment procedure
maintain the integrity of the biosensor prototype, modifications of the pretreatment procedure were were
needed in
needed in comparison
comparison to to the
the bulk
bulk gold
gold nanoparticles
nanoparticles oror to
to the
the treated
treated particles.
particles. This
This cleaning
cleaning process
process
would decrease
would decreasethetheelectrode
electrodecharge transfer
charge resistance,
transfer thus improving
resistance, the sensitivity
thus improving of the biosensor.
the sensitivity of the
biosensor. In a typical pretreatment procedure, a row of 5 or 7 biosensors was immersed in a 2 M
KOH solution for 15 min. After rinsing with copious amount of DI water, the biosensors were placed
Biosensors 2017, 7, 10 4 of 11

Biosensors 2017, 7, 10 4 of 11
In a typical pretreatment procedure, a row of 5 or 7 biosensors was immersed in a 2 M KOH solution
in a1520-fold
for diluted
min. After concentrated
rinsing H2SOamount
with copious 4 solutionof(95.0 to 98.0
DI water, thew/w %) for another
biosensors 15 min.
were placed in aDI20-fold
water
was then used to rinse the biosensor prototypes. The biosensors were then placed in a 20-fold
diluted concentrated H2 SO4 solution (95.0 to 98.0 w/w %) for another 15 min. DI water was then used diluted
concentrated
to HNO3 solution
rinse the biosensor (70%The
prototypes. w/wbiosensors
%) for another
were15 min.
then The biosensors
placed in a 20-foldwere rinsed
diluted once more
concentrated
and then
HNO 3
dried
solution by a
(70% gentle
w/w flow
%) of
for nitrogen
another gas.
15 This
min. pretreatment
The biosensors procedure
were resulted
rinsed once in a
more significant
and then
decrease in electrode charge transfer resistance, enhancing the sensitivity and reproducibility
dried by a gentle flow of nitrogen gas. This pretreatment procedure resulted in a significant decrease of the
biosensor.
in electrode charge transfer resistance, enhancing the sensitivity and reproducibility of the biosensor.

2.4. Characterization of
2.4 Characterization of the
the Surface
Surface Area
Responses
Responses of of biosensors
biosensors were
were characterized
characterized by by assessing
assessing thethe stability
stability and
and reproducibility
reproducibility ofof the
the
electrochemical
electrochemical surface area. CyclicCyclicvoltammograms
voltammogramswere wereobtained
obtainedatatscan
scanrates
rates ranging
ranging from
from 3030to
to
100100 mV/s
mV/s in ainsolution
a solution
of Kof3Fe(CN)
K3 Fe(CN) 6 and
6 and K4 Fe(CN)
K4Fe(CN) 6 , 5 mM
6, 5 mM in each
in each component,
component, having
having 0.1 M0.1KCl
M
(Figure
KCl 2a). As
(Figure 2a).presented in Figure
As presented 2b, oxidation
in Figure peak current
2b, oxidation presented
peak current a lineara relationship
presented versus
linear relationship
the square
versus root of root
the square the scan rate.
of the Assuming
scan the diffusion
rate. Assuming coefficient
the diffusion of ferricyanide
coefficient ion to be ion
of ferricyanide constant,
to be
this linearthis
constant, relationship demonstrated
linear relationship the stability
demonstrated theof electro-active
stability surface area
of electro-active based
surface on based
area the Randles–
on the
Sevcik equation.equation.
Randles–Sevcik Using theUsing
equation, the calculated
the equation, electro-active
the calculated surface area
electro-active showed
surface less thanless
area showed 2%
relative
than 2% standard deviation
relative standard from sensor
deviation fromtosensor
sensorto(nsensor
= 3), signifying high reproducibility.
(n = 3), signifying high reproducibility.

Figure 2.
Figure 2. (a) Cyclic
Cyclicvoltammograms
voltammogramsobtained
obtainedinina asolution
solutionofof
K3K
Fe(CN) 6 and
3 Fe(CN) K4Fe(CN)
6 and 6, 5 mM
K4 Fe(CN) in each
6 , 5 mM in
component, having 0.1 M KCl at scan rates ranging from 30 to 100 mV/s and (b) oxidation
each component, having 0.1 M KCl at scan rates ranging from 30 to 100 mV/s and (b) oxidation peak peak current
plotted plotted
current versus square
versus root of scan
square rate.
root of scan rate.

2.5. Immobilization
2.5. Immobilization of
of T-Tau
T-TauAntibody
Antibodyonto
ontothe
theGold
GoldWorking
WorkingElectrode
Electrodeofofthe
theBiosensor
Biosensor
In aa typical
In typical experiment,
experiment, 55 to to 77 biosensors
biosensors were subjected to
were subjected to surface
surface modification
modification in in aa single
single batch.
batch.
SAM of MPA was employed to covalently immobilize anti-T-Tau on the surface of the gold electrode.
SAM of MPA was employed to covalently immobilize anti-T-Tau on the surface of the gold electrode.
TheMPA
The MPAmolecule
moleculeconsisted
consistedofofa athiol
thiolfunctional
functionalgroup
group atat one
one end,
end, which
which processed
processed a great
a great affinity
affinity to
to gold, and a carboxylic group at another end which was suitable for bonding
gold, and a carboxylic group at another end which was suitable for bonding covalently to proteins covalently to proteins
throughaa peptide
through peptide bond
bond after
after an
an activation
activation procedure.
procedure. ThiolThiol modification
modificationof of the
the gold
gold electrode
electrode surface
surface
for protein
for protein immobilization
immobilization was was aa well-developed
well-developed technique
technique [22–24].
[22–24]. The
The biosensors
biosensors were
were immersed
immersed
in 1 mM solution of MPA in pure ethanol for 24 h, rinsed with DI water,
in 1 mM solution of MPA in pure ethanol for 24 h, rinsed with DI water, and dried in a steam ofand dried in a steam of N
N22..
The MPA-modified biosensors were incubated in 0.1 M PBS (pH = 7.4)
The MPA-modified biosensors were incubated in 0.1 M PBS (pH = 7.4) containing 0.25 M EDC and containing 0.25 M EDC and
0.05 M
0.05 M NHS
NHS for for 55 hh to
toactivate
activateMPAMPAcarboxylic
carboxylicgroups.
groups.The Theactivated
activatedbiosensors
biosensors were
werethen
thenrinsed by
rinsed
0.1 M PBS and dried by N 2 flow. 5 µL of 0.05 mg/mL anti-T-Tau was then casted on the sensing area
by 0.1 M PBS and dried by N2 flow. 5 µL of 0.05 mg/mL anti-T-Tau was then casted on the sensing
of each
area biosensor
of each biosensorandandleftleft
to dry
to dry overnight
overnight atat4 4°C.
◦ C.Antibody-immobilized
Antibody-immobilizedbiosensors
biosensors were rinsed
were rinsed
with 0.1 M PBS to remove loosely bonded proteins. The biosensors were then dried again undera
with 0.1 M PBS to remove loosely bonded proteins. The biosensors were then dried again under
asteam
steamofofNN 2 and stored at 4 °C. ◦ This immobilization process was very similar to one described
2 and stored at 4 C. This immobilization process was very similar to one described
elsewhere [19]. XPS was used to characterize the MPA-SAM coverage of the gold working electrode
(results not shown). Identical to our previous study [19], the surface coverage of MPA-SAM was high
and the formation of Au–S covalent bond together with the upward orientation of MPA-SAM
Biosensors 2017, 7, 10 5 of 11

elsewhere [19]. XPS was used to characterize the MPA-SAM coverage of the gold working electrode
(results not shown). Identical to our previous study [19], the surface coverage of MPA-SAM was
high and the formation of Au–S covalent bond together with the upward orientation of MPA-SAM
carboxylic groups, were confirmed. This showed that the immobilization procedure was effective.
The XPS results were in agreement with those reported by others [25–28]. Therefore, the anti-T-Tau
bonded biosensor was ready for T-Tau antigen detection.

2.6. Differential Pulse Voltammetry (DPV) Measurement

DPV is a well-established electroanalytical technique [29]; however, its applications to biomedical
measurement has not been fully exploited. Cyclic voltammetry (CV) and chronoamperometry (CA)
are generally used in biomedical measurements. Both CV and CA provide sufficient sensitivity in
practical biomedical applications. The required electronic interface for CV and CA are relatively simple.
However, DPV applies a series of regular potential pulse superimposed on the potential stair steps.
The current is then measured immediately prior to each potential change. Consequently, the charging
current can be minimized, resulting in a higher sensitivity. It is based on this technical advantage
that DPV was used for the detection of T-Tau using our biosensor system. The anti-T-Tau was first
bonded and functionalized as described above. The biosensors were then incubated in solutions of
T-Tau with different concentrations for 3 h at room temperature. Antigen solutions were prepared both
in 0.1 M PBS and undiluted human serum. After the incubation, biosensors were rinsed with 0.1M PBS
removing any unbonded T-Tau. A solution of K3 Fe(CN)6 and K4 Fe(CN)6 , 5 mM in each component,
was prepared in 0.1 M PBS, 20 µL of this redox probe was dropped to the sensing area of the biosensor,
and the DPV measurement then took place.
In this study, an Electrochemical Workstation (CHI 660 model C) was used. The software of the
Workstation provided direct experimental setting for the DPV measurement. In a typical measurement,
the initial potential was set at −0.3V and the final potential was set at +0.3 V. The potential increase was
set at 0.004 V, the amplitude at 0.05 V, and the pulse width at 0.05 s. The pulse period was set at 0.2 s.

3. Results and Discussion

3.1. Evaluation of the Pretreatment Procedure by Electrochemical Impedance Spectroscopy (EIS)

In order to validate the enhancement of the sensor response, reproducibility, and electron charge
transfer due to the cleaning procedure, electrochemical impedance spectroscopy (EIS) was employed
for two groups of sensors consisting of four sensors each. Sensors in Group 1 were subjected to the
cleaning protocol described above, whereas sensors in Group 2 were cleaned by ethanol and deionized
water (DIW) sequentially. A solution of K3 Fe(CN)6 and K4 Fe(CN)6 , 5 mM of each component, was
prepared in 0.1 M PBS and used for EIS tests. Twenty microliters of redox couple solution was casted
on the sensing area of each sensor for EIS. Figure 3 presents the EIS results obtained for the two groups
of sensors in the form of a Nyquist plot using a frequency range of 10−2 to 104 Hz with 5 mV voltage
amplitude. An equivalent electrical circuit model was fitted to EIS data using EC-lab software. Randles
equivalent circuit was selected to model the experimental data. In a typical EIS measurement, the
initial potential was set at 0.0 V. The high frequency was set at 10,000 Hz and the low frequency was
set at 0.01 Hz. The amplitude was set at 0.005 V and the quiet time was set at 2 s.
Considering the physical structure of the interface, each component in the Randles circuit
represents an element in the actual electrode/analyte physical interface. The semicircular region
of the Nyquist plots associated with the electron transfer processes was modeled by a parallel
circuit representation of a resistor (R2 ) and the constant phase element (CPE). The tail at the lower
frequencies indicated the presence of diffusion limited electrochemical processes, represented using
the Warburg element (W2 ). The solution resistance was represented by R1 . Table 1 presents the
calculated R2 values from Randles model data fitting for all the sensors tested. According to Table 1,
the charge transfer resistance (R2 ) that characterizes the interfacial electron transfer resulting from
Biosensors 2017, 7, 10 6 of 11

the K3 Fe(CN)6 /K4 Fe(CN)6 redox couple, decreases significantly after the cleaning process when
comparing the Group 2 sensors to the Group 1 sensors. Moreover, the data scattering, which could be
Biosensors 2017, 7, 10 6 of 11
observed for the electrodes in Group 2, was minimized by the cleaning procedure used for the
Group
Group 1 sensors.
1 sensors.Thus,Thus, the
the EIS
EIS test
test successfully validated the
successfully validated theprofound
profoundeffect
effectofofthis
thiscleaning
cleaning
procedure, demonstrating the excellent reproducibility of the sensors and the decrease
procedure, demonstrating the excellent reproducibility of the sensors and the decrease in sensor in sensor
charge
chargetransfer
transfer resistance.
resistance.

Figure 3. Nyquist
3. Nyquist plots
plots obtainedfrom
obtained fromEIS
EISininpresence
presenceofofKK3Fe(CN)
Fe(CN)6/K4Fe(CN) 6 redox couple for the
Figure 3 6 /K4Fe(CN)6 redox couple for the
twotwo groups
groups of of sensors
sensors (group1 1subjected
(group subjectedtotocleaning
cleaning procedure),
procedure), and
and equivalent
equivalentRandles
Randlescircuit.
circuit.

Table 1. Calculated R2 (charge transfer resistance) values from Randles model data fitting.
Table 1. Calculated R2 (charge transfer resistance) values from Randles model data fitting.
Sensor #1 Sensor #2 Sensor #3 Sensor #4
Sensor #1 Sensor #2 Sensor #3 Sensor #4
Group 1 198 Ω 200 Ω 201 Ω 204 Ω
Group 1 198 Ω 200 Ω 201 Ω 204 Ω
6150 Ω
Group 2 Group 2 6150 Ω 6913Ω
6913 Ω Ω Ω
79417941 11346 Ω11346 Ω

3.2.3.2. Measurement
Measurement of of T-Tau
T-Tau ProteinsininPBS
Proteins PBSSolution
Solution

TheTheTauTau protein
protein ladder,human
ladder, humanrecombinant
recombinant(Cat.(Cat. No. T7951,
T7951,Sigma
SigmaAldrich,
Aldrich,St.St.Louis,
Louis,MO,
MO,
USA)
USA) was
was used
used inin thisstudy.
this study.Adult
Adultbrain
brainTau
Tauproteins
proteins are
are varied
varied in insize
sizefrom
from352 352toto441
441amino
amino acids
acids
(approximately
(approximately 36.8toto45.9
36.8 45.9kDa).
kDa). This
This protein
protein ladder
ladder contained
contained66recombinant
recombinantTau Tauproteins
proteinswith
with
molecular masses of 36.8, 39.7, 40.0, 42.6, 42.9, and 45.9 kDa, respectively [30,31].
molecular masses of 36.8, 39.7, 40.0, 42.6, 42.9, and 45.9 kDa, respectively [30,31]. In the purchased In the purchased T-
Tau protein ladder, 50 µL contained 0.25 µg of each of the six isoforms. In
T-Tau protein ladder, 50 µL contained 0.25 µg of each of the six isoforms. In this study, we did not this study, we did not
intend to measure each isoform separately and only assessed that the parameter was the total T-Tau
intend to measure each isoform separately and only assessed that the parameter was the total T-Tau
protein in the test medium. We used 0.1 M PBS as test medium (pH = 7.4), and dissolved the T-Tau
protein in the test medium. We used 0.1 M PBS as test medium (pH = 7.4), and dissolved the T-Tau
protein ladder in PBS over the range of 1000 pg/mL to 100,000 pg/mL. The anti-T-Tau concentration
protein ladder in PBS over the range of 1000 pg/mL to 100,000 pg/mL. The anti-T-Tau concentration
used was 500,000 pg/mL. This higher antibody concentration was used in order to minimize the
used was 500,000 pg/mL. This higher antibody concentration was used in order to minimize the
possibility of its becoming a rate-limited component in this bio-recognition mechanism. It was
possibility of its becoming a rate-limited component in this bio-recognition mechanism. It was feasible
feasible to modify and optimize this antibody concentration. This future investigation is beyond the
to modify
scope ofand
this optimize this antibody concentration. This future investigation is beyond the scope of
presentation.
this presentation.
In order to explain the detection mechanism of the T-Tau biosensor, EIS measurements were
In order
carried out toforexplain
biosensorsthe detection
incubated mechanism
in solutions of thedifferent
with T-Tau biosensor,
concentrationsEIS measurements
of T-Tau protein. were
carried
Figureout for biosensors
4 presents incubated
the EIS Nyquist in obtained
plots solutionsinwith different
presence concentrations
of K3Fe(CN) 6/K4Fe(CN) of6 redox
T-Taucouple
protein.
Figure 4 presentsimmobilized
for antibody the EIS Nyquist plots obtained
biosensors incubatedinin presence of K3 Fe(CN)
T-Tau solutions /K4 Fe(CN)
of 61000 6 redox
and 100,000 couple
pg/ml.
forAntigen
antibodysolutions
immobilized werebiosensors
prepared incubated in T-Tau
in 0.1 M PBS. solutionsequivalent
A Randles of 1000 and 100,000
circuit waspg/mL. Antigen
fitted to the
solutions were prepared
experimental data using in EC-lab
0.1 M PBS. A Randles
software. The dataequivalent
obtained circuit
fromwas fitted tofitting
the circuit the experimental
is presented data
in
Table
using 2. Ret software.
EC-lab value in Randles
The data equivalent
obtainedcircuit
from theis defined as the resistance
circuit fitting is presented to charge
in Table transfer of the in
2. Ret value
electrochemical
Randles equivalentinterface.
circuit is defined as the resistance to charge transfer of the electrochemical interface.
Biosensors 2017, 7, 10 7 of 11
Biosensors 2017, 7, 10 7 of 11

Biosensors 2017, 7, 10 7 of 11

4. Electrochemical
FigureFigure 4. Electrochemical impedance
impedancespectroscopy (EIS) Nyquist
spectroscopy (EIS) Nyquistplots
plots obtained
obtained in presence
in presence of of
K3Fe(CN)
K3 Fe(CN) 6 /K 4 /K 4Fe(CN)
6Fe(CN)
6 6 redox
redox couple
couple for
for antibody
antibody immobilized
immobilized biosensors incubated
biosensors in
incubated T-Tau
in solutions
T-Tau solutions
for 3 hfor
at3room
h at room temperature.
temperature. Antigen
Antigen solutions wereprepared
solutionswere prepared inin 0.1
0.1MMPBS.
PBS.
Figure 4. Electrochemical impedance spectroscopy (EIS) Nyquist plots obtained in presence of
K3Fe(CN)
Table 2.6/K 4Fe(CN)
Data 6 redox
obtained couple
from for antibody
Randles immobilized
equivalent biosensors
circuit modeling incubated
of EIS Nyquistinplots
T-Tauinsolutions
Figure 3.
Table 2. Data obtained from Randles equivalent circuit modeling of EIS Nyquist plots in Figure 4.
for 3 h at room temperature. Antigen solutions were prepared in 0.1 M PBS.
T-Tau (pg/ml)
Table 2. DataT-Tau (pg/mL)
obtained RandlesQ(µF)
1000
from (Ωmodeling
Zw1480
1.33 circuit
equivalent ) RofetEIS
1154 125Rplots
(Ω)Nyquist s (Ω)in Figure 3.

1000 100,000 1.33 120.8 1480 1865 1154

201.2 68.8 125
T-Tau (pg/ml) 120.8
100,000 1865 201.2 68.8
According to Table 2, 1000
resistance to1.33 1480
charge transfer (R1154 125
et) of the sensing interface decreased

significantly from 1154 Ω100,000 120.8

for the sensor incubated 1865
in a T-Tau 201.2
solution 68.8of 1000 pg/ml to 201.2 Ω for
According to Table 2, resistance to charge transfer (Ret ) of the sensing interface decreased
the one incubated in a T-Tau solution of 100,000 pg/ml. As reported previously [32], the formation of
significantly
T-Tau
from to
According 1154
protein film TableΩ for
on the
the sensor
2, surface
resistance leads
incubated
to to
charge in a T-Tau
transfer
the development (Retof
) of solution
the sensing
positive
of 1000
charges,
pg/mL
interface to 201.2
decreased
which enhance the
Ω
for the one incubated
significantly
charge permeability in
from 1154 a
ofΩT-Tau
thefor solution
the sensor
electrode of 100,000
incubated
to the pg/mL.
in acharged
negatively As reported
T-Tau solution
redox probe previously
of 1000 pg/ml 6to
of [Fe(CN) [32], 201.2
]3-/4- the formation
Ω for
. Therefore,
the
of T-Tau one
bindingincubated
protein Tauinon
morefilm a T-Tau
the to
protein solution
surface
the surface of 100,000
leads to thepg/ml.
dramatically As reported
development
decreased ofpreviously
the [32],
positiveresistance
electrode the formation
charges, which
to of
enhance
the charge
T-Tau protein film on6]the 3−/4− .
the charge permeability
transfer of [Fe(CN) of .surface
3−/4−the This leads totothe
electrode
phenomenon thedevelopment
negatively
was exploitedof positive
charged
as the redoxcharges,
sensing which
probe ofenhance
mechanism [Fe(CN) the
6]
in DPV
charge permeability
measurements.
Therefore, binding more of the
Figure Tau5 electrode
shows
proteinthe to
tothe
thenegatively
DPV charged
measurement
surface redoxdecreased
results
dramatically ofprobe
T-Tau of proteins
[Fe(CN) 6]
in a. Therefore,
3-/4-
the electrode 0.1resistance
M PBS to
binding
solutionmore
test Tau protein
medium as to the
well 3
as−surface
/4
its− dramatically
calibration curve.decreased
According the
to electrode
the figure, resistance
the anodic topeak
the charge
current
the charge transfer of [Fe(CN) 6] . This phenomenon was exploited as the sensing mechanism in
transfer of with
associated [Fe(CN)
one 6]3−/4−. This phenomenon was exploited
electron transferthe reaction as the sensing mechanism in DPV
DPV measurements. Figure 5 shows DPV of [Fe(CN)6]4- to results
measurement [Fe(CN) 6]3- was increased by increasing
of T-Tau proteins in a 0.1 M PBS
measurements. Figure
the concentration of T-Tau. 5 shows the DPV measurement results of T-Tau proteins in a 0.1 M PBS
solution testtest
solution medium
medium asas well
wellasasits itscalibration curve.According
calibration curve. According to the
to the figure,
figure, the anodic
the anodic peak peak current
current
associated with one electron transfer reaction of [Fe(CN) ] 4- to [Fe(CN) ]3- was increased by increasing
associated with one electron transfer reaction of [Fe(CN)66] to [Fe(CN)6] 6was increased by increasing
4- 3-

the concentration
the concentration of of
T-Tau.
T-Tau.

Figure 5. (a) Differential pulse voltammetry (DPV) measurement of T-Tau proteins over the
concentration range of 1000 pg/mL to 100,000 pg/mL in 0.1 M PBS solution. (b) Calibration curve of
the DPV outputs and T-Tau protein concentration in 0.1 M PBS solution. Anti-T-Tau concentration is
Figure
500,0005.pg/mL.
(a) Differential pulse voltammetry (DPV) measurement of T-Tau proteins over the
Figure 5. (a) Differential pulse voltammetry (DPV) measurement of T-Tau proteins over the
concentration range of 1000 pg/mL to 100,000 pg/mL in 0.1 M PBS solution. (b) Calibration curve of
concentration range of 1000 pg/mL to 100,000 pg/mL in 0.1 M PBS solution. (b) Calibration curve of
the DPV outputs and T-Tau protein concentration in 0.1 M PBS solution. Anti-T-Tau concentration is
the DPV outputs
500,000 pg/mL.and T-Tau protein concentration in 0.1 M PBS solution. Anti-T-Tau concentration is
500,000 pg/mL.
 Biosensors 2017, 7, 10 8 of 11
Biosensors 2017, 7, 10 8 of 11

Figure
Figure 5a5a shows
shows thethe typical
typical DPV
DPV measurements
measurements of of a T-Tau
a T-Tau protein
protein concentration
concentration ofof 1000
1000 pg/mL
pg/mL
to 100,000 pg/mL. Each measurement was accomplished using a single-use,
to 100,000 pg/mL. Each measurement was accomplished using a single-use, disposable biosensor. disposable biosensor.
TheThe current
current outputs
outputs obtained
obtained werefree-of-noise,
were free-of-noise, asas demonstrated
demonstrated in in Figure5a.5a.Figure
Figure Figure 4b5bis is
thethe
calibration curve based on the DPV measurements of multiple experimental runs (n > 3). The axis forfor
calibration curve based on the DPV measurements of multiple experimental runs (n > 3). The axis
thetheT-Tau
T-Tau protein
protein concentration
concentrationis in is logarithmic scale, covering
in logarithmic a wide range
scale, covering a wideof T-Tau
range concentrations.
of T-Tau
The calibration curve is a linear least-square-fitting based on experimental
concentrations. The calibration curve is a linear least-square-fitting based on experimental data. A linear relationship
data. A
of Y = 2.6X − 4.7 was obtained, where Y is the current output of the biosensor
linear relationship of Y = 2.6X − 4.7 was obtained, where Y is the current output of the biosensor and X is the and
T-Tau
2
X protein concentration.
is the T-Tau The R value of
protein concentration. The this
R2linear
valuefitting
of thisis linear
0.85. One must
fitting recognize
is 0.85. that this
One must detection
recognize
covered a very large concentration range of T-Tau protein and the modification
that this detection covered a very large concentration range of T-Tau protein and the modification step of the biosensor
wasofcarried
step out individually
the biosensor was carried andout
manually. Thus, and
individually the uniformity
manually. Thus, of eachthe biosensor
uniformitywas of noteach
100%
identical.was
biosensor Refinement
not 100%could further
identical. enhance this
Refinement linear
could relationship
further enhance between
this linear the current outputs
relationship of the
between
biosensor and the T-Tau concentration.
the current outputs of the biosensor and the T-Tau concentration.
3.3. Measurement of T-Tau Proteins in Undiluted Human Serum
3.3. Measurement of T-Tau Proteins in Undiluted Human Serum
DPV measurements for different T-Tau protein concentrations in undiluted human serum were
DPV measurements for different T-Tau protein concentrations in undiluted human serum were
also carried out in this study. Human serum (Cat No.3667, Sigma Aldrich, St. Louis, MO, USA) was
also carried out in this study. Human serum (Cat No.3667, Sigma Aldrich, St. Louis, MO, USA) was
used to prepare antigen solutions.
used to prepare antigen solutions.
Figure 6a shows the typical DPV measurements of various T-Tau protein concentrations in the
Figure 6a shows the typical DPV measurements of various T-Tau protein concentrations in the
serum. The T-Tau protein concentration was tested between 1000 pg/mL and 100,000 pg/mL. Similar
serum. The T-Tau protein concentration was tested between 1000 pg/mL and 1,000,000 pg/mL.
to the test in 0.1 M PBS, a single-use, disposable biosensor was used for each measurement. Figure 6b
Similar to the test in 0.1 M PBS, a single-use, disposable biosensor was used for each measurement.
shows the corresponding calibration curve of DPV measurements in serum based on results shown in
Figure 6b shows the corresponding calibration curve of DPV measurements in serum based on results
Figure 6a with n = 3. The anti-T-Tau concentration used in this phase of study was 500,000 pg/mL.
shown in Figure 6a with n = 3. The anti-T-Tau concentration used in this phase of study was 500,000
Figure 6b yields a least-square fitted calibration equation of Y = 2.8X − 6.9, where Y is the current
pg/mL. Figure 6b yields a least-square fitted calibration equation of Y = 2.8X −2 6.9, where Y is the
outputs of the biosensor and X is the T-Tau concentration in blood serum. The R value of this equation
current outputs of the biosensor and X is the T-Tau concentration in blood serum. The R2 value of this
was 0.88. It is feasible to optimize operational parameters further in this T-Tau detection system,
equation was 0.88. It is feasible to optimize operational parameters further in this T-Tau detection
such as the antibody concentration, the range of the detecting T-Tau proteins, concentration, and
system, such as the antibody concentration, the range of the detecting T-Tau proteins, concentration,
others. Our purpose of this presentation is to demonstrate that the basic designed biosensor with DPV
and others. Our purpose of this presentation is to demonstrate that the basic designed biosensor with
transduction mechanism can be effectively used for T-Tau protein detection.
DPV transduction mechanism can be effectively used for T-Tau protein detection.

Figure 6. (a) DPV measurement of T-Tau proteins over the concentration range of 1000 pg/mL to
Figure 6. (a) DPV measurement of T-Tau proteins over the concentration range of 1000 pg/mL to
100,000 pg/mL in undiluted human serum. (b) Calibration curve obtained from DPV measurements
100,000 pg/mL in undiluted human serum. (b) Calibration curve obtained from DPV measurements
for T-Tau protein concentration range of 1000 to 1,000,000 pg/mL in undiluted human serum. Anti-T-
for T-Tau protein concentration range of 1000 to 100,000 pg/mL in undiluted human serum. Anti-T-Tau
Tau concentration is 500,000 pg/mL.
concentration is 500,000 pg/mL.

3.4. Interference Study of T-Tau Proteins Measurement of This Biosensor

3.4. Interference Study of T-Tau Proteins Measurement of This Biosensor
The bio-recognition mechanism of this biosensor was based on the interaction of the antibody
The bio-recognition mechanism of this biosensor was based on the interaction of the antibody
and antigen of T-Tau protein, and this detection mechanism was very specific. However, in order to
and antigen of T-Tau protein, and this detection mechanism was very specific. However, in order to
examine any potential interference, β-amyloid 42, another important biomarker of neuro-
degenerative disorders, was used in this interference study. The T-Tau detection biosensor was
 Biosensors 2017, 7, 10 9 of 11

Biosensors 2017, 7, 10 9 of 11
examine any potential interference, β-amyloid 42, another important biomarker of neuro-degenerative
disorders,
prepared aswas used in this
described interference
previously as study. The T-Tau
in Section 2.4. detection biosensor
The anti-T-Tau was preparedused
concentration as described
was
previouslyatas
maintained in Section
500,000 pg/mL 2.4.Recombined
The anti-T-Tauhumanconcentration
β-amyloid used
42 at was maintained of
a concentration at 50,000
500,000pg/mL
pg/mL
Recombined
with human
an incubation of 2 hours,42identical
timeβ-amyloid at a concentration of 50,000
to a previous study pg/mL withthen
in PBS, was an incubation
used in thistime of 2 h,
study.
identical to a previous study in PBS, was then used in this study. Figure 7 shows the
Figure 7 shows the testing results. Both the β-amyloid 42 antigen and the zero concentration of T-Tau testing results.
PBSBoth the β-amyloid
solution show the42 sameantigen
baseand
linethe zero concentration
as comparing to otherof T-Tauprotein-contained
T-Tau PBS solution show thesolutions
PBS same base
as line as comparing
presented in Figureto 5.
other
ThisT-Tau
studyprotein-contained
confirmed positivelyPBS that
solutions as presented
this T-Tau in Figure
biosensor 5. Thistostudy
was specific T-
Tauconfirmed positively that this T-Tau biosensor was specific to T-Tau protein only.
protein only.

Figure 7. Interference study using β-amyloid 42 of 50,000 pg/mL as the biomarker.

Figure 7. Interference study using β-amyloid 42 of 50,000 pg/ml as the biomarker.

4. Conclusions
5. Conclusions
AA cost-effective
cost-effective single-use,
single-use, in vitro
in vitro biosensor
biosensor fordetection
for the the detection of a biomarker
of a biomarker of neuro- of
degenerative disorder, T-Tau protein, has been designed, manufactured, and evaluated in in
neuro-degenerative disorder, T-Tau protein, has been designed, manufactured, and evaluated
phosphate-buffer
phosphate-buffer saline
saline andand undiluted
undiluted human
human serum.
serum. DPVDPV was
was used
used as the
as the measurement
measurement technique.
technique.
Measurements of T-Tau protein in both 0.1 M PBS and undiluted human
Measurements of T-Tau protein in both 0.1 M PBS and undiluted human serum over the serum over the concentration
range of 1000
concentration pg/mL
range to 100,000
of 1000 pg/mLpg/mL showed
to 100,000 pg/mLexcellent
showedresults and good
excellent linearity
results and goodof the calibration
linearity of
thecurves. This biosensor
calibration curves. Thisplatform technology
biosensor platformcan be furthercan
technology optimized andoptimized
be further can be applied
and canto detect
be
other biomarkers
applied of neuro-degenerative
to detect other disorders, including
biomarkers of neuro-degenerative P-TV.au
disorders, protein P-TV.au
including and β-amyloid
protein42.and
β-amyloid 42.
Author Contributions: Chung-Chiun Liu and Alireza Molazemhosseini conceived and designed the experiments;
Yifan Dai
Author performed the
Contributions: experiments;Liu
Chung-Chiun Yifanand
Dai Alireza
and Chung-Chiun Liu analyzed
Molazemhosseini the data;
conceived andChung-Chiun
designed theLiu
experiments; Yifan Dai performed the experiments; Yifan Dai and Chung-Chiun Liu analyzed theLiu
contributed reagents/materials/analysis tools; Yifan Dai, Alireza Molazemhosseini and Chung-Chiun wrote
data;
the paper.
Chung-Chiun Liu contributed reagents/materials/analysis tools; Yifan Dai, Alireza Molazemhosseini and
Conflicts ofLiu
Chung-Chiun wroteThe
Interest: the authors
paper. declare no conflict of interest.

Conflicts of Interest: The authors declare no conflict of interest.

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