Research Article

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Multiplexed electrochemical detection of

three cardiac biomarkers cTnI, cTnT and BNP
using nanostructured ZnO-sensing platform
Nandhinee Radha Shanmugam1 , Sriram Muthukumar2 , Ambalika Sanjeev Tanak1 & Shalini
Prasad*,1
1
Department of Bioengineering, University of Texas at Dallas, Richardson, TX 75080, USA
2
Enlisense LLC, 1813 Audubon Pond Way, Allen, TX 75013, USA
* Author for correspondence: Tel.: +972 883 4247; shalini.prasad@utdallas.edu

Aim: Development of a label-free multiplexed point-of-care diagnostic device for a panel of cardiac
biomarkers – cardiac troponin-T (cTnT), troponin-I (cTnI) and B-type natriuretic peptide (BNP). Methods: A
nonfaradaic electrochemical immunoassay designed with anisotropic high surface area ZnO nanostruc-
tures grown using low-temperature hydrothermal methods was selectively immobilized with capture
antibodies. Multiplexed detection in human serum using ZnO nanostructures based on complementary
electrochemical measurement techniques – electrochemical impedance spectroscopy and Mott–Schottky.
Results: Linear signal response for detection of three biomarkers in human serum with dynamic range of
1 pg/ml–100 ng/ml and limit of detection at 1 pg/ml and low signal response to background interferences
was achieved. Conclusion: First demonstration of simultaneous detection of three cardiac biomarkers in
clinically relevant range with sensor’s analytical performance and linear response of detection showed po-
tential utility in screening clinical samples for early diagnosis of acute myocardial infarction and chronic
heart failure.

First draft submitted: 3 August 2017; Accepted for publication: 9 January 2018; Published online:
1 February 2018

Keywords: cardiac biomarkers (cTnI, cTnT, BNP) • electrochemical technique • multiplexed detection • near-patient
test • point-of-care • rapid • zinc oxide nanostructure

Biomarker detection using nanostructured sensing surfaces represent newly emerging platforms for biological
sensing applications. Nanostructures of metal oxides due to their interesting nanomorphological, catalytic and
functional properties are explored extensively as matrices for reliable immobilization of biomolecules [1]. ZnO due
to its high isoelectric point of pH 9.5 and exciting surface-charge properties in addition to their fast electron transfer
kinetics is a promising candidate for fabrication of novel nanostructure-based electroanalytical biosensors [2,3].
Surface engineering, such as ZnO-nanostructured surfaces, provides signal amplification that enables detection of
target biomarkers with enhanced sensitivity and selectivity. Although pH sensitivity of ZnO may be of concern while
dealing with body fluids which have wide pH variability such as tears, sweat and saliva, it would have significant
impact on the sensing capability (signal response) while detecting it in human serum (pH lower than isoelectric
point). For capacitive impedance change-based electrochemical biosensors, the changes that occur at the electrode–
electrolyte interfaces are fundamental to biomarker detection [4]. When ZnO nanostructure-based biosensor is
brought in contact with test sample, biomolecules binding to sensor surface cause imbalance in electrochemical
potential at the interface leading to Fermi level pinning events. Such events lead to shift in surface potential which
causes modifications to capacitive impedance proportional to the amount of biomolecule binding at both ZnO
space charge layer (SCL) and Helmholtz or electrical double layer (EDL) [5,6]. Electrochemical characterization
methods – Mott–Schottky analysis and electrochemical impedance spectroscopy (EIS) – respectively help exploit
the electrochemical processes occurring at SCL and EDL. In the current scenario, due to the complexity and
heterogeneity of diseases, there is incredible emphasis on the development of portable diagnostic devices capable of
detecting multiple biomarkers simultaneously in real-time at point of care (POC) [7]. By leveraging nanofabrication
technologies, small footprint-multiplexed sensor arrays with each array consisting of selective capture probe for

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Research Article Shanmugam, Muthukumar, Tanak & Prasad

highly sensitive detection of specific biomarker can be successfully designed at low cost [8,9]. Combinatorial sensing
approach using complementary electrochemical techniques provides additional insight into electro-ionic changes
at the interface and can help in accurate determination of target biomarker both quantitatively and qualitatively.
In our previous studies, we have demonstrated sensitive detection of cardiac troponin-T (cTnT) and cardiac
troponin-I (cTnI) biomarkers using nanostructured ZnO-sensing platform [10,11]. Electrochemical characterization
using above-mentioned techniques has been recognized to provide complementary information regarding binding
events and thus helps in achieving selectivity of detection. In addition, ZnO-nanostructured surfaces are more
favorable for performing Mott–Schottky analysis due to its semiconducting properties than metal surfaces where
charge build-up is localized exclusively at the surface. Though screening for cardiac troponins helps in risk
stratification of myocardial-infarcted patients, combining B-type natriuretic peptide (BNP) and N-terminal pro
BNP (NT-proBNP) screening tests guides physicians to ‘rule out’ any misdiagnosis, that is, cardiovascular patients
scheduled to undergo noncardiac surgery, patients to be treated with COX-2 inhibitors or patients with malignant
disease being treated with cardiotoxic chemotherapeutic agents. In this work, we present a novel, flexible and
multiplexed sensor with ZnO nanostructures synthesized hydrothermally for electrochemical detection of three
cardiac biomarkers simultaneously (cTnT, cTnI and BNP) in human serum. This is the first demonstration of
simultaneous detection of three cardiac biomarkers in a rapid manner suitable for POC detection. This work can
achieve translatability for single-drop finger-prick diagnostics for assessing cardiac function. Here we show that
selective functionalization of arrays enable multiplexed detection, and small feature size with large surface area-to-
volume ratio enables detection of ultra-low concentrations with small sample volume. Nanostructured ZnO-based
biosensor thus shows high potential for POC cardiac biomarker diagnosis in clinical samples being label-free,
sensitive, selective and having low cost of manufacturability.

Materials & methods

Chemicals & materials
Zinc nitrate hexahydrate (MW = 297.49), hexamethyeletetramine (HMTA; MW = 140.19), deionized water
(DI), dimethyl sulfoxide (DSP), human serum and superblock were purchased from Thermo Fisher Scientific
(MA, USA). Phosphate-buffered saline (PBS) and bovine serum albumin (BSA) were obtained from Sigma-Aldrich
(MO, USA). Stock concentrations of cTnI and BNP monoclonal antibodies and antigen aliquots were purchased
from Abcam (MA, USA) and cTnT from US Biologicals (MA, USA). All reagents purchased were of reagent grade
and were used without any purification.

Growth of ZnO nanostructures

ZnO seed deposition on gold working electrodes was carried out under Ar plasma of 12 sccm and 15 mTorr at
50 W deposition power in radio frequency (RF)-magnetron sputter reaction chamber for 30 min. ZnO-seeded
samples were then subjected into a hydrothermal reaction bath consisting of equimolar concentrations of 0.05 M
zinc nitrate and HMTA dissolved in 500 ml of deionized water and heated at 80◦ C for another 30 min [10].
ZnO nanostructure grown samples were then rinsed with DI water to remove any growth residues and used for
electrochemical characterization studies.

Infrared analysis
The fourier transform infrared (FTIR) spectra were recorded using Nicolet 6700 spectrometer (Thermo Fisher
Scientific) equipped with germanium attenuated total reflectance accessory and spectral corrections were performed
in OMNIC software. The individual spectroscopic signature of target biomarkers in human serum were obtained
with resolution of 4 cm-1 in the infrared region between 4000 and 600 cm-1 by performing 256 scans per sample.
About 30 μl of sample containing target biomarkers diluted to a concentration of 1 μg/ml in human serum was
used for measurements.

Immunoassay protocol
The first step in an immunoassay involves functionalization of nanostructured ZnO-sensing electrodes with DSP
and thiol-based linker molecule. This chemisorption-based approach gives more stability when compared with
biomolecule immobilization based on physisorption. The next step is antibody immobilization (α-cTnI or α-cTnT
or α-BNP on respective arrays), which is achieved by formation of amide linkage with NHS or NH2 terminal ends
of linker molecules. The unbound NHS sites of the linker molecules are blocked using Superblock to eliminate

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 Triplex cardiac biomarker sensor Research Article

Nanostructured ZnO multiplexed sensor array Metal ZnO nanorods

CE RE CE RE CE RE
WE WE WE

Gold electrode Anti-cTnT immobilized ZnO electrode

Anti-cTnl immobilized ZnO electrode Anti-BNP immobilized ZnO electrode EC

Gold EF

Band gap, Eg
SEM of nanostructured ZnO electrode

EV

100 nm

SCR EDL

500
EIS capacitance
Mott–Schottky capacitance
400
RSC
(1/C)2 (1/μF)2

RS 300
RSS CSS Rct

200

CSC Cdl
100

0
-0.6 -0.4 -0.2 0.0 0.2 0.4 0.6
Applied voltage (V)

Figure 1. Nanostructured ZnO multiplexed sensor characteristics. (A) Nanostructured ZnO-multiplexed sensor array configuration for
simultaneous detection of cTnI, cTnT and BNP biomarkers (top). SEM micrograph of ZnO nanostructures grown selectively at working
electrodes by hydrothermal growth method. (B) Schematic representation of charges at nanostructured ZnO electrode–electrolyte
interface due to biomolecular binding interactions. (C) Equivalent electrical circuit model for nanostructured ZnO biosensor. (D)
Capacitance measured at the interface using EIS and MS as a function of applied potential.
BNP: B-type natriuretic peptide; CE: Counter electrode; cTnI: Cardiac troponin-I; cTnT: Cardiac troponin-T; EDL: Electrical double layer;
EIS: Electrochemical impedance spectroscopy; MS: Mott–Schottky; RE: Reference electrode; SCR: Semiconducting region; SEM: Scanning
electron microscope; WE: Working electrode.

the nonspecific interactions with antigen molecules. The sensor is then washed thrice with PBS followed by
treatment with zero concentration of complex biological buffer solution, human serum. Starting with the lowest
concentration, varying aliquots of target biomarker (cTnI or cTnT or BNP) diluted in human serum were tested
on respective arrays with 15 min incubation time followed by three human serum washes in between each dose
concentration. The tested doses of cTnI antibody are 0.1, 1, 10, 100, 1E3, 1E4 and 1E5 pg/ml. Error bars on the
calibration dose response graph represent 1.65 standard deviation over mean for n = 3 replicates.

Results
Nanostructured ZnO biosensor
Nanostructured ZnO/gold microelectrode-multiplexed arrays in Figure 1A were fabricated using conventional
semiconductor fabrication processes on flexible polyimide substrates. Nanostructured sensing region consisted
of ZnO nanorods synthesized by two-step wet chemical hydrothermal method using conditions as described in
our previous work. In addition, the stability of nanostructured ZnO platform was established through open-

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Research Article Shanmugam, Muthukumar, Tanak & Prasad

cTnI biomarker
1.0

0.8
Absorbance

0.6

0.4

0.2

0.0
cTnT biomarker
1.0

0.8
Absorbance

0.6

0.4

0.2

0.0
BNP biomarker
1.0

0.8
Absorbance

0.6

0.4

0.2

0.0
4000 3500 3000 2500 2000 1500 1000
Wavenumbers (cm-1)

Figure 2. Infrared absorption spectra of cTnI, cTnT and BNP biomarkers between 4000 and 600 cm-1 using germanium attenuated total
reflectance.
BNP: B-type natriuretic peptide; cTnI: Cardiac troponin-I; cTnT: Cardiac troponin-T.

circuit potential and impedance measurements. Supplementary Figure 1A shows that open-circuit potential of
nanostructured ZnO sensor in KCl is at -0.08 ± 0.17 V versus Ag/AgCl reference electrode and that it becomes
stationary with time. Similarly, single-frequency impedance measurement at 100 Hz in Supplementary Figure 1B
indicates stable performance of nanostructured ZnO in a complex physiological testing buffer, human serum.
Following this, the individual arrays were selectively immobilized with respective capture antibodies (i.e., α-cTnT,
α-cTnI and α-BNP) and were used toward detection of its target biomarker (i.e., cTnT, cTnI and BNP).

Enhanced capacitance from ZnO nanostructures

We employed an individual nanostructured ZnO multiplexed-sensing array to understand the contribution of CSC
and CH to total capacitance based on impedimetric Mott–Schottky and EIS measurements. A physiological buffer
solution of 0.1 M PBS was introduced on to the sensing area and electrical response was measured as a function of
applied electrode potential, V, using both the techniques.

Infrared spectroscopy analysis of biomarkers

We studied the infrared spectroscopic signatures of target biomarkers (cTnI, cTnT and BNP) diluted in human
serum as shown in Figure 2. The spectrum reveals a broad band at 3300 cm-1 and sharp band at 1641 cm-1
attributed to ν(O–H) and ν(N–H) stretches in biomarkers. The constructive deconvolution of FTIR spectra is
shown in Supplementary Figure 2. The peaks at 1642 and 1688 cm-1 are attributed to the intermolecular amide
I stretches whereas 1613 cm-1 is due to contribution from O–H stretch. Similarly, we observe contribution from
N–H, O–H stretches at 3326, 3422 and 3555 cm-1 . The results show no significant variation in their signature
that may be attributed to lower sensitivity and inability to spectroscopically distinguish due to low concentration
of target biomarkers.

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 Triplex cardiac biomarker sensor Research Article

Sensitivity of ZnO biosensor

The n-type nanostructured ZnO-multiplexed sensor array immobilized with respective capture antibodies were
then used to test different concentrations of cTnI, cTnT and BNP biomarkers prepared in human serum. Prior to
inoculation with biomarkers, immobilized ZnO surfaces were subjected to treatment with blocking buffer. This step
is important to prevent nonspecific binding of biomarkers on to DSP sites. EIS and MS measurements taken with
human serum containing zero concentration of target biomarker post-blocking step were considered the zero-dose
measurement for further analysis. This range of output response observed is represented in Figure 3.
Supplementary Figure 3 shows the Bode analysis of the impedance response obtained with multiplexed sensor
array toward specific detection of cTnI, cTnT and BNP biomarkers at 10 pg/ml concentration level. The phase angle
denotes the contribution of resistive and capacitive elements to the total impedance of the system. At frequencies
lower than 1000 Hz, the phase angle approaches -90◦ that is indicative of successful detection of biomarkers and
denotes dominant capacitive impedance. Similarly, we observe that both magnitude and phase response differ for
different target biomarkers which is attributed to various characteristics of cTnI, cTnT and BNP such as its overall
charge.
When aliquots of varying concentrations of target biomarkers (cTnI in array 1, cTnT in array 2 and BNP in
array 3) were sequentially tested, a rapid change to electrostatic charge environment at the interface was observed
using both EIS and MS techniques as shown in Figure 3. The decrease in capacitive impedance measured using EIS
at 100 Hz and Mott–Schottky at 0.7 V indicates charge perturbation due to target biomarker binding interactions
and coincides well with our previous reports [11]. Binding of charged cTnI or cTnT or BNP on to ZnO surfaces
results in depletion of majority carriers in SCL and accumulation of opposite polarity ionic charges in EDL and
thus capacitive changes dominates. The electro-ionic charge modulation due to increasing concentrations of tested
doses measured using EIS resulted in dominating capacitive impedance change between 4.8 and 2.1 k for cTnI
detection in array 1 as in Figure 3A, 5.3 and 2.1 k for cTnT detection in array 2 as in Figure 3C and 2.8 and
1.2 k for BNP detection in array 3 as in Figure 3E. This resulted in dynamic range of detection at 55% for cTnI,
59% for cTnT and 54% for BNP. Similarly, capacitive changes observed using MS represented in Figure 3B for
cTnI detection, Figure 3D for cTnT detection and Figure 3F for BNP detection shows a dynamic range of 50, 61
and 53%, respectively.

Selectivity of ZnO biosensor

Specificity of capture antibody for target biomarker was further evaluated to understand electrical output signal
response due to any nonspecific interactions as shown in Figure 4. A signal noise threshold for nanostructured
ZnO platform was established at 9.8% using EIS and 13.6% using MS. It is measured as three-times changes
to output signal response between zero dose and blocking step. A cross-reactivity control experiment with BSA
(most abundant protein found in human plasma) diluted in human serum and in absence of target biomarkers was
established as shown in Figure 4A using EIS and Figure 4B using MS. There were no significant changes to output
response and maximum change observed were well within established noise threshold. The maximum impedance
change observed was at 6, 5 and 7%, respectively, for α-cTnI, α-cTnT and α-BNP immobilized on individual
nanostructured ZnO arrays. Similarly, 11, 11 and 9% were maximum changes to space charge capacitance observed
respectively for α-cTnI, α-cTnT and α-BNP immobilized on to ZnO.
In Figure 4A & B, dose response of BSA to the immobilized capture probe appears to be random and below
specified signal noise threshold. The observed response can be attributed due to the nonspecific adsorption of the
BSA on the sensing site. This demonstrates the specificity of the immobilized capture probe to the specific target
biomarker which shows a linear response as seen in Figure 3.
Whereas in Figure 4C, we see a linear response for capture probe immobilized with anti-cTnI and anti-cTnT
with EIS. This phenomenon could be due to the similar structure of the cTnI and cTnT which shows affinity
toward the complementary immobilized capture probe within the EDL. Since structure of BNP could differ, this
response is nonlinear. However, the detection using MS shows a nonlinear trend due to the effect of nonspecific
charge distribution at surface charge layer. The signal obtained from nonspecific and cross-reactive experiments
falling below the specified signal noise threshold indicates the specificity and sensitivity of the immobilized capture
probe to the specific target biomarker.
A second control experiment to establish the nonspecific interactions between specific capture antibody and
testing biomarkers were conducted by mixing together 1:1 concentration ratio of other two biomarkers. The
signal response observed was well within noise threshold at low concentrations. However, at higher concentrations,

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Research Article Shanmugam, Muthukumar, Tanak & Prasad

7 180
Detection of cTnl in human serum using EIS Detection of cTnl in human serum using Mott–Schottky

160
6 Zero dose

Mott-Schottky capacitance (1/μF)2

140
Zero dose
5
120
Impedance (kΩ)

4 100

3 80

60
2
40
1
20

0 0
1E-02 1E-01 1E+00 1E+01 1E+02 1E+03 1E+04 1E+05 1E+06 1E-02 1E-01 1E+00 1E+01 1E+02 1E+03 1E+04 1E+05 1E+06
Concentration (pg/ml) Concentration (pg/ml)

7 180
Detection of cTnT in human serum using EIS Detection of cTnT in human serum using Mott–Schottky
175
6 100
Mott-Schottky capacitance (1/μF)2

Zero dose

5
80 Zero dose
Impedance (kΩ)

4
60

3
40
2

20
1

0 0
1E-02 1E-01 1E+00 1E+01 1E+02 1E+03 1E+04 1E+05 1E+06 1E-02 1E-01 1E+00 1E+01 1E+02 1E+03 1E+04 1E+05 1E+06
Concentration (pg/ml) Concentration (pg/ml)

7.0 180
Detection of BNP in human serum using EIS Detection of BNP in human serum using Mott–Schottky
6.5 175
4.0 120
Mott-Schottky capacitance (1/μF)2

100 Zero dose

3.0 Zero dose
Impedance (kΩ)

80

2.0 60

40
1.0
20

0.0 0
1E-02 1E-01 1E+00 1E+01 1E+02 1E+03 1E+04 1E+05 1E+06 1E-02 1E-01 1E+00 1E+01 1E+02 1E+03 1E+04 1E+05 1E+06
Concentration (pg/ml) Concentration (pg/ml)

Figure 3. Detection of target biomarkers diluted in human serum using EIS and MS measurements on nanostructured ZnO-based
multiplexed sensing array. Calibration dose response for (A & B) cTnI; (C & D) cTnT and (E & F) BNP.
BNP: B-type natriuretic peptide; cTnI: Cardiac troponin-I; cTnT: Cardiac troponin-T; EIS: Electrochemical impedance
spectroscopy; MS: Mott–Schottky.
 Triplex cardiac biomarker sensor Research Article

100 100
Crossreactivity studies with BSA using EIS Crossreactivity studies with BSA using Mott–Schottky

Percentage change in Mott-Schotky capacitance

90 a-cTnl 90 a-cTnl
Percentage change in impedance

a-cTnT a-cTnT
a-BNP a-BNP

20 20

15 15 Signal noise threshold

Signal noise threshold

10 10

5 5

0 0
1E-02 1E-01 1E+00 1E+01 1E+02 1E+03 1E+04 1E+05 1E+06 1E-02 1E-01 1E+00 1E+01 1E+02 1E+03 1E+04 1E+05 1E+06
Concentration (pg/ml) Concentration (pg/ml)

100 Non-specificity studies using EIS 100 Non-specificity studies using Mott–Schottky

90
a-cTnl vs cTnT & BNP Percentage change in Mott-Schotky capacitance 90 a-cTnl vs cTnT & BNP
a-cTnT vs cTnT & BNP a-cTnT vs cTnl & BNP
Percentage change in impedance

a-BNP vs cTnT & cTnl a-BNP vs cTnT & cTnl

20 20

15 15 Signal noise threshold

Signal noise threshold

10 10

5 5

0 0
1E-02 1E-01 1E+00 1E+01 1E+02 1E+03 1E+04 1E+05 1E+06 1E-02 1E-01 1E+00 1E+01 1E+02 1E+03 1E+04 1E+05 1E+06
Concentration (pg/ml) Concentration (pg/ml)

Figure 4. Detection of cross-reactive and non-specific biomarkers diluted in human serum using EIS and MS measurements on
nanostructured ZnO based multiplexed sensing assay. Cross-reactivity of capture antibody immobilized at ZnO multiplexed sensor array
for BSA diluted in human serum using (A) EIS and (B) MS. Nonspecific interactions of capture antibody for other two test biomarkers
diluted together in human serum at 1:1 concentration ratios using (C) EIS and (D) MS.
BNP: B-type natriuretic peptide; BSA: Bovine serum albumin; cTnI: Cardiac troponin-I; cTnT: Cardiac troponin-T; EIS: Electrochemical
impedance spectroscopy; MS: Mott–Schottky.

nonspecificity observed using EIS was higher than noise threshold, whereas this is not the case with MS. These
studies show that detection using complementary techniques may help address the issues due to nonspecific
interactions and achieve high specificity and sensitivity. Thus, nanostructured ZnO-based multiplexed sensor array
shows detection limit at 1 pg/ml for simultaneous detection of cTnI, cTnT and BNP in human serum and feasibility
of testing complex biological samples. The way ZnO nanostructures are processed during hydrothermal growth
plays an important role to attain stability of the immunoassay, as the linker could hydrolyze due to insufficient
binding sites due to deficient oxygen vacancies.

Discussion
As seen in Figure 1, uniform, vertically aligned and self-organized growth of ZnO nanorods are due to availability
of nucleation sites from presputtered ZnO seed layer and its preferential growth is along its basal plane (0001).
Nucleation is a growth process that begins when the chemical bath containing precursor molecules is supersaturated
with ZnO nuclei resulting in formation of hexagonal c-axis oriented crystalline growth of ZnO nanostructures
as shown by SEM micrograph in Figure 1A. The large surface area offered by ZnO nanostructures selectively

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Research Article Shanmugam, Muthukumar, Tanak & Prasad

grown at working electrodes of multiplexed array allows microenvironment for biomolecular confinement. Thus,
to explore the sensing characteristics of developed nanostructured multiplexed-sensing platform, the ZnO surfaces
were functionalized with established thiol linker conjugation forming Zn–S bridges [12,13].
The mechanism of charge transfer between the nanostructured ZnO electrode and the sample containing the
target biomarkers is fundamental to the development of biosensor discussed in this work. An interfacial potential
is generated due to distribution of charges at the interface when ZnO electrode is in contact with sample solution
to equilibrate the electrochemical potential. Development of charges within ZnO constitute an SCL formed at
the surface of the electrode and an EDL or Helmholtz layer (HL) – formed by inner Helmholtz plane and outer
Helmholtz plane [14] at the solution interface. The charge distribution at the interface is dependent on the material
and structural characteristics of the electrode. Either electrons or holes can be aligned near ZnO surface with ions
or species of opposite charge aligned at the surface on electrolyte side. Similarly, inner Helmholtz plane and outer
Helmholtz plane are formed by charges of opposite polarity. In both the above descriptions, the structure resembles
that of a parallel plate capacitor and hence, the distribution of charges at the ZnO electrode/electrolyte interface
can be measured in terms of capacitance [15]. The capacitance associated with HL is termed double layer capacitance
Cdl or Helmholtz capacitance CH and that associated with SCL correspond to space-charge capacitance, CSC . If
QH and QSC respectively denote the charge density in HL and SCL, then the differential capacitance per unit area
in respective layers is given by the equation:

QH  0
CH or Cdl  
VH WH

In the above equation, ε0 represents the permittivity of free space, ε represents the dielectric constant of medium
and WH is the thickness of the double layer.

QSC  2  kT  
CSC    VSC  
VSC  q 0 s N D  q 

In the above equation, εs is the dielectric constant of the semiconductor electrode, q is the elementary charge,
ND is the charge carrier density or dopant density, k is the Boltzmann constant and T is the temperature. Then,
the capacitance at the semiconductor electrode electrolyte interface can be modeled as two capacitors in series and
the total capacitance is given by the following equation [12,13].

1 1 1
 
C CSC CH

In our conditions of nanostructured electrode surface, each of ZnO nanorods acts as individual capacitor that
contributes to its own CSC and CH . The sum of individual capacitances provides signal amplification that allows for
higher sensitivity of detection. A schematic representation of charge distribution due to immunoassay design (see
’Materials & methods’ section) is shown in Figure 1B. The binding of DSP/capture antibody/target biomarker to
n-type ZnO results in depletion of its charge carriers (electrons) and thus changes to CSC . Due to more positive
charge associated with SCL, an equal and opposite charge develops at the surface causing changes to CH . This
electrostatic charge environment gets modulated with change in target biomarker binding and hence capacitive
changes can be monitored with the help of electrical readouts. When an electric potential is applied, an electric
field is developed both in SCL and EDL that results in interfacial potential drop across both the regions [16]. EIS
is a surface characterization technique employed to characterize changes to CH whereas Mott–Schottky is used to
measure CSC . The electrical nature of ZnO electrode/electrolyte interface can be modeled using capacitive and
resistive elements as described in Figure 1C. The resistive components include the solution resistance (RS ), charge
transfer resistance (Rct ), space-charge resistance (RSC ) and surface state resistance (RSS ). The conductance of the
buffer and its ionic concentration impedes the flow of current through bulk of the solution which mainly affects
RS . Rct represents the ability of the electrode to give up or accept charges from the electrolyte and is affected by

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 Triplex cardiac biomarker sensor Research Article

Table 1. Comparison on various cardiac biomarkers and their performance metrics.

Biomarker Technique for detection Test medium Range Limit of detection Ref.
cTnT Voltammetry + EIS Human serum 0.1–1 ng/ml 0.076 ng/ml [19]
cTnT Voltametry Human serum 0.001−70.3 ng/ml 0.009 ng/ml [20]
cTnI FET PBS 0.096−46 ng/ml 0.092 ng/ml [21]
BNP Voltammetry Human serum 0.001–100 ng/ml 3.3 ng/ml [22]
BNP Voltammetry and EIS Plasma 0.1–5 ng/ml 1E-04 ng/ml [23]
BNP Electrochemical Human serum 0.57–193 ng/ml 0.47 ng/ml [24]
magnetoimmunosensor
cTnT, cTnI and BNP EIS and Mott–Schottky Human serum 1E-03–100 ng/ml 1 pg/ml This work
BNP: B-type natriuretic peptide; cTnI: Cardiac troponin-I; cTnT: Cardiac troponin-T; EIS: Electrochemical impedance spectroscopy; FET: Field-effect transistor; PBS: Phosphate-buffered saline.

charge distribution at EDL. Any modifications to electrode surface alters Cdl . A parallel resistor-capacitor (RC)
combination of Rct and CH is used to represent the surface phenomenon or EDL at the electrode interface [5,17].
Similarly, for ZnO with no surface states, RSS and surface state capacitance (CSS ) in parallel combination represents
the physical structure of the electronic charge distribution at the interface. However, in the presence of surface
states, series combination of RSS and CSS should be considered parallel to space-charge elements (RSC and CSC )
to represent the charges at semiconductor interface [18]. Capacitance derived from both the measurements as in
Figure 1D shows the dependence between C-2 and V. Hence, measured electrochemical data were fitted to circuit
model and obtained results indicate that both CH and CSC are of same magnitude and hence cannot be ignored in
total capacitance. The highly sensitive performance of the electrochemical biosensor by EDL modulation extracted
using EIS and Mott–Schottky techniques has been compared in Table 1.

Conclusion
In summary, we present nanostructured ZnO biosensor for label-free and multiplexed detection of cTnI, cTnT
and BNP in human serum with high sensitivity and selectivity using complementary detection approaches. A
linear signal response for detection and low signal response to background interferences with the limit of detection
at 1 pg/ml for tested biomarkers shows credibility of developed sensor arrays for operations in more complex
biological samples. With more focused future works on improving the dynamic range of detection and reliability,
the demonstrated ZnO-multiplexed sensor arrays show great promise for testing clinical samples at POC settings.

Future perspective
Personalized medicine is emerging as an important area of patient care in cardiology. In this context, near-patient
testing devices will play a major role in changing the paradigm of patient care. Hence, there is a need for developing
technologies that enable rapid, near-patient testing that can screen and quantify cardiac biomarker panels and
connect them to specific chronic health conditions. The sensor technology presented in this article demonstrates
proof of feasibility for detecting three specific cardiac biomarkers that are implicated in the diagnosis of acute
myocardial infarction or congestive heart failure. The developed technology can detect cardiac protein biomarkers,
cTnI, cTnT and BNP simultaneously in human serum at clinically significant concentrations in the pg/ml range.
The lower level of detection showcases the ability of the device to be used as a tool for evaluating cardiac risk in
clinical diagnostics. This lower limit of detection was achieved due to the novel device architecture comprising of
ZnO nanostructures selectively grown on to the working electrode of the sensor resulting in increased S/N. Further
testing directly on patient samples can establish robustness and ability of the device to detect essential biomarkers
in a multiplexed manner. By coupling the sensor to a portable smart device, a near-patient test can easily designed.
Thus, the presented demonstration shows proof-of-feasibility of a sensor for multiplexed detection of a panel of
cardiac biomarkers which can translate to a clinical setting.

Supplementary data
To view the supplementary data that accompany this paper please visit the journal website at: www.futuremedicine.com/doi/suppl/
10.2217/fca-2017-0074.

Acknowledgements
The authors would like to thank the Cecil and Ida A Green Endowment at UT Dallas (TX, USA) for supporting this work.

future science group 10.2217/fca-2017-0074

Research Article Shanmugam, Muthukumar, Tanak & Prasad

Summary points
Multiplexed biosensing of a panel of cardiac biomarkers
r Demonstration of point-of-care biosensing capability for three cardiac biomarkers – cardiac troponin-I, cardiac
troponin-T and B-type natriuretic peptide in human serum.
Rapid detection enables feasibility for developing a near-patient test
r The sensor has response time in the order of minutes for detecting and quantifying multiple biomarkers which
makes it highly attractive for developing a near-patient test.
Zinc oxide nanostructured platform enhances sensor response
r The zinc oxide nanostructures played an important role in enhancing the S/N, which in turn enabled 1 pg/ml
sensitivity for all three biomarkers in human serum.
r Enhanced signal response was captured using electrochemical technique – electrochemical impedance
spectroscopy and Mott–Schottky.
r ZnO nanostructured platform demonstrated high specificity and selectivity to the target biomarkers by reducing
nonspecific signal.

Financial & competing interests disclosure

S Prasad and S Muthukumar have a significant interest in Enlisense LLC, a company that may have a commercial interest in
the results of this research and technology. The potential individual conflict of interest has been reviewed and managed by The
University of Texas at Dallas (TX, USA), and played no role in the study design; in the collection, analysis, and interpretation of data;
in the writing of the report, or in the decision to submit the report for publication. S Muthukumar was supported by Enlisense LLC.
The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in
or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined
in the Declaration of Helsinki for all human or animal experimental investigations.

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