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M. A. Ali, R. Kumar, A. Sharma, B. D. Malhotra and R. John, J. Mater. Chem. B, 2019, DOI:
10.1039/C9TB00126C.
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Page 1 of 31 Journal of Materials Chemistry B
Hollow-nanospheres-based microfluidic biosensors for biomonitoring ofView Article Online

DOI: 10.1039/C9TB00126C
cardiac troponin I
Nawab Singha, Prabhakar Raib,c, Md. Azahar Alid, Rudra Kumarb, Ashutosh Sharmab, B. D. Malhotrae*, and Renu
Journal of Materials Chemistry B Accepted Manuscript

Johna*
aDepartment of Biomedical Engineering, Indian Institute of Technology Hyderabad, 502285 Telangana, India.
bDepartment of Chemical Engineering, Indian Institute of Technology Kanpur, Kanpur, 208016, India.
cWildlife Section, Zoological Survey of India, Kolkata 700053, India.
dDepartment of Electrical and Computer Engineering, Iowa State University, Ames, Iowa-50011, USA.
eDepartment of Biotechnology, Delhi Technological University, Shahbad Daulatpur, Main Bawana Road, Delhi-
Published on 13 April 2019. Downloaded by UNIVERSITE PARIS SUD on 4/13/2019 10:23:27 AM.
110042, India.
Abstract
Cardiovascular diseases (CVD) are the leading causes of death worldwide. Cardiac troponin I (cTnI) is
a cardiac biomarker exploited extensively for the early diagnosis of acute myocardial infarction (AMI).
We report a highly sensitive microfluidic biosensor for electrochemical detection of human cTnI in real
patient samples. In this device, a patterned mesoporous nickel vanadate hollow-nanospheres
modified chitosan (Ch-Ni3V2O8) was integrated with microfluidic structure. With excellent redox
activity and biocompatibility, larger surface, tunable oxidation states (V5+, V4+, and V3+), the Ch-Ni3V2O8
matrix was used to functionalize the cardiac antibodies of troponin I (cAb) and amplify the
electrochemical readouts. This device offered a high sensitivity of 38.88 µA/ng mL-1/cm2 for a wide
range of cTnI concentration (0.005‒100 ng mL‒1). A low limit-of-detection of 5 pg mL‒1 and high
stability, high reproducibility and good selectivity were achieved due to integration of microfluidic
elements with hollow-nanosphere Ch-Ni3V2O8. This microfluidic device can be implemented to detect
B-type natriuretic peptide, myoglobin, cardiac troponin C, and cardiac troponin T by functionalizing
the specific antibody recognition elements and used for real point-of-care application in bio-medicine.
Keywords: Cardiac Troponin I, Ni3V2O8 Hollow-nanosphere, Microfluidics, Acute Myocardial Infarction,

Electrochemical.
*Corresponding Authors: renujohn@iith.ac.in*, Ph: +91-40-23016097, bansi.malhotra@gmail.com*
1
Journal of Materials Chemistry B Page 2 of 31
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DOI: 10.1039/C9TB00126C
1. Introduction
Quantification of cardiac biomarkers such as cardiac troponins (I, T and C) found in heart muscle can

be highly beneficial towards the prognosis of cardiovascular diseases (CVD) or acute myocardial
infarction (AMI). cTnI has been found to be a prime biomarker owing to its excellent sensitivity and
superior specificity for myocardial injuries.1–3 CVD are related to atherosclerosis of the heart arteries
that can occur due to accumulation of a fatty material (e.g cholesterol plaque) inside the artery walls.
The AMI happens when a heart muscle gets damaged owing to the lack of blood supply to heart that
may cause blockage of a coronary artery. Thus, primary precise diagnosis of AMI and its immediate
treatment are presently a serious issue for the treatment of heart stroke. Cardiomyocyte necrosis
occurs due to myocardial cell damage resulting in expression of the cardiac muscle proteins such as
cTnI that is released in the blood serum. cTnI in blood serum is indicative of AMI.4 The detection of
cTnI is, thus, extremely important for medical diagnostics of AMI.
Many techniques such as enzyme-linked immunosorbent assay (ELISA)5,6, chemiluminescent

immunoassay7 and radioimmunoassay (RIA)8,9, have been used for quantification of cardiac troponins
(cTns). The major issue with the conventional assays for the detection of cTns relates to poor
sensitivity and limit of detection. The commercial available assays such as “Abbot i-STAT CTnI” and
“Xpand CTnI Flex” by i-STAT Handheld, Abbott Point of Care Inc. and Dimension Xpand Plus by Siemens
Healthcare have been found to have low limit of detections such as 0.02 and 0.04 ng/mL) for blood
serum cTns monitoring.10 In addition, some of the issues such as handling of samples, long detection
times and the high cost, make these techniques unattractive.1,11,12
With recent advancement of micro/nanofabrication technology, the microfluidic structured

devices including microfluidic has gained much attention to quantify cTns at point-of-care setting.
Microfluidic devices have been predicted to be a remarkable technique for application in point-of-care
(POC) diagnostics due to numerous advantages such as portability, low reagent consumption, reduced
time of analysis and these can be integrated with nano-scale analysis, and nano-biosensors.13,14
Advanced functional materials enabled microfluidic biosensors have a potential for increased
biomolecules sensing abilities15 due to high aspect ratio, ultra-sensitivity and low limit of detection.15–
17 Cadmium selenide quantum dots (QCdSe), carbon nanotubes (CNTs), zinc oxide nanowires, boron-
doped diamond and microporous nanocomposite, were incorporated into microfluidic structures for
quantification of chronic myelogenous leukemia18, cholesterol19, environmental pH17, pesticide
(atrazine)20 and cardiac biomarker21 with high sensitivity and selectivity. In spite of these interesting
2
developments, it is still challenging to interface desired biomolecules with functional nanomaterials

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DOI: 10.1039/C9TB00126C
in microfluidic devices.
Owing to the multiple oxidation states and sites, nanostructured binary metal oxides (nBMO)

can be used as interfaces for organic electronic components, energy storage, supercapacitors and as
electrode materials for improved electrochemical performance compared to those based on single
component oxides.22–26 The nBMO have been found to contain porous NiCo2O4 hollow nanospheres27
and hollow polyhedron CuCo2O4.28 The zinc cobaltite (ZnCo2O4) nanoparticles29, sphere-like zirconium
molybdate (ZrMo2O8) nanostructure30 and spinel NiCo2O4 nanosheet arrays31 have been utilized as
biosensor electrode materials to obtain enhanced electrochemical biosensing performances due to
their higher electronic conductivity and fast heterogeneous electron transfer. The nickel vanadate
(Ni3V2O8) exhibits interesting oxidation states and has a better conductivity than that of single NiO.25,26
In addition, V2O5 with tunable oxidation states (V5+, V4+, and V3+) have received significant attention as
an electrode material for high electrochemical signal.25 Hollow and mesoporous features of Ni3V2O8
may help to bind with antibody molecules. Hollow-nanostructures allowed the loading of more
number of antibodies in its hollow space inside the spheres that may facilitate larger number of active
sites on a transducer surface to enable improved electrochemical biorecognization event.27,28
In this paper, we report on the development of a microfluidic immunosensor at point of care

setting for the detection of cTnI in human serum samples using electrochemical technique. In this
microfluidic device, the sensing electrode acted as immunotransducer having a hierarchical
mesoporous hollow-nanospheres structure modified with chitosan (Ch-Ni3V2O8) and immobilized with
cardiac antibody (cAb). The hollow-nanosphere Ch-Ni3V2O8 microfluidic biosensor offered high
sensitivity, low detection limit, superior accuracy and minimal interference yielding better
quantification of bio and chemical molecules compared to conventional techniques. To the best of our
knowledge, this is the first report on development of an electrochemical microfluidics biosensor based
on chitosan modified mesoporous Ni3V2O8 hollow-nanospheres for cTnI detection.
2. Experimental section
Chemicals and reagents
Nickel chloride (Ni(NO3)2.6H2O), sodium metavanadate (NaVO3), were procured from Loba Chemie
and ammonium hydroxide was bought from Fisher Scientific. Chitosan (low molecular weight), bovine
serum albumin (BSA) and aqua regia (HNO3+HCl) were procured from Sigma Aldrich, USA. The
polydimethylsiloxane (PDMS) and SU8-2050 negative photoresists were purchased from Microchem
3
(Newton, MA), USA. Mouse monoclonal antibodies of cTnI (cAb) and cTnI antigen were procured from
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DOI: 10.1039/C9TB00126C
Abcam, USA and phosphate buffer saline (PBS) pH 7.4 was used in this microfluidic biosensor.
Synthesis of Ni3V2O8 hollow-nanospheres

Ni3V2O8 hollow-nanospheres were synthesized following a previously reported method with a
modification.26 In this procedure, Ni(NO3)2.6H2O and NaVO3 (3:2) molar ratio solution were prepared
in 60 mL of deionized (DI) water. A clear solution of Ni(NO3)2.6H2O was prepared using 30 mL of DI
water. NaVO3 was dissolved in 30 mL of DI water after which 4 mL of ammonium hydroxide solution
was added to form a clear solution at pH 8.5. Finally, both the solutions were mixed using a stirrer to
form a homogeneous solution and then transferred into a hydrothermal autoclave reactor that was
kept at 185 oC for about 22 h. After centrifugation the precipitate was washed many times with ethyl
alcohol and water. Next, the sample was dried at 70 oC overnight and calcinated at 600 oC for 3 h to
obtain Ni3V2O8. First ammonium hydroxide (NH4OH) was added to sodium metavanadate suspension
solution to obtain ammonium orthovanadate, this reaction can be illustrated as follows.
NaVO3 + 3NH4OH → (NH4)3VO4 + NaOH + H2O (1)
This ammonium orthovanadate reacted with nickel nitrate and as a result nickel vanadate was formed.
3Ni(NO3)2 + 2(NH4)3VO4 → Ni3V2O8 + 6NH4NO3 (2)
Ni3V2O8 modification with chitosan
Chitosan (molecular weight: 90,000 Da) was dissolved in 10 mL of 2% (w/v) acetic acid solution with a
magnetic stirrer at constant stirring (500 rpm) for about 1h until the solution became transparent after
which 20 mg of Ni3V2O8 was added. This mixture was stirred at 600 rpm by adding dropwise 1%
tripolyphosphate (TPP) as a cross-linker and was kept overnight at 30 oC. The resulting solution was
centrifuged at 8000 rpm for 25 min and was washed with DI water and then freeze-dried. We
investigated the surface electrokinetic potential for Ni3V2O8 and Ch-Ni3V2O8 via zeta potential studies.
The zeta potential was found to be as 26.07 mV for Ni3V2O8 (Fig. S1 a) and it increased to 41.04 mV in
case of the Ch-Ni3V2O8 (Fig. S1 b) indicating the formation of a stable nanocomposite material. The
proposed mechanism relating to the preparation of Ch-Ni3V2O8 hollow-nanospheres is shown in Fig.
1.
An aqueous colloidal solution containing Ni3V2O8 has been found to exhibit weak conjugation
with biomolecules and is presently a major concern for the development of efficient biosensors.
Ni3V2O8 hollow-nanospheres can perhaps be modified by a natural biopolymer such as chitosan (Ch)
to overcome this problem. Chitosan is known to provide improved dispersion quality, reduced
4
agglomeration of particles and can be used to engineer functional groups on the surface of Ni3View
V2OArticle
8
Online
DOI: 10.1039/C9TB00126C
hollow-nanospheres for conjugation of desired biomolecules. Ch has been found to facilitate stable
thin film formation and has excellent biocompatibility owing to its good adhesion capability. An

efficient loading of bioreceptor molecules is expected due to the existence of hydroxy and amino
clusters in Ch-Ni3V2O8 nanocomposite, making it an ideal candidate for biosensing applications.
Besides this, Ch modified nanostructured metal oxides are known to have high surface absorption
capability, strong affinity for antibodies/enzymes and good stability resulting in excellent biosensing
performance.32,33
Serum sample preparation
The blood serum sample of cardiac patient was collected and pre-treated at Kamineni Hospital, LB
Nagar, Hyderabad, India as per approved Biosafety standards and protocols at Kamineni Hospital, LB
Nagar, Hyderabad, India. Prior to conducting the real sample analysis experiments, approvals were
obtained from the hospital biosafety committee of Kamineni Hospital, LB Nagar, Hyderabad, India.
The concentration of cTnI present in the serum sample of cardiac patient was validated against the
Beckman Coulter chemiluminescence immunoassay (CLIA) detection technique. With the known
concentration of cTnI in serum sample, we prepared various concentrations of cTnI by diluting it with
PBS (pH 7.4) solution. Then, these different concentrations of serum samples were used for sensing
of cTnI.
Device fabrication
The microfluidic biosensor was nanoengineered using soft lithography technique and integration of
hollow-nanospheres. This was fabricated using a three-electrode system containing patterned gold
(Au) and silver (Ag) electrode on a glass substrate. The integration of Ch-Ni3V2O8 composite onto Au
electrode was accomplished for loading of the cTnI antibodies and bonding of PDMS microchannel
(Fig. 2).
The Au coated glass substrate was first cleaned with ethanol and water to prepare
microelectrode patterned with a size of 2mm × 1.5 cm using wet chemical etching. For this, a chemical
etching resistant shadow mask was applied onto the Au substrate and then dipped in the etching
solution of (1:3, HNO3/HCL) for 2 min at 30 oC. The substrate was washed with DI water and the mask
was peeled off from the substrate. The patterned Au working electrode was then treated with a
solution of (3:1, H2SO4/H2O2) at 30 oC for 8 min and was washed with DI water. The Au electrode
surface was modified with uniformly deposited Ch-Ni3V2O8 gel solution via spin-coating technique and
5
was kept overnight at 37 oC in a closed chamber. Owing to chitosan, Ch-Ni3V2O8 was uniformly
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DOI: 10.1039/C9TB00126C
deposited of Au surface.
For the reference electrode (RE), a 600 nm-thick Ag layer was deposited on a glass substrate

using e-beam evaporation by placing a shadow mask on top of the surface. To obtain Ag/AgCl RE, the
Ag electrode was treated with 0.2 M potassium chloride (KCl) for 100 s. A bare Au electrode acted as
the counter electrode (CE). The various fabrication steps followed for fabrication of gold (Au)
microelectrodes on gold substrate are shown in Fig. S2a. The soft lithography technique was used to
fabricate PDMS microchannels and plasma bonding with glass substrate containing all the three
electrodes (Ch-Ni3V2O8, Au and Ag/AgCl). The fabricated two microchannels (height and width, 200
µm each) were connected via single inlet and outlet. The stepwise illustration for fabrication of
microfluidic channels and bonding with sensor electrode is shown in Fig. S2b (Supplementary
Information). For immobilization, cAb solution was injected through microfluidic channels on the
surface of Ch-Ni3V2O8/Au electrode. The amine (−NH2) and hydroxy (−OH) functional groups present
on the Ch-Ni3V2O8/Au electrode surface facilitated their binding with carboxyl (−COOH) groups of the
cAb via electrostatic interaction. And the Ni3V2O8 that was positively charged (as is evident by zeta
potential Fig. S1) could undergo electrostatic interaction with a negatively charged Fc portion of the
cAb.33 Finally, the cAb immobilized cAb/Ch-Ni3V2O8/Au bioelectrode was treated with BSA molecules
to block the non-specific sites on the sensor electrode surface. A flow rate (20 μL/min) of cTnI solution
with PBS was achieved using a syringe pump through microfluidic device and stored at 4 oC.
3. Results and discussion
3.1 Spectroscopic Analysis
Fig. 3a shows results of the X-ray diffraction (XRD) studies carried out to confirm the crystallinity of
synthesized Ni3V2O8. The peaks found at 2θ = 35.96, 43.78, and 63.66, pertained to (122), (151), and
(442) planes, respectively. These results indicated the presence of orthorhombic crystal structure of
Ni3V2O8 with lattice parameter such as a = 8.24 Å, b = 11.38 Å and c = 5.90 Å, respectively, (JCPDF card
no. 01-074-1484). The crystallite size of Ni3V2O8 was determined using the Scherrer equation (3).
kλ
D = β cos θ (3)
where D represents the crystallite size, k is a constant (0.9), λ is the wavelength of the source (Cu K1,
1.54 Å), β is the full width at half maximum (FWHM) and θ is a peak location in radians. The crystallite
size for highest intensity peak seen at 43.78o, pertaining to (151) plane was found to be 10.4 nm.
6
The porosity and the surface area of Ni3V2O8 hollow-nanospheres were determined usingViewthe
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DOI: 10.1039/C9TB00126C
Brunauer–Emmett–Teller (BET) studies. The pore size and adsorption-desorption isotherm obtained
for Ni3V2O8 hollow-nanospheres are shown in Fig. 3b. The Ni3V2O8 exhibited mesoporous structure

with good surface area of 40.042 m2 g-1. The average pore size and total pore volume of Ni3V2O8 were
determined to be as 7.54 nm and 0.073 cc g-1, respectively. The distribution of pore size (inset of Fig.
3b) revealed that the Ni3V2O8 was mesoporous. The high BET surface area of this mesoporous
structure facilitated increased loading of the biomolecules onto the sensor transducer surface.
Raman studies were conducted to investigate the qualitative composition of Ch-Ni3V2O8

composite (Fig. 3c). For Ni3V2O8, the peaks were obtained at 350, 480, 552, 761 and 818 cm–1. The
peaks at 350 and 480 cm–1 related to the bending vibrations of V–O–V bonds, while peaks at 761 and
818 cm–1 were assigned to asymmetric and symmetric stretching mode of V–O bonds, respectively.34,35
The peak observed at 552 cm–1 was assigned to the first order phonon (1P) scattering in Ni–O.36 In the
spectrum of Ch-Ni3V2O8, the peaks at 889, 975 and 1082 cm–1 were assigned to ν(C–O–C), ν(CH) rings
and ν(C–H) band, respectively whereas peaks obtained at 1196, 1297 and 1406 cm–1 were due to ν(C–
N), ν(C–C) stretching band and –CH2–OH bond, respectively. The peaks found at 1511 and 1628.5 cm–1
pertained to the amide II and amide I while the peak region of 1740-1847 cm–1 was related to ν(C=O)
due to acetic acid solvent, respectively. The peaks at 2838.6 and 3200-3330 cm–1 belonged to ν(CH2)
stretching and –OH bonds respectively.37,38 These results clearly showed the successful
functionalization of Ch-Ni3V2O8 nanocomposite and were consistenet with the results of XRD, FTIR,
XPS and EDX mapping. Intensity of Ch-Ni3V2O8/Au Raman spectrum was slightly increased as compared
to Ch-Ni3V2O8 spectrum resulting in formation of a stable thin film of Ch-Ni3V2O8 composite on Au
electrode.
Further, the Fourier transform infrared (FTIR) measurements were conducted to examine the loading
of antibodies on the Ch-Ni3V2O8 composite (Fig. 3d). The FTIR peaks seen in the region, 649–885 cm−1
pertained to metal oxygen (M–O) vibrational bands in the Ch-Ni3V2O8 matrix.39,40 The peaks found at
1030–1092, 1156–1314, and 1406 cm−1 related to the epoxy group (C–O–C stretching vibrations), –
CH2–OH stretching and –CH2 bending, respectively while the peaks obtained at 1567 and 1634 cm−1
were due to –NH bending vibrations confirming the presence Ch in Ch-Ni3V2O8 composite matrix. The
peaks seen in the region of 2865-2928 and 3446 cm−1 were due to –CH stretching vibrations and –OH
stretching vibrations, respectively.41 The absorption peaks obtained at 1260 and 1309 cm−1 were
assigned to C–N (amine) stretching vibration in the cAb/Ch-Ni3V2O8. The sharp peaks obtained at 1574
and 1651 cm−1 were attributed to N–H bending and C=O stretching vibration, respectively, revealing
the presence of amide bond arising due to attachment of the antibodies on the Ch-Ni3V2O8 matrix via
electrostatic interaction.
7
The XPS studies were conducted to confirm the surface elemental structure and electronic
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DOI: 10.1039/C9TB00126C
configuration of atoms in the Ch-Ni3V2O8 nanocomposite. A wide-range survey XPS spectrum of Ch-
Ni3V2O8 (Fig. 4a) indicated the presence of trace elements peaks of C 1s ( 284.5), N 1s ( ̴397.7), V 2p

( ̴527.4), O 1s ( ̴530.8), and Ni 2p ( ̴853.5). The Ni 2p spectra (Fig. 4b) contained two spin-orbit peaks
at 854.3 and 872.2 eV that were due to presence of Ni 2p3/2 and Ni 2p1/2, respectively. These results
revealed that the Ni species were present in the Ni2+ state in Ch-Ni3V2O8 structure. The two satellite
peaks found at 860.3 and 878.5 eV were also found. The high-resolution peaks seen at 515.1 and 522.2
eV arising due to the V 2p3/2 and V 2p1/2 spin-orbit states revealed the existence of vanadium in the V5+
state in Ch-Ni3V2O8 structures (Fig. 4c). The O 1s spectra of Ch-Ni3V2O8 showed two sharp peaks at
529.6 eV that were attributed to the metal-oxygen bond, while the peak at 531.32 eV was ascribed to
metal-hydroxyl bond on the surface of Ni3V2O8 arising due to chemisorption and physisorption of
H2O22,25,26,42 (Fig. 4d). After modification of Ni3V2O8 with chitosan, the C 1s spectra showed binding
energies at 283.5, 285 and 286.5 eV pertaining to C−C and C−H bonds, C−NH and C–O groups and C–
O–C=O, C=O/C−O−C (alkoxy/epoxy), and N–C=O groups, of chitosan in the Ch-Ni3V2O8 nanocomposite
(Fig. 4e). Besides this, the deconvoluted N 1s peak was found at 398 and 399.9 eV. The peak at 398 eV
indicated the presence of amine group while the peak at 399.9 eV was ascribed to the protonated
amine species43,44 (Fig. 4f).
3.2 Microscopic studies
The morphological shape and structure of Ni3V2O8 hollow-nanospheres and chitosan wrapped
Ch-Ni3V2O8 composite were investigated using FESEM (Fig. 5). Fig. 5(a, b and c) reveals the FESEM
images of the synthesized Ni3V2O8 via hydrothermal process revealing the formation of hollow-
nanospheres that were inter-connected to each other. The nanospheres were composed of
nanosheet-like structures resulting in the formation of urchin-shaped structure (Fig. 5a). At high
magnification, the structure of nanospheres was found to be hollow (Fig. 5b). Fig. 5c shows clearly
hollow-nanosphere in the structure of Ni3V2O8. The hollow-nanospheres in the Ni3V2O8 structure were
attributed to self-assembly of the nanosheets. Fig. (d and e) shows uniformly wrapped Ni3V2O8 hollow-
nanosphere via chitosan with the formation of stable nanocomposite. This result was consistent with
the observed increase in the zeta potential (Fig. S1) arising due to formation of the highly stable
mesoporous Ch-Ni3V2O8 nanocomposite. After immobilization of cAb on the Ch-Ni3V2O8 surface (Fig.
f), a structural modification at the surface perhaps occurred due to the electrostatic interaction
between cAb and Ch-Ni3V2O8 resulting in creation of an amide bond.
The energy-dispersive X-ray (EDX) analysis were conducted to approve the presence of nickel
(Ni), vanadium (V) and oxygen (O) elements in Ni3V2O8 (Fig. S4). The weight and atomic percentage of
8
Ni, V and O in Ni3V2O8 were obtained to be 56.31, 21.59, 22.09 and 34.7, 15.33, 49.96%,DOI:
respectively
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10.1039/C9TB00126C
(inset Fig. S4, d). The presence of O (Fig. a), Ni (Fig. b) and V elements were confirmed by the EDX
mapping (Fig. c) in Ni3V2O8. Fig. d shows the EDX spectra. In the mapping of Ch-Ni3V2O8 composite

exhibited additional peaks for C and N element (Fig. S5) owing to functional groups of chitosan
polysaccharide (like C−NH2, C−O−C, −OH etc.). The weight and atomic percentage of C, N, O, V, and Ni
in Ch-Ni3V2O8 sample were obtained to be 25.27, 12.38, 32.05, 8.29 and 22% and 35.96, 13.22, 38.87,
3.67 and 8.27%, respectively (inset of Fig. S5, f). The results of mapping confirmed the existence of C
(Fig. a), N (Fig. b), O (Fig. c), V (Fig. d) and Ni elements (Fig. e) in Ch-Ni3V2O8 composite. Fig. f pertained
to the EDX spectra of Ch-Ni3V2O8.
Transmission electron microscopic (TEM) studies were conducted on Ni3V2O8 and Ch-Ni3V2O8
nanocomposite (Fig. 6). The presence of urchin-shaped nanospheres of Ni3V2O8 containing a hollow
space inside the spheres can be seen in Fig. 6 (a, b and c). The occurrence of lattice fringes (Fig. 6d)
corresponded to (122) and (151) planes containing 0.25 and 0.205 nm interplanar space, respectively,
indicating that the separate nanosheets were crystalline in nature. Hence, the growth of the Ni3V2O8
was typically across the (151) and (122) planes that were strongly supported by the XRD studies. The
nanospheres were polycrystalline in nature as was revealed by the selected area electron diffraction
(SAED) array of Ni3V2O8 with three diffraction rings of dissimilar radii equivalent to (151), (122) and
(442) planes (inset in Fig. 6d). The Ni3V2O8 Hollow-nanospheres were wrapped by Ch as depicted in
Fig. 6(e and f). It appeared that the anionic hydroxyl groups of Ch resulted in the creation of a strong
bond between the cationic Ni3V2O8 and chitosan.33 The wrapping layers of chitosan on Ni3V2O8
nanosphere can be clearly seen in Fig. 6f.
3.3 Surface plasmon resonance (SPR) studies
The SPR studies were conducted to confirm the immobilization of cardiac antibodies (cAb) on the Ch-
Ni3V2O8/Au matrix (Fig. 7). The self-assembled monolayer of Ch-Ni3V2O8 on Au surface was utilized as
a baseline (Fig. 7a, step 1). Subsequently, the surface was washed with PBS after which cAb molecules
interacted with the Ch-Ni3V2O8/Au electrode surface for about 15 minutes resulting in change in the
SPR angle (Step 2). This can be clearly seen in the SPR curve (Fig. 7b) indicating the immobilization of
cAb on the Ch-Ni3V2O8/Au sensor electrode. Prior to deactivation of the cAb/Ch-Ni3V2O8/Au sensor
surface with BSA (Step 4), the surface was regenerated with PBS (Step 3). The immobilized cAb/Ch-
Ni3V2O8/Au sensor surface was washed with PBS (Step 5). The surface density of the immobilized cAb
on the Ch-Ni3V2O8/Au sensor electrode surface was calculated via kinetic evaluation software using
equation (4).
9
∆ Response [mo] View Article Online

DOI: 10.1039/C9TB00126C
D= (4)
Conversion factor[mo.(mm2 ng)]
where D is the surface density of antibodies. The value of surface density of cAb on the cAb/Ch-

Ni3V2O8/Au sensor electrode was obtained as 4.41 ng mm–2. The amount of cAb loaded onto the
cAb/Ch-Ni3V2O8/Au sensor electrode surface was determined as 34.88 ng by the following equation
(5).
𝐴mount = Surface density (ng mm2) × Surface area (mm2)

(5)
The number of cAb bound to the cAb/Ch-Ni3V2O8/Au sensor electrode surface was calculated using
equation (6).
Amont(ng) 10 ―9(g)
N= × × 6.022 × 1023(mol ―1) (6)
Molar mass(g mol) (ng)
where N is the number of binding molecules of cAb on the electrode surface, was found to be 8.75×
1014.
Prior to sensing, the surface of microfluidic device was stabilized by injecting PBS into the
microfluidic system using a constant flow rate at 20 µL/min via a syringe pump and repeated CV
measurement graph was recorded till the peak current got saturated. CV measurements were carried
out to examine the effect of flow rate of the fabricated cAb/Ch-Ni3V2O8/Au microfluidic device at a
flow rate from 1 to 30 μL/min. We found that the peak current increased with increasing flow rate
(Fig. S5a). The diffusion (diffusivity) of redox species at different flow rates were calculated using the
Randles‒Sevcik equation (7) and the analogous plot in between diffusivity and flow rate has been
shown in Fig. S5b. The diffusivity (diffusion coefficient) was increased with increasing the flow rate
owing to improved mass transport at higher flow rates and it was found that the diffusivity saturated
almost at a flow rate of 20 μL/min. Therefore, all the electrochemical studies were carried out at this
optimal flow rate.
4. Electrochemical characterization
The electrochemical studies were conducted to investigate the redox properties of fabricated
electrodes in presence of 0.05 molar (M) PBS (pH 7.4) with 0.005 M [Fe (CN)6]3-/4- as redox electrolyte
(Fig. 8a). The anodic peak current of Ni3V2O8/Au electrode observed as 69.66 µA was ascribed to the
positively charged Ni2+ and tunable oxidation states of (V5+, V4+, and V3+) ions on the Ni3V2O8 matrix
that received electrons from [Fe (CN)6]3-/4- redox reaction. Alternately, this could perhaps be ascribed
to the high surface area of the Ni3V2O8/Au electrode resulting in increased rate of diffusion of
electrolytic ions due to increased heterogeneous electron transfer at the Ni3V2O8/Au electrode
10
surface.25,33 After modification of Ni3V2O8 by Ch, the magnitude of anodic peak current ofViewCh-
Article Online
DOI: 10.1039/C9TB00126C
Ni3V2O8/Au electrode decreased to 49.68 µA due to the development of cross-linked network with the
–OH, –NH2 groups of Ch and sluggish electron transfer process from the electrolytic medium to the

electrode surface.33,45 On immobilization of the cAb with BSA on Ch-Ni3V2O8/Au electrode surface,
peak current of cAb/Ch-Ni3V2O8/Au immunoelectrode further decreased to 32.51 μA and the potential
slightly shifted to higher potential owing to the insulating environment of cAb and BSA molecules.
Macromolecular structure of the antibodies and BSA on the Ch-Ni3V2O8 surface, hindered the
movement of electrons generated in the electrolytic medium. Increasing the scan rate of cyclic
voltammetry (CV) measurements of different electrodes, the peak currents of both anodic (𝐼𝑃𝑎 ) and
cathodic (𝐼𝑃𝑐) were simultaneously enhanced alongwith the linear shift of the peak potential (∆𝐸𝑃 =
𝐸𝑃𝑎‒ 𝐸𝑃𝑐). Thus, the Ni3V2O8/Au, Ch-Ni3V2O8/Au electrodes and cAb/Ch-Ni3V2O8/Au bioelectrode
revealed diffusion-controlled processes. The diffusion coefficient (D) for ferro-ferricyanide electrolyte
solution was determined using the Randles‒Sevcik equation (7).46
32
IP = k n A DC ν (7)
where k is a constant, n is the number of electrons appearing in the redox couple response
(stoichiometry = 1), D is the electrolyte diffusion coefficient, C is the concentration of electrolyte (5
mM), ν is the CV measured scan rate (20 mV s‒1), A represents the area of transducer surface (0.008
cm2) and IP is the anodic and cathodic peak current. The Ni3V2O8/Au, Ch-Ni3V2O8/Au electrodes and the
cAb/Ch-Ni3V2O8/Au bioelectrode revealed the value of the diffusion coefficient as 20.96 × 10‒4, 10.66
× 10‒4 and 4.56 × 10‒4 cm2s−1, respectively. The increased D value of the Ni3V2O8/Au electrode was due
to fast electrons transfer rate and electrochemically mediated nature as compared to the Ch and cAb
functionalized bioelectrode. The surface concentrations of the electrolyte ions for Ni3V2O8/Au, Ch-
Ni3V2O8/Au and cAb/Ch-Ni3V2O8/Au electrodes were determined by the Brown-Anson model as given
equation (8).46
I ∗ A V n2F2
IP = 4RT (8)
where I* is the electrode surface concentration in mole cm‒2, A is the surface area (0.008 cm2) of
microfluidic electrode, V is the scan rate, n is the number of electrons transported in CV measurement
(1), F is the Faraday constant and IP is the anodic peak current. For the Ni3V2O8/Au, Ch-Ni3V2O8/Au, and
cAb/Ch-Ni3V2O8/Au electrodes, the value of surface concentrations of electrolyte ions was obtained
as 4.66 × 10‒7, 3.29 × 10‒7 and 2.17 × 10‒7 mole cm‒2, respectively. The different values of surface
11
concentration obtained for electrodes indicated the modification of the electrode DOI:
with10.1039/C9TB00126C
ChView
and
Article Online
antibodies.
Electrochemical impedance spectroscopy (EIS) studies were conducted to examine the

interfacial features of microfluidic device in the frequency range of (0.01‒105 Hz). The interfacial
properties of electrodes were examined by calculating the charge transfer resistance (RCT) and double
layer capacitance (Cdl) at the conductive electrode and the ionically electrolyte interface (Fig. 8b and
c). RCT is the diameter of semicircle portion in the impedance spectra, semicircle portion resembled to
the finite procedure of electron transfer kinetics of redox study at the interface of a conductive
electrode. Dielectric and insulating nature of materials may result in change in RCT at the
electrode/electrolyte interface. The frequency and time constant can be associated with RCT and Cdl
using equation (9).
𝟏
𝟐𝛑𝐟𝐦𝐚𝐱 = RCT Cdl = 𝛕 (9)
where τ is the time constant and fmax is the maximum frequency. The observed decreased RCT value of
the Ni3V2O8/Au electrode (2.57 kΩ) can be related to the Ch-Ni3V2O8/Au electrode (3.96 KΩ) owing to
high surface area, positively charged Ni2+ and tunable oxidation states of (V5+, V4+, and V3+) ions on the
Ni3V2O8 matrix that increased the electron transportation from the [Fe(CN)6]3-/4- medium as a result
reduction of RCT value.25 The increased RCT value of the Ch-Ni3V2O8/Au electrode (3.96 KΩ) was due to
formation of the cross-linked network of the –OH, –NH2 groups of Ch on the Ni3V2O8/Au electrode
surface that influenced the polarity on the surface of electrode. Consequently, the decreased ability
of ferro-ferricyanide ions resulted in low RCT value.47 After immobilization of cAb and BSA on the Ch-
Ni3V2O8/Au sensor electrode, the RCT value was found to be increased (5.99 KΩ). This was due to the
formation of insulating layers of cAb on the sensor electrode surface that obstructed the flow rate of
charge transfer process of redox species. In EIS electrochemical charges transfer among the electrode
and electrolyte, the heterogeneous electron transfer (Ket) constant was calculated using equation (10)
RT
Ket = 2 2
n F A RCT C
(10)
where R is the gas constant, T is the absolute temperature, n is the number of electrons participating
in redox process, F is the Faraday constant, A is the active area of the sensor surface and C is the
electrolyte concentration. The Ket value for the Ch-Ni3V2O8/Au bioelectrode found to be as 1.69 × 10‒6
cm s‒1 was greater than that of the cAb/Ch-Ni3V2O8/Au immunoelectrode (1.12 × 10‒6 cm s‒1) indicating
rapid electrons transport.
4.1 Response studies
12
We measured the electrochemical response of the fabricated microfluidic biosensor

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DOI: 10.1039/C9TB00126C
(cAb/Ch-Ni3V2O8/Au) as a function of cTnI concentration (0.005‒100 ng/mL) (Fig. 9a). In this method,
the detection of the analyte at the sensor electrode was accomplished by exploring the transfer of

electrons from the sensor electrode surface to the electrolytic medium. The magnitude of the current
changed at the electrode/electrolyte interface once the target analytes were captured at the sensor
electrode of microfluidic device. For cTnI detection, its several concentrations were injected into the
microfluidic sensor by maintaining a constant flow rate at 20 µL/minute using the syringe pump. The
magnitude of the anodic peak current of this microfluidic sensor was found to decrease with increasing
concentration of cTnI (Fig. 9a). The observed decrease in current arising due to the interaction of cAb
and cTnI at the sensor electrode surface resulted in the formation of an insulating layer of the antigen-
antibodies complex that hindered the transport of electrons through [Fe(CN)6]3‒/4‒ redox conversion.48
For each concentration, the sensor was washed with PBS to regenerate the electrode surface for the
detachment of antigen-antibody complex. The calibration plot obtained for cAb/Ch-Ni3V2O8/Au-based
sensor among the anodic peak current and cTnI concentration, followed equation (11) with a
regression coefficient of 0.982, [Fig. 9b].
Ipa (A) = 1.434 µA ‒ 0.311 µA × cTnI (ng mL‒1) (11)
The fabricated sensor revealed a high sensitivity of 38.88 µA/(ng mL‒1)/cm2 with the low detection
limit (LOD) of 5 pg mL‒1 with the detection time of 2.57 min (Fig. S5c). To calculate LOD, we used the
3SD/m criteria, wherein the SD is the standard deviation of blank sample which obtained from three
repeated measurement and m is the sensitivity of the calibration graph. The sensitivity and the
detection limit of this sensor were significantly higher as compared to those reported for cTnI
detection.49–56 This may perhaps be due to greater interaction of the antigens with the antibodies at
the surface of cAb/Ch-Ni3V2O8/Au and increased loading of the antibodies at the sensor surface. This
was ascribed to the integration of the mesoporous Ch-Ni3V2O8 hollow-nanosphere into the microfludic
structure. The Ni3V2O8 provided the desired electronic properties, mesoporous hollow-
nanostructured, high surface area and multiple oxidation states led to improved electrochemical
response. Besides this, the presence of Ch biopolymer with Ni3V2O8 provided a highly biocompatible
and stable functionalized sensor platform to enhance the loading of antibodies on the sensor surface
resulting in improved sensitivity and low limit of detection. Table 1 shows results of these studies
alongwith those reported in literature.
A PDMS-Au microfluidic chip-CdTe quantum dot-based biosensor reported a LOD of 4.0 pg mL‒1
with linear range of 0.01 ng mL‒1–50 ng mL–1 and a detection time of 3.7 h57. A fluorescent
europium(III)-chelate-dyed nanoparticles-based biosensor showed a LOD of 2.0 pg mL‒1 with
13
detection time as 15 min58 . An LOD of 0.1 pg mL‒1 with linear range of (0.1 pg mL‒1–100 DOI:
ng mL –1)View
wasArticle Online
10.1039/C9TB00126C
reported using a TiO2 nanotube array-based biosensor with a detection time of 2 h11. In another work,
Au nanoparticles Ru-peptide revealed a LOD 0.4 pg mL–1 with the detection time of 2 h59. Compared

to the above works, the detection time reported in this work is much lower (about 2.57 minutes).
The developed microfluidics biosensor was used for cTnI detection in human blood serum
sample procured from a Kamineni Hospital, LB Nagar, Hyderabad, India. The various serum
concentrations were injected onto the sensor via microchannels onto the sensor surface and
electrochemical CV response was recorded (Fig. 9c). The CV response was determined with a standard
sample of cTnI by cumulative concentration in the range, 0.02−50 ng mL‒1. Similarly, we conducted
the testing using numerous cTnI concentration in the blood serum. The cTnI present in the serum
sample interacted with cAb on the sensor electrode resulting in an improved surface concentration of
cTnI on the sensor electrode surface. The magnitude of current was found to decrease with increasing
serum concentration of cTnI owing to greater communication of serum sample and cAb at
electrode/electrolyte boundary, leading to trapping of the cTnI serum at the cAb/Ch-Ni3V2O8/Au
immunosensor electrode surface of device (Fig. 9d). The peak current at higher concentration of
serum sample was found to be less than that of standard sample (Fig. 9d) perhaps due to the presence
of other biomolecules in the serum sample. The obtained relative standard deviation (RSD) for the
produce current was less than ±5%, ±3.5% and ±2% for the cTnI serum concentration of 50, 5 and 0.02
ng mL‒1, respectively. We found the RSD value to be less than ±1% for 20 ng mL‒1 concentration (Table
S1). As a result, the fabricated device could be used to detect the cTnI in the human blood serum
samples with low concentration of 0.02 ng mL‒1.
We conducted experiments relating to the recovery of serum samples with the standard
samples. The recovery values of serum samples were in the range of (92-104%) revealing good
evaluation of the fabricated microfluidic device. The recovery data of the sensor is shown to the Table
S1 (Supporting Information).
4.2 Control and reproducibility studies
We carried out the control experiments with Ch-Ni3V2O8/Au electrode in the absence of cAb.
The injection of cTnI concentration into the Ch-Ni3V2O8/Au electrode did not result in any significant
variation in the output of amperometric signal (Fig. 10a) due to non-specific interaction of cTnI
biomolecule at the Ch-Ni3V2O8/Au electrode surface in the absence of cAb. In the presence of cAb, the
cAb/Ch-Ni3V2O8/Au electrode exhibited significant change in the output signal (Fig. 10a) indicating that
observed change in current was due to interaction of cAb with cTnI biomolecule. The minor instability
in the response current of the Ch-Ni3V2O8/Au electrode was owing to the non-specific interactions of
14
cTnI (1 to 60 ng mL‒1). The reproducibility test for cAb/Ch-Ni3V2O8/Au-based sensor wasDOI:

also carried
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10.1039/C9TB00126C
out. For this study, the four sets of cAb/Ch-Ni3V2O8/Au bioelectrodes were fabricated under similar
conditions. The four sets of electrodes revealed electrochemical response of 5 ng mL‒1 concentration

of cTnI with 2.59 and 7.43% standard deviation and relative standard deviation respectively, (Fig. 10b)
indicating high reproducibility
4.3 Selectivity and shelf-life stability

The selectivity of the microfluidic device was carried out in the presence of interferents (30
ng mL‒1), B-type natriuretic peptide (BNP), myoglobin (Mb), cardiac troponin C (cTnC), and cardiac
troponin T (cTnT) and cTnI (30 ng mL‒1). The CV response of the sensor during the detection of cTnI
Fwas not significantly influenced owing to the existence of interferents (Fig. 10c). In this experiment,
cTnI revealed the lowest magnitude of current response value as compared to all interferents due to
increased interaction of cTnI with cAb/Ch-Ni3V2O8/Au surface resulting in decrease of current
response. However, cTnc and cTnT exhibited a minor influence indicating high selectivity. The stability
of the sensor was determined by measuring CV response at a consistent interval of time up to 10
weeks. The microfluidic sensor was stored at 4 oC after the experiments. This device revealed 90% of
response up to 6 weeks after which the current value of CV response was found to be decreased (Fig.
10d)
5. Conclusion
A high sensitive microfluidic sensor has been fabricated using hollow-nanosphere structures
of Ni3V2O8 for human cardiac troponin I detection. The mesoporous structure and specific surface area
have been analysed by BET and the conjugation of cAb on the surface of Ch-Ni3V2O8/Au biointerface
has been confirmed by FTIR and surface plasmon response studies. In this device, Ch-Ni3V2O8
composite material has been incorporated into the microfluidic structure. The distinctive structure of
Ch-Ni3V2O8 composite like mesoporosity, hollow-nanosphere structure, good biocompatibility and the
redox performance of binary Ni3V2O8 has been found to facilitate the loading of more antibodies with
improved detection performance such as high selectivity, low limit of detection and high sensitivity.
Also, this device exhibited a LOD of 5 pg mL−1 of cTnI, which is much lower than the previously reported
cTnI biosensors and extensive stability, highlighting the advantage of the proposed biomolecule
detection platform. The mesoporous hollow-nanosphere structure of Ch-Ni3V2O8 biomaterial in the
sensor has been found to offer biophysical microenvironment for the antigen and antibodies
interaction resulting in higher affinity for cTnI. Also, the high surface area and high electronic
properties of this microfluidic device facilitate faster electron transport. The efforts have also been
made to test human serum samples of cTnI using this microfluidic sensor. The efforts should be made
15
to obtain improved limit of detection of this proposed microfluidic biosensor platform and to utilize
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DOI: 10.1039/C9TB00126C
for detection of other clinically important biomolecules (e.g. low density lipoprotein, B-type natriuretic
peptide, myoglobin, cardiac troponin C, and cardiac troponin T etc.).

Table 1. The characteristics of the cAb/Ch-Ni3V2O8/Au biosensors alongwith those reported in

literature for cTnI detection
Working electrodes Sensing techniques Linear range (ng LOD (ng Sensitivity References
mL−1) mL−1)
2-ABA CV 0 .01–1 0.01 … 49
functionalized
graphene
interdigitated
Aptamer-based SPCE Chronoamperometry 0.024–2.4 0.024 … 50
electrode
PDMS CV 0.2–10000 0.148 … 51
microchannel−IDA
gold electrode
Vacrel® 8100 Amperometry … 0.025 … 52
microfluidic
immunochip
Carbon nanofiber EIS/CV 0.1–100 0.2 … 53
GNPs−Screen Capacitive 0.2–12.5 0.2 … 54
printed electrode
APTES/n-WO3/ITO EIS 1–250 16.75 26.56 Ω ng−1mL 55
cm−2)
Si nanowire Field effect transistor 0.092–46 0.092 … 56
cAb/Ch-Ni3V2O8/Au CV 0.005−100 0.005 38.88 µA/(ng Present

microfluidic chip mL‒1)/cm2 work
16
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DOI: 10.1039/C9TB00126C
Acknowledgements

P Rai is thankful to DST, India for the financial support vide Inspire Faculty Award (IFA14-MS20). The
authors are thankful to Dr. Inayatullah Ghori, Kamineni Hospital, LB Nagar, Hyderabad, India
for providing the clinical sample.
Associated Content
Supplementary Information
Zeta potential studies, Microfluidic device fabrication, Instrumentation, EDX mapping of Ni3V2O8 and
Ch-Ni3V2O8, CV response of the microfluidic device at different flow rate and a Comparison table for
output CV signals in percentage standard deviation and sensor recovery of the microfluidic sensor
responding to standard and blood serum samples of cTnI.
Figure Captions
Schematic: Graphical abstract.
Fig. 1. Proposed mechanism for the modification of Ni3V2O8 with chitosan.
Fig. 2. Graphic illustration of the fabrication of microfluidic device for quantification of cTnI. (a)
Schematic demonstration of the microfluidic device with inserted cAb/Ch-Ni3V2O8/Au as a transducer.
(b) Antibodies were immobilized on the surface of Ch-Ni3V2O8/Au electrode inside the microfluidics
channels. (c) Formation of immunocomplex after interaction of cTnI with the cAb inside the
microfluidic channels for the detection of cTnI. (d) Photograph of a microfluidic device for sensing of
human cardiac troponin I. (e) Enlarged view of the optical microscopic image of the microfluidic
sensor. (f) FESEM image of the Ni3V2O8.
Fig. 3. Characterization of Ni3V2O8 nanospheres, Ch-Ni3V2O8 and cAb/Ch-Ni3V2O8 bioelectrode: (a) X-

ray diffraction spectra of Ni3V2O8 nanospheres. (b) Nitrogen adsorption-desorption isotherms of
Ni3V2O8. Inset: pore size distribution arrangement. (c) Raman spectra of Ni3V2O8, Ch-Ni3V2O8 and Ch-
Ni3V2O8/Au. (d) FTIR spectra of Ch-Ni3V2O8 and cAb/Ch-Ni3V2O8.
Fig. 4. XPS studies for Ch-Ni3V2O8 biomaterial. (a) Wide-scan spectra, shows the presence of Ni, V, O C
and N elements, (b) Ni 2p spectra, (c) V 2p spectra, (d) O 1s spectra, (e) C 1s spectra, after
deconvolution and (f) N 1s spectra for Ch-Ni3V2O8 composite.
17
Fig. 5. FESEM characterization of the cAb/Ch-Ni3V2O8 biomaterial. (a, b and c) FESEM images of
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DOI: 10.1039/C9TB00126C
synthesized Ni3V2O8 hollow-nanospheres in hydrothermal reaction environments such as 185oC, 22 h.

(d and e) FESEM images of Ch-Ni3V2O8 nanocomposite. (f) cAb immobilized Ch-Ni3V2O8

nanocomposite.
Fig. 6. TEM characterization of the synthesized Ch-Ni3V2O8 biomaterial. (a, b and c) TEM images of
Ni3V2O8 hollow-nanospheres. (d) Atomic-scale image of Ni3V2O8 hollow-nanospheres. Inset: SAED
pattern. (e and f) images of Ch-Ni3V2O8 nanocomposite.
Fig. 7. Surface plasmon resonance (SPR) study. (a) SPR spectra for the immobilization of cardiac
antibodies (cAb) on the Ch-Ni3V2O8/Au electrode. (b) SPR curve (black) of Ch-Ni3V2O8/Au as baseline
and blue of cAb/Ch-Ni3V2O8/Au.
Fig. 8. Electrochemical characterization: (a) CV response of the microfluidic sensor based on

Ni3V2O8/Au, Ch-Ni3V2O8/Au electrode and cAb/Ch-Ni3V2O8/Au immunoelectrode in the presence of
0.05 M PBS (pH 7.4) having 0.005 M [Fe (CN)6]3−/4− solution as redox species at 20 mV s−1 scan rate. (b)
Nyquist plot and relating fitting for Ni3V2O8/Au electrode via EIS technique to calculate Rct and Cdl
values. The semicircle and straight regions indicated kinetic and mass transfer control. Inset shows the
equivalent circuit model for EIS spectra. The frequency range was set to (0.01–105 Hz) as a function of
potential (0.03V). (c) EIS spectra for Ni3V2O8/Au, Ch-Ni3V2O8/Au electrode and cAb/Ch-Ni3V2O8/Au
immunoelectrode.
Fig. 9. Cyclic voltammetry response: (a) CV response of the cAb/Ch-Ni3V2O8/Au microfluidic sensor as
a function of cTnI (0.005–100 ng/mL) at 20 mV s−1 scan rate in a PBS solution of (pH 7.4). (b)
Microfluidic sensor calibration curve presenting the output current versus cTnI concentration. The
inset reveals the connection among the logarithm of cTnI concentration and output current. (c) CV
response of the microfluidic sensor to blood serum samples of cTnI. (d) Comparison plots for output
CV signals of the microfluidic sensor responding to standard and blood serum samples of cTnI. The
standard deviation of three repeated measurements for all concentrations were calculated with error
bars.
Fig. 10. (a) Control study of the cAb/Ch-Ni3V2O8/Au microfluidic electrode in a PBS solution of (pH 7.4).
(b) Histogram plot of reproducibility test carried out at 5 ng/mL cTnI concentration for four sets of
microfluidic electrode for the current output. (c) The selectivity of the cAb/Ch-Ni3V2O8/Au microfluidic
electrode. (d) Shelf-life stability of the cAb/Ch-Ni3V2O8/Au microfluidic electrode was determined by
measuring CV response at a consistent interval of time up to 10 weeks. It is observed that the co-
efficient of variances are in between 0.48 to 8.4%.
18
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DOI: 10.1039/C9TB00126C
References

1 M.-I. Mohammed and M. P. Y. Desmulliez, Lab. Chip, 2011, 11, 569–595.
2 K. Kim, C. Park, D. Kwon, D. Kim, M. Meyyappan, S. Jeon and J.-S. Lee, Biosens. Bioelectron., 2016,
77, 695–701.
3 X. Han, S. Li, Z. Peng, A. M. Othman and R. Leblanc, ACS Sens., 2016, 1, 106–114.
4 B. McDonnell, S. Hearty, P. Leonard and R. O’Kennedy, Clin. Biochem., 2009, 42, 549–561.
5 I.-H. Cho, E.-H. Paek, Y.-K. Kim, J.-H. Kim and S.-H. Paek, Anal. Chim. Acta, 2009, 632, 247–255.
6 J. Wang, A. Ibáñez, M. P. Chatrathi and A. Escarpa, Anal. Chem., 2001, 73, 5323–5327.
7 C. A. Marquette, F. Bouteille, B. P. Corgier, A. Degiuli and L. J. Blum, Anal. Bioanal. Chem., 2009, 393,
1191–1198.
8 F. S. Apple, A. Falahati, P. R. Paulsen, E. A. Miller and S. W. Sharkey, Clin. Chem., 1997, 43, 2047–
2051.
9 B. Cummins, M. L. Auckland and P. Cummins, Am. Heart J., 1987, 113, 1333–1344.
10 R. H. Christenson, E. Jacobs, D. Uettwiller-Geiger, M. P. Estey, K. Lewandrowski, T. I. Koshy, K.
Kupfer, Y. Li and J. C. Wesenberg, J. Appl. Lab. Med. AACC Publ., 2017, 1, 544–561.
11 P. Kar, A. Pandey, J. J. Greer and K. Shankar, Lab. Chip, 2012, 12, 821–828.
12 K. Thygesen, J. Mair, H. Katus, M. Plebani, P. Venge, P. Collinson, B. Lindahl, E. Giannitsis, Y. Hasin,
M. Galvani, M. Tubaro, J. S. Alpert, L. M. Biasucci, W. Koenig, C. Mueller, K. Huber, C. Hamm and A.
S. Jaffe, Eur. Heart J., 2010, 31, 2197–2204.
13 S. K. Sia and L. J. Kricka, Lab. Chip, 2008, 8, 1982–1983.
14 E. K. Sackmann, A. L. Fulton and D. J. Beebe, Nature, 2014, 507, 181–189.
15 M. Medina-Sánchez, S. Miserere and A. Merkoçi, Lab. Chip, 2012, 12, 1932–1943.
16 L. Reverté, B. Prieto-Simón and M. Campàs, Anal. Chim. Acta, 2016, 908, 8–21.
17 J. Kim, Z. Li and I. Park, Lab. Chip, 2011, 11, 1946–1951.
18 A. S. Ghrera, C. M. Pandey, M. A. Ali and B. D. Malhotra, Appl. Phys. Lett., 2015, 106, 193703.
19 A. Wisitsoraat, P. Sritongkham, C. Karuwan, D. Phokharatkul, T. Maturos and A. Tuantranont,
Biosens. Bioelectron., 2010, 26, 1514–1520.
20 M. Medina-Sánchez, C. C. Mayorga-Martinez, T. Watanabe, T. A. Ivandini, Y. Honda, F. Pino, K.
Nakata, A. Fujishima, Y. Einaga and A. Merkoçi, Biosens. Bioelectron., 2016, 75, 365–374.
21 N. Singh, M. A. Ali, P. Rai, A. Sharma, B. D. Malhotra and R. John, ACS Appl. Mater. Interfaces, 2017,
9, 33576–33588.
22 Y. Lei, J. Li, Y. Wang, L. Gu, Y. Chang, H. Yuan and D. Xiao, ACS Appl. Mater. Interfaces, 2014, 6,
1773–1780.
23 W. Kang, Y. Tang, W. Li, X. Yang, H. Xue, Q. Yang and C.-S. Lee, Nanoscale, 2014, 7, 225–231.
24 A. K. Mondal, D. Su, S. Chen, X. Xie and G. Wang, ACS Appl. Mater. Interfaces, 2014, 6, 14827–
14835.
25 C. Wang, D. Fang, H. Wang, Y. Cao, W. Xu, X. Liu, Z. Luo, G. Li, M. Jiang and C. Xiong, Sci. Rep., 2016,
6, 20826.
26 R. Kumar, P. Rai and A. Sharma, J. Mater. Chem. A, 2016, 4, 9822–9831.
27 W. Huang, Y. Cao, Y. Chen, J. Peng, X. Lai and J. Tu, Appl. Surf. Sci., 2017, 396, 804–811.
28 J. Yang, H. Ye, Z. Zhang, F. Zhao and B. Zeng, Sens. Actuators B Chem., 2017, 242, 728–735.
29 F. S. Omar, A. Numan, N. Duraisamy, S. Bashir, K. Ramesh and S. Ramesh, J. Alloys Compd., 2017,
716, 96–105.
30 J. Vinoth Kumar, R. Karthik, S.-M. Chen, N. Raja, V. Selvam and V. Muthuraj, J. Colloid Interface Sci.,
2017, 500, 44–53.
31 K. K. Naik, S. Kumar and C. S. Rout, RSC Adv., 2015, 5, 74585–74591.
32 K. Kamil Reza, N. Singh, S. K. Yadav, M. K. Singh and A. M. Biradar, Biosens. Bioelectron., 2014, 62,
47–51.
19
33 P. R. Solanki, M. K. Patel, M. A. Ali and B. D. Malhotra, J. Mater. Chem. B, 2015, 3, 6698–6708. View Article Online
DOI: 10.1039/C9TB00126C
34 L. Niu, Y. Wang, F. Ruan, C. Shen, S. Shan, M. Xu, Z. Sun, C. Li, X. Liu and Y. Gong, J. Mater. Chem. A,
2016, 4, 5669–5677.
35 R. Kumar, P. K. Gupta, P. Rai and A. Sharma, New J. Chem., 2018, 42, 1243–1249.

36 Z. Qiu, D. He, Y. Wang, X. Zhao, W. Zhao and H. Wu, RSC Adv., 2017, 7, 7843–7856.
37 V. Sanna, A. M. Roggio, S. Siliani, M. Piccinini, S. Marceddu, A. Mariani and M. Sechi, Int. J.
Nanomedicine, 2012, 7, 5501–5516.
38 A. Zając, J. Hanuza, M. Wandas and L. Dymińska, Spectrochim. Acta. A. Mol. Biomol. Spectrosc.,
2015, 134, 114–120.
39 M. A. Ali, P. R. Solanki, M. K. Patel, H. Dhayani, V. V. Agrawal, R. John and B. D. Malhotra, Nanoscale,
2013, 5, 2883–2891.
40 M. Wu, X. Zhang, S. Gao, X. Cheng, Z. Rong, Y. Xu, H. Zhao and L. Huo, CrystEngComm, 2013, 15,
10123–10131.
41 R. Nanda, A. Sasmal and P. L. Nayak, Carbohydr. Polym., 2011, 83, 988–994.
42 M. Xing, L.-B. Kong, M.-C. Liu, L.-Y. Liu, L. Kang and Y.-C. Luo, J. Mater. Chem. A, 2014, 2, 18435–
18443.
43 Maachou Hamida, Genet Michel J., Aliouche Djamel, Dupont-Gillain Christine C. and Rouxhet Paul
G., Surf. Interface Anal., 2013, 45, 1088–1097.
44 J. F. B. Barata, R. J. B. Pinto, V. I. R. C. Vaz Serra, A. J. D. Silvestre, T. Trindade, M. G. P. M. S. Neves,
J. A. S. Cavaleiro, S. Daina, P. Sadocco and C. S. R. Freire, Biomacromolecules, 2016, 17, 1395–1403.
45 Q. Sheng, K. Luo, L. Li and J. Zheng, Bioelectrochemistry, 2009, 74, 246–253.
46 A. J. Bard, L. R. Faulkner, J. Leddy and C. G. Zoski, Electrochemical methods: fundamentals and
applications, wiley New York, 1980, vol. 2.
47 J. Singh, A. Roychoudhury, M. Srivastava, P. R. Solanki, D. W. Lee, S. H. Lee and B. D. Malhotra,
Nanoscale, 2013, 6, 1195–1208.
48 A. Kaushik, A. Yndart, R. D. Jayant, V. Sagar, V. Atluri, S. Bhansali and M. Nair, Int. J. Nanomedicine,
2015, 10, 677–685.
49 S. K. Tuteja, M. Kukkar, C. R. Suri, A. K. Paul and A. Deep, Biosens. Bioelectron., 2015, 66, 129–135.
50 H. Jo, J. Her, H. Lee, Y.-B. Shim and C. Ban, Talanta, 2017, 165, 442–448.
51 S. Ko, B. Kim, S.-S. Jo, S. Y. Oh and J.-K. Park, Biosens. Bioelectron., 2007, 23, 51–59.
52 J. Horak, C. Dincer, E. Qelibari, H. Bakirci and G. Urban, Sens. Actuators B Chem., 2015, 209, 478–
485.
53 A. Periyakaruppan, R. P. Gandhiraman, M. Meyyappan and J. E. Koehne, Anal. Chem., 2013, 85,
3858–3863.
54 V. Bhalla, S. Carrara, P. Sharma, Y. Nangia and C. Raman Suri, Sens. Actuators B Chem., 2012, 161,
761–768.
55 D. Sandil, S. Kumar, K. Arora, S. Srivastava, B. D. Malhotra, S. C. Sharma and N. K. Puri, Mater. Lett.,
2017, 186, 202–205.
56 T. Kong, R. Su, B. Zhang, Q. Zhang and G. Cheng, Biosens. Bioelectron., 2012, 34, 267–272.
57 F. Zhou, M. Lu, W. Wang, Z.-P. Bian, J.-R. Zhang and J.-J. Zhu, Clin. Chem., 2010, 56, 1701–1707.
58 M.-L. Järvenpää, K. Kuningas, I. Niemi, P. Hedberg, N. Ristiniemi, K. Pettersson and T. Lövgren, Clin.
Chim. Acta, 2012, 414, 70–75.
59 M. Shan, M. Li, X. Qiu, H. Qi, Q. Gao and C. Zhang, Gold Bull., 2014, 47, 57–64.
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Graphical abstract
1
DOI: 10.1039/C9TB00126C
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Materials Chemistry B: Journal of

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acceptance, before technical editing, formatting and proof reading.

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Hollow-nanospheres-based microfluidic biosensors for biomonitoring ofView Article Online

Journal of Materials Chemistry B Accepted Manuscript

Keywords: Cardiac Troponin I, Ni3V2O8 Hollow-nanosphere, Microfluidics, Acute Myocardial Infarction,

*Corresponding Authors: renujohn@iith.ac.in*, Ph: +91-40-23016097, bansi.malhotra@gmail.com*

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Journal of Materials Chemistry B Accepted Manuscript

Many techniques such as enzyme-linked immunosorbent assay (ELISA)5,6, chemiluminescent

With recent advancement of micro/nanofabrication technology, the microfluidic structured

developments, it is still challenging to interface desired biomolecules with functional nanomaterials

Journal of Materials Chemistry B Accepted Manuscript

In this paper, we report on the development of a microfluidic immunosensor at point of care

Chemicals and reagents

Synthesis of Ni3V2O8 hollow-nanospheres

Journal of Materials Chemistry B Accepted Manuscript

NaVO3 + 3NH4OH → (NH4)3VO4 + NaOH + H2O (1)

3Ni(NO3)2 + 2(NH4)3VO4 → Ni3V2O8 + 6NH4NO3 (2)

Ni3V2O8 modification with chitosan

Journal of Materials Chemistry B Accepted Manuscript

Serum sample preparation

Journal of Materials Chemistry B Accepted Manuscript

3. Results and discussion

3.1 Spectroscopic Analysis

Journal of Materials Chemistry B Accepted Manuscript

Raman studies were conducted to investigate the qualitative composition of Ch-Ni3V2O8

Journal of Materials Chemistry B Accepted Manuscript

3.2 Microscopic studies

Journal of Materials Chemistry B Accepted Manuscript

3.3 Surface plasmon resonance (SPR) studies

∆ Response [mo] View Article Online

Journal of Materials Chemistry B Accepted Manuscript

𝐴mount = Surface density (ng mm2) × Surface area (mm2)

Journal of Materials Chemistry B Accepted Manuscript

Electrochemical impedance spectroscopy (EIS) studies were conducted to examine the

Journal of Materials Chemistry B Accepted Manuscript

4.1 Response studies

We measured the electrochemical response of the fabricated microfluidic biosensor

Journal of Materials Chemistry B Accepted Manuscript

Ipa (A) = 1.434 µA ‒ 0.311 µA × cTnI (ng mL‒1) (11)

Journal of Materials Chemistry B Accepted Manuscript

4.2 Control and reproducibility studies

cTnI (1 to 60 ng mL‒1). The reproducibility test for cAb/Ch-Ni3V2O8/Au-based sensor wasDOI:

Journal of Materials Chemistry B Accepted Manuscript

4.3 Selectivity and shelf-life stability

Journal of Materials Chemistry B Accepted Manuscript

Table 1. The characteristics of the cAb/Ch-Ni3V2O8/Au biosensors alongwith those reported in

2-ABA CV 0 .01–1 0.01 … 49

PDMS CV 0.2–10000 0.148 … 51

Carbon nanofiber EIS/CV 0.1–100 0.2 … 53

GNPs−Screen Capacitive 0.2–12.5 0.2 … 54

APTES/n-WO3/ITO EIS 1–250 16.75 26.56 Ω ng−1mL 55

Si nanowire Field effect transistor 0.092–46 0.092 … 56

cAb/Ch-Ni3V2O8/Au CV 0.005−100 0.005 38.88 µA/(ng Present

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Journal of Materials Chemistry B Accepted Manuscript

Schematic: Graphical abstract.

Corresponding Authors: renujohn@iith.ac.in, Ph: +91-40-23016097, bansi.malhotra@gmail.com*