Accepted Manuscript
Title: Graphene oxide functionalized long period fiber grating
for highly sensitive hemoglobin detection
Authors: C. Liu, B.J. Xu, L. Zhou, Z. Sun, H.J. Mao, J.L.
Zhao, L. Zhang, X. Chen
PII: S0925-4005(18)30123-0
DOI: https://doi.org/10.1016/j.snb.2018.01.117
Reference: SNB 23966
To appear in: Sensors and Actuators B
Received date: 31-8-2017
Revised date: 10-1-2018
Accepted date: 11-1-2018
Please cite this article as: C.Liu, B.J.Xu, L.Zhou, Z.Sun, H.J.Mao, J.L.Zhao,
L.Zhang, X.Chen, Graphene oxide functionalized long period fiber grating
for highly sensitive hemoglobin detection, Sensors and Actuators B:
Chemical https://doi.org/10.1016/j.snb.2018.01.117
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1
Graphene oxide functionalized long period fiber grating for
highly sensitive hemoglobin detection
C. Liu
a
, B.J. Xu
b,*
, L. Zhou
a,b
, Z. Sun
c
, H.J. Mao
b
, J.L. Zhao
b
, L. Zhang
c
, X. Chen
a,*
a
School of Electronic Engineering, Bangor University, Bangor LL57 1UT, United Kingdom
b
Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, Shanghai,
200050, P.R. China
c
Aston Institute of Photonic Technologies, Aston University, Birmingham B4 7ET, United Kingdom
*
Corresponding authors:
Email address: xbj@mail.sim.ac.cn (B.J. Xu),x.chen@bangor.ac.uk (X. Chen). Tel: +44 1248 382480
Research Highlights:
Label-free biosensor based on graphene oxide (GO)-long period grating (LPG)
GO-LPG configuration for strong light-matter interaction
A new deposition technique for the cylindrical substrate
Ultrahigh sensitivity with detectable concentration of 0.05 mg/mL, far below hemoglobin threshold value for anemia
defined by WHO
ABSTRACT
We present graphene oxide (GO) nanosheets functionalized long period grating (LPG) for ultrasensitive hemoglobin sensing. The
sensing mechanism relies on the measurement of LPG resonant intensity change induced by the adsorption of hemoglobin molecules
onto GO, where GO as a bio-interface linkage provides the significant light-matter interaction between evanescent field and target
molecules. The deposition technique based on chemical-bonding associated with physical-adsorption was developed to immobilize
GO nanosheets on cylindrical fiber device. The surface morphology was characterized by scanning electron microscope, atomic
force microscopy, and Raman spectroscopy. With relatively thicker GO coating, the refractive index (RI) sensitivity of GO-LPG
was extremely enhanced and achieved -76.5 dB/RIU, -234.2 dB/RIU and +1580.5 dB/RIU for RI region of 1.33-1.38, 1.40-1.44 and
1.45-1.46, respectively. The GO-LPG was subsequently implemented as an optical biosensor to detect human hemoglobin giving a
sensitivity of 1.9 dB/(mg/mL) and a detectable concentration of 0.05 mg/mL, which was far below the hemoglobin threshold value
for anemia defined by World Health Organization. The proposed GO-LPG architecture can be further developed as an optical
biosensing platform for anemia diagnostics and biomedical applications.
Keywords: Anemia; Biosensor; Graphene oxide; Hemoglobin; Long period grating; Optical sensor.
1. Introduction
For decades, a variety of efforts have been made to develop accurate and low expense chemosensors and biosensors for the
applications in food safety, environmental monitoring, clinical analysis, and healthcare sectors. Anemia is a common concern in
geriatric health with estimated prevalence increasing with advancing age [1,2]. Anemia is typically defined
using the World Health Organization (WHO) criteria of hemoglobin levels lower than 130 mg/mL for men and
120 mg/mL for women [3]. Anemia has serious consequences for some clinical and functional outcomes in the
elder population. Abnormal blood hemoglobin concentrations always relate to other diseases, such as
thalassemia, stroke and diabetes [4,5]. It has been reported that an almost 2-fold increase in the occurrence
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of Alzheimer disease among patients with anemia [6]. The impact of anemia on quality of life, functional
abilities and recovery from illness must be received much clinical attention.
The electrochemical biosensors assisted with curcumin nanoparticles and carbon dots have been developed
to determine the hemoglobin concentrations [7-9]. However the traditional biosensors are usually time
consuming, labeling required, complicated and expensive. Recent years, optical fiber sensors based on fiber Bragg
gratings (FBGs), long period gratings (LPGs) and tilted fiber gratings (TFGs) have been widely investigated for chemical and
biomedical applications [10-15]. Fiber sensors own the advantages of electromagnetic interference immunity, miniaturized size,
multiplex, label-free, and real-time detection. However, the major challenge is the lack of high sensitivity for applications with low
concentration of analyte. The sensitization techniques have been developed to improve the refractive index (RI) sensitivity by
coating the fiber device with thin-film materials, such as Langmuir-blodgett, carbon nanotube (CNT), zinc oxide (ZnO), titanium
dioxide (TiO
2
) as well as metallic coatings [16-21].
To date, 2D-nanomaterials including graphene, transition metal dichalcogenide and phosphorene have received significant
attentions due to their unique mechanical, electronic, optical and chemical properties [22-24]. Grephene is a novel carbon
nanomaterial that contains a flat monolayer of carbon atoms tightly packed into a 2D honeycomb lattice. As an oxidized derivative
of graphene, graphene oxide (GO) is strongly hydrophilic and water soluble due to the presence of oxygen-containing groups such
as hydroxyl, epoxy, carboxyl on its basal plane and sheet edges. GO exhibits favorable biocompatibility which provides the
capability to install biomolecular linkages on the surface as a biosensing platform for the detection of DNA, glucose, and protein
[25-28]. The enriched oxygen-containing functional groups on GO causes it to be served as sites for immobilization of various
biomolecules through covalent bonding. In addition, GO could adsorb biomolecules by noncovalent interactions such as hydrogen
bonding, π-π stacking, and electrostatic interaction [29]. The noncovalent interactions make GO as an ideal sensing material for
hemoglobin detection. GO/hemoglobin composite hydrogel based on electrostatic interaction was reported for enzymatic catalysis
[30]. GO-metal organic framework composites were developed for the selective isolation of hemoglobin based on π-stacking
interaction between GO and hemoglobin [31].
In this work, we report a highly sensitive biosensor based on GO-coated LPG for hemoglobin detection. In our experiment, a new
deposition method based on chemical-bonding associated with physical-adsorption has been developed. By this approach, the GO
nanosheets were deposited over the cylindrical surface of LPG. The surface morphology and the coated material were characterized
by Scanning Electron Microscope (SEM), Atomic Force Microscopy (AFM), and Raman spectroscopy. With GO deposition, the
sensitivity of LPG has been extremely enhanced both in lower and higher RI regions. In further, the GP-LPG has been implemented
as optical biosensor for the detection of human hemoglobin showing a high sensitivity.
2. Materials and methods
2.1. Materials
The aqueous dispersion of graphene oxide, sodium hydroxide (NaOH), (3-Aminopropyl)triethoxysilane (APTES), and human
hemoglobin were purchased from Sigma-Aldrich (United Kingdom). Methanol, ethanol, acetone, and deionized (DI) water were
purchased from Thermo Fisher Scientific Inc. (United Kingdom).
2.2. Principle of LPG
A long period grating is typically formed by a periodic refractive index modulation in the order of hundreds of micrometers in
fiber core. The perturbation of RI in fiber core results in light coupling from the fundamental core mode to the forward-propagating
cladding modes, yielding a series of attenuation bands in transmission spectrum with corresponding wavelength satisfying the phase
matching condition [11]:
)(
,
res
eff
icl
eff
co
nn
(1)
where
eff
co
n
and
eff
icl
n
,
are the effective refractive indices of core and ith cladding mode, Λ is the grating period. The RI sensitivity of
LPG is dependent on the phase matching condition, in which the effective refractive indices of the cladding modes depend upon the
difference between cladding refractive index (CRI) and surrounding-medium refractive index (SRI). For a conventional bare LPG,
the highest sensitivity occurs at the SRI value approaching the CRI by the higher order mode [13]. However, when the SRI exceeds
the CRI, the core mode couples with radiation modes and the phase matching condition will no longer be satisfied.
The transmission power T of the attenuation bands is given by [12,20]:
L)(sin1
2
T
(2)
where L is the length of LPG, and κ is the coupling coefficient between LP
νj
and LP
µk
mode:
2
0 0
0
),(),(),,(
4
r
kvj
rdrdrrzr
P
(3)
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where
is the FWHM of grating profile, P
0
is the power of the mode,
( , , )rz
is the permittivity variation,
( , )r
is the
transverse field of the cladding mode, and r and ϕ represent radial and angular field, respectively. The coupling coefficient is
determined by the overlap integral of core and cladding modes and by the amplitude of the periodic modulation of the mode
propagation constants.
In the case of a conventional bare LPG, the intensity T changes slowly with the increase of SRI, providing poor sensitivity as
intensity sensor [13]. In the case of a coated-LPG, due to the electric field distribution varies rapidly, the coupling strength would
change and consequently enable film coated LPG as intensity sensor with good sensitivities [16-21]. The coating layer could make
a transition from cladding guided modes to overlay guided modes as well as radiation modes.
2.3. Deposition of GO nanosheets
A 15 mm-long LPG with a period of 400μm was UV-inscribed in H
2
-loaded single mode fiber with point-by-point fabrication
technique. After the UV exposure, the grating was annealed at 85 °C for 24h to remove residual hydrogen and stabilize its optical
properties.
In this work, the deposition technique based on chemical-bonding associated with physical-adsorption was developed to deposit
GO nanosheets on cylindrical fiber device. As shown in Fig. 1, the LPG was initially cleaned with acetone to remove the residual
contaminant on fiber surface. Then the LPG was alkaline treated by immersion in 1.0 M NaOH solution for 1 h at room temperature
to enrich the number of hydroxyl group (-OH) on the surface, washed with deionized (DI) water thoroughly and dried (Fig. 1a).
For the silanization (Fig. 1b), the alkaline-treated LPG was firstly immersed in 5% APTES solution (v/v in ethanol) for 1 h at
room temperature to form Si-O-Si bonding on the surface, followed by washing with ethanol to remove unbound monomers and
baked in an oven at 70
o
C for 30 min to improve the stability of APTES monolayer.
After APTES silanization, the LPG was incubated in 1ml of 1.0 mg/mL GO aqueous solution contained in a custom-made
mini-bath, which was placed on a hot plate at 42 °C for 3 h. The epoxy group of GO reacted with amino group of APTES-silanized
fiber surface, hence GO nanosheets were gradually deposited on the fiber surface (Fig. 1c) while the aqueous solution was being
slowly evaporated by heating. Afterwards, another two cycles of physical-adsorption procedures were conducted. The GO solution
(1 ml of 1.0 mg/mL) was added into mini-bath to immerse the LPG. The aqueous solution was being slowly evaporated by heating
at 42 °C for 3 h while solid GO nanosheets were gradually and physically adsorbed onto fiber surface. When the entire LPG was
emerged out of the solution, a brownish coating was observed on the fiber surface (Fig. 1d).
2.4. Measurement system and data analysis
The interrogation system (Fig. 2) was employed for the measurement of optical properties of GO-LPG and biochemical sensing.
A broadband light source (BBS) was used to launch the light into fiber device and the transmission spectra were captured by an
optical spectrum analyzer (OSA, Agilent HP86140, Agilent Technologies Inc.). The data was analyzed using a customized program
which automatically defined the resonant wavelength and intensity by the centroid calculation method. To avoid the thermal and
bend cross-talk effects, the fiber device was mounted on a homemade straight V-groove container with two ends fixed and all
chemical experiments were performed in a fume cupboard at a controlled room temperature of 22.0 ± 0.1 °C.
3. Results and discussion
3.1. Surface morphological characterization
The surface morphology of GO-deposited fiber was characterized by optical microscope, SEM, and AFM. The initial microscope
image (Fig. 3a) shows a clear boundary between bare and GO-coated sections, demonstrating a successful deposition on fiber
surface. The surface coverage was examined by SEM with the magnification of 1000×. The detailed texture in Fig. 3b indicates that
the GO nanosheets have been deposited onto fiber to form a homogeneous layer over the entire cylindrical surface. Moreover, the
thickness of GO overlay after 3 cycles’ deposition was determined by AFM. As shown in AFM tapping mode topographic image
(Fig. 3c) and the height profile (Fig. 3d), the thickness of GO overlay is identified as around 501.8 nm.
3.2. Optical characterization of GO-LPG
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The measurements of Raman spectra and LPG transmission were carried out to verify the coating material as well as the optical
property of LPG. Both bare and coated fiber samples were measured by using Renishaw Raman Microscope 1000 (with 632.8 nm
light) with the Raman spectra depicted in Fig. 4a. In comparison with bare sample, the GO-coated fiber shows three typical
characteristic peaks (D, G and 2D) in Raman spectrum indicating the presence of GO. The D peak at 1335 cm
-1
was assigned to local
defect and disorder of GO caused by attachment of hydroxyl and epoxide groups on the carbon basal plane and edges. The G peak
at 1599 cm
-1
was due to the first order scattering of the E
2g
plane of sp
2
carbon atoms [32].
The LPG transmission spectra of the 7
th
cladding mode [14] with the central wavelength at 1591.6nm (in the air) were monitored
before and after the GO deposition. It can be seen from Fig. 4b that the GO coating induces a blue-shift of 3.8 nm in wavelength and
an increase of 12 dB in intensity of LPG attenuation band.
3.3. Enhanced sensitivity of GO coated-LPG
The GO-LPG was implemented for RI sensing of the test solutions. A series of aqueous sucrose solutions were prepared with the
measured RI values from 1.3315 to 1.4615. The GO-LPG was placed in a straight V-groove with two ends fixed and the solution
was added into V-groove by careful pipetting to cover the entire grating region. After each measurement, both grating device and
V-groove were rinsed with methanol and DI water thoroughly.
Fig. 5a plots the transmission spectra of GO-LPG with test solutions of different RIs. There are two different trends for the RI
region below and above the CRI (
1.445). For the RI lower than the CRI, the intensity of the attenuation band decreases as the RI
increases. This is consistent with the theoretical analysis in Section 2.2. Due to the coating layer, the coupling coefficient Eq.(3)
between core and cladding modes decreases as the RI increases. Once the RI is equal to the CRI, the cladding modes are no longer
confined by the cladding layer which is acting as an infinite medium and supports no discrete cladding modes, hence a broadband
radiation mode coupling occurs with no distinct attenuation bands. When the RI is higher than the CRI, the fiber does not support
any bound cladding mode and the re-appeared attenuation band corresponds to leaky mode coupling [18,19]. By increasing the RI,
the resonant intensity increases because the leaky mode is better confined by the Fresnel reflection while its wavelength is
influenced with a blue-shift.
The intensity change of the attenuation band against the RI is plotted in Fig. 5b. For the RI region below the CRI, the intensity
shows a nonlinear behavior for a gradual decrease with RI which is consistent with those LPGs coated with CNT, ZnO, TiO
2
[17-19].
The sensitivity achieves -76.5 dB/RIU and -234.2 dB/RIU for RI range of 1.33-1.38 and 1.40-1.44, respectively, exhibiting 2.5
times and 5 times higher than that of CNT-deposited LPG for the corresponding RI ranges [17]. For the RI higher than the CRI, the
resonant intensity increases dramatically against RI. The sensitivity approaches +1580.5 dB/RIU which is 7.3 times higher than that
of ZnO-coated LPG [18]. For the case of RI=1.4615, the resonant intensity reaches -28.35 dB, indicating that around 99.8 % of core
mode has been coupled into leaky mode, which is extremely high than that of metal oxide-coated LPGs [18,19]. The enhanced
sensitivity of GO-LPG may be caused by the unique features of GO material, such as extraordinary large surface-to-volume ratio,
high carrier mobility, and excellent optical properties. GO provides strong light-matter interaction between evanescent field and
external medium.
3.4. Detection of human hemoglobin
The GO-LPG was performed as a biosensor to detect human hemoglobin. A set of hemoglobin concentrations ranging from 0.0 mg/mL, 0.2
mg/mL, 0.4 mg/mL, 0.6 mg/mL, 0.8 mg/mL and 1.0 mg/mL were prepared with sucrose solution (RI=1.4610) acting as RI buffer. Fig.6a shows
the spectra of GO-LPG under different hemoglobin concentrations and Fig. 6b plots the evolution of resonant intensity. It can be seen that the
resonant intensity increases by 1.91 dB when the hemoglobin concentration changes from 0.0 mg/mL to 1.0 mg/mL. Defining the concentration
sensitivity as the change induced by 1 mg/mL hemoglobin, we have the device sensitivity of 1.9 dB/(mg/mL). If use a low-noise interrogation
system with a resolution of 0.1 dB, the GO-LPG could detect a hemoglobin concentration change as small as 0.05 mg/mL, which is far below
the hemoglobin threshold value for anemia.
The increase of resonant intensity could attribute to the local refractive index change caused by the adsorption of hemoglobin
molecules onto GO, where the measured pH values of hemoglobin concentrations are around 7.0, hence the strongest π−π
interactions lead to the most effective adsorption of proteins onto GO [30,31]. Taking into account the advantages of both enhanced
RI sensitivity and favorable biocompatibility, GO provides a significant sensing linkage between evanescent field and target
biomolecules with enhanced light-matter interaction, consequently exhibiting ultrahigh sensitivity for hemoglobin detection.
4. Conclusions
In conclusion, we have presented GO-LPG based optical biosensor for ultrasensitive human hemoglobin detection. The deposition technique
based on chemical-bonding associated with physical-adsorption has been developed to immobilize GO nanosheets on cylindrical fiber device
with the desirable thickness of 501.8 nm and good uniformity. Surface morphology of GO overlay was characterized by SEM, AFM, and
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