sensors

Article
A Complete Optical Sensor System Based on a
POF-SPR Platform and a Thermo-Stabilized
Flow Cell for Biochemical Applications
Nunzio Cennamo 1, *,† , Francesco Chiavaioli 2,† , Cosimo Trono 2, *, Sara Tombelli 2 ,
Ambra Giannetti 2 , Francesco Baldini 2 and Luigi Zeni 1
1 Department of Industrial and Information Engineering, Second University of Naples, Via Roma 29,
Aversa 81031, Italy; luigi.zeni@unina2.it
2 Institute of Applied Physics “Nello Carrara”, CNR, Via Madonna del Piano 10, Sesto Fiorentino 50019, Italy;
f.chiavaioli@ifac.cnr.it (F.C.); s.tombelli@ifac.cnr.it (S.T.); a.giannetti@ifac.cnr.it (A.G.);
f.baldini@ifac.cnr.it (F.B.)
* Correspondences: nunzio.cennamo@unina2.it (N.C.); c.trono@ifac.cnr.it (C.T.); Tel.: +39-0815-010-367 (N.C.);
+39-0555-226-359 (C.T.)
† These authors contributed equally to this work.

Academic Editor: Alexander Star

Received: 2 December 2015; Accepted: 1 February 2016; Published: 4 February 2016

Abstract: An optical sensor platform based on surface plasmon resonance (SPR) in a plastic optical
fiber (POF) integrated into a thermo-stabilized flow cell for biochemical sensing applications is
proposed. This device has been realized and experimentally tested by using a classic receptor-analyte
assay. For this purpose, the gold surface of the POF was chemically modified through the formation
of a self-assembling monolayer. The surface robustness of the POF-SPR platform has been tested
for the first time thanks to the flow cell. The experimental results show that the proposed device
can be successfully used for label-free biochemical sensing. The final goal of this work is to achieve
a complete, small-size, simple to use and low cost optical sensor system. The whole system with
the flow cell and the optical sensor are extensively described, together with the experimental results
obtained with an immunoglobulin G (IgG)/anti-IgG assay.

Keywords: flow cell; surface plasmon resonance; plastic optical fiber; sensors system; biosensors

1. Introduction
A fast and reliable detection of analytes (such as proteins, viruses, bacteria, toxins, DNA
and chemicals in general) at very small concentration is essential in many fields, ranging from
medical diagnostics, pharmaceutical research, environmental monitoring and security to quality
control in the food industry. One of the most used approaches to detect a chemical/biochemical
interaction taking place on a functionalized surface is based on the measurement of the related
refractive index (RI) change [1]. In this context, optical sensing platforms, such as surface plasmon
resonance (SPR) [2], localized SPR (LSPR), interferometric configurations [3], resonating structures
and optical fiber gratings [4,5], represent valid and widespread options with respect to electronic and
silicon-based devices.
SPR-based biochemical sensors using optical fibers have been shown to be able to play an
important role in RI sensing, especially where fast, portable, low cost and rugged units are needed for
early detection and identification [6–11]. Among the existing device configurations, those based on fiber
tips along with the use of nanoparticles or other nanomaterials are one of the most investigated [12].
These small-scale objects allow a wide range of possibilities to modify the device in the sensitive area,

Sensors 2016, 16, 196; doi:10.3390/s16020196 www.mdpi.com/journal/sensors

Sensors 2016, 16, 196 2 of 9

enabling also unique effects such as LSPR. Another mostly-studied configuration involves the use
of fiber tapers [13]. Nowadays this technology has reached enough maturity to be a good choice for
any kind of chemical and biological SPR-based sensor. In any event, the performance of the existing
SPR-based optical fiber sensors generally depends on the adhesion of the metallic layer (commonly
gold) to the fiber surface [14]. SPR is known to be a very sensitive technique for determining RI
variations at the interface between a metallic layer and a dielectric medium (buffer solution or real
fluid containing the analyte under investigation). In practical implementations, the biological targets
are usually transported through a microfluidic system [15] by means of a buffer solution or a carrier
fluid. In general, the optical fiber employed can be either a glass optical fiber or a plastic one.
For low-cost SPR-based sensing systems, plastic optical fibers (POFs) are especially advantageous
due to their excellent flexibility, easy manipulation, great numerical aperture, large diameter, and the
fact that plastic is able to withstand smaller bend radii than glass. The same optical sensor platform
based on SPR in a POF has already been tested in different biochemical applications and in low
molecular weight substance detection [16–18], using different receptors, without the integration into a
thermo-stabilized flow cell system.
Here, the first example of the POF-SPR biosensor with a flow cell system for selective detection of
a specific biological target (analyte) is presented. In this work, IgG/anti-IgG assay was implemented
as exemplifying bioassay, with the IgG biolayer deposited on the gold surface and the biological
target, anti-IgG, transported through a new thermo-stabilized flow cell by means of a buffer fluid.
As a preliminary feasibility test, human serum with IgG spiked in was used to mimic a more real
and effective measuring environment. This complete optical sensor system can be used for the
future reduction of the device cost and dimension, with the possibility of integrating the POF-SPR
sensing platform with microfluidic and optoelectronic devices, eventually leading to a “lab-on-a-chip”
device [15,19].

2. Experimental Section

2.1. Materials
Ethanol (EtOH), 11-mercaptoundecanoic acid, bovine serum albumin (BSA) and all the reagents
for buffer preparation (phosphate-buffered saline (PBS), 40 mmol/L, pH 7.4) were purchased
from Sigma-Aldrich (Milan, Italy). Mouse IgG and goat anti-mouse-IgG were purchased from
Zymed Laboratories, Invitrogen Immunodetection (Milan, Italy). 1-Ethyl-3-[3-dimethylaminopropyl]
carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from EuroClone
(Milano, Italy). Human serum (C Reactive Protein Free Serum) was purchased from HyTest Ltd. (Turku,
Finland). Polydimethylsiloxane (PDMS, Sylgard®184 silicone elastomer kit) was purchased from Dow
Corning (Wiesbaden, Germany).

2.2. Optical Sensor Platformand Experimental Setup

A picture of the POF-SPR platform is shown in Figure 1. The plastic optical fiber has a PMMA
core of 980 µm and a fluorinated polymer cladding of 20 µm. The previously-reported experimental
results indicate that this configuration with a fiber of 1000 µm in diameter exhibits good performances
in terms of RI sensitivity and resolution but it is less performing in terms of signal to noise ratio (SNR)
with respect to the same platform exploiting smaller diameter of the fiber [20–22]. However, as stated
in our previous work [16], the choice of a multimode plastic fiber having a larger core diameter, derives
from the need to realize an easy-to-handle and low-cost device. The fabricated optical sensor platform
was realized removing the cladding of the fiber along half the circumference, spin coating on the
exposed core a buffer of Microposit S1813 photoresist (MicroChem Corp, Westborough, MA, USA)
with a thickness of about 1.5 µm achieved with a spin speed of 3000 rpm, and, finally, sputtering a
thin film (60 nm) of gold using a SCD 500 sputtering machine (Leica Microsystems, Wetzlar, Germany)
ensuring its uniformity and repeatability, as described in previous literature [20,23].
Sensors 2016, 16, 196 3 of 9

The photoresist buffer layer between the gold film and the POF core is necessary to obtain an
Sensors 2016, 16, 196 3 of 9
enhancement of the plasmonic resonance, which is due to the presence of the high refractive index
Sensors 2016, 16, 196 3 of 9
photoresist layer [23]. Actually, in the visible range of interest, the refractive index is about 1.49
The photoresist buffer layer between the gold film and the POF core is necessary to obtain an
for PMMA The(POF
enhancement core),
of
photoresist 1.41 for
the plasmonic
buffer layer fluorinated
resonance,
between which thepolymer
gold film(POF
is due to
and cladding)
thethepresence
POF core ofand
the 1.61 refractive
high
is necessaryfortothe Microposit
index
obtain an
S1813photoresist
photoresist
enhancement layerbuffer
of the layer.
[23].plasmonic
Actually, resonance,
in the visible range
which of interest,
is due the refractive
to the presence of theindex
high isrefractive
about 1.49 for
index
The
PMMA realized
photoresist (POF sensing
core),
layer [23]. region
1.41 was in about
for fluorinated
Actually, 10 mm
polymer
the visible in
range(POF length. In order
cladding)
of interest, theand to1.61
obtain
refractive a good
forindex
the robustness
Microposit
is about S1813
1.49 forof the
gold photoresist
film
PMMA it is(POF buffer
necessary
core), layer.
to sputter
1.41 the goldpolymer
for fluorinated film after (POFhaving waited
cladding) andat1.61
leastforonetheday after theS1813
Microposit spinning
The realized
photoresist
of Microposit buffer
S1813 sensing
layer. region
photoresist, which was isabout 10 mm in length. In
an electronic-grade order to
product obtain a agood
ensuring good robustness
adhesionofof the
metal film to its surface, after the complete evaporation of the solvent (propylene glycol after
the gold
The film
realizedit is necessary
sensing to
region sputter
was the
about gold
10 mm filmin after
length. having
In orderwaited
to at
obtainleast
a one
good day
robustness the
of
monomethyl
spinning
the gold of Microposit
film it is S1813tophotoresist,
necessary sputter the whichfilm
gold is an electronic-grade
after having waited product
at least ensuring
one day a good
after the
ether acetate [24]).
adhesion
spinning of the metal film
Microposit S1813to its surface, after
photoresist, whichthe is complete evaporation of
an electronic-grade the solvent
product ensuring(propylene
a good
In this paper, the robustness of the gold film has been tested by implementing a bioassay under
glycol
adhesion monomethyl
of the metal ether
filmacetate
to its [24]).
surface, after the complete evaporation of the solvent (propylene
flow conditions,
Inmonomethyl
as it willrobustness
this paper, the
be presented in the next sections. The experimental setup was arranged
of the gold film has been tested by implementing a bioassay under
glycol ether acetate [24]).
to measure
flow In the
conditions,light
this paper, spectrum
asthe
it will transmitted
be presented
robustness of thein thethrough
gold nexthas
film the POF
sections.
been and was characterized
The experimental
tested by implementing setup awas by under
a halogen
arranged
bioassay to
lampmeasure
as optical
flow the source
conditions, itand
lightasspectrum
will bebytransmitted
a spectrum
presented analyzer
inthrough
the nextthe [20,23].
POF
sections. and The
Thewas employed
characterized
experimental halogen
by was
setup lamplamp
a halogen
arranged exhibits
to
a wavelength
as opticalthe
measure emission
source and
light spectrumrange from
by atransmitted
spectrum360 nm to 1700
analyzer
through nm,
POF while
[20,23].
the The was
and thecharacterized
employeddetection
halogen range
bylamp ofexhibits
the spectrum
a halogen lamp a
wavelength
analyzer emission
(USB2000+VIS-NIR
as optical range
source and by spectrometer, from 360 nm
a spectrum analyzer to 1700
Ocean Optics, nm, while
[20,23]. Dunedin, the
The employed detection
FL, USA) range
halogen of
waslamp the spectrum
fromexhibits
about 330 a nm
analyzer
wavelength
to 1100 nm. The (USB2000+VIS-NIR
emission
spectrometer rangeisfrom spectrometer,
finally nm toOcean
360connected 1700tonm, aOptics,
whileDunedin,
computer thefor FL,
detection
the USA)
datarange was
of the
processing from about
spectrum
and analysis.
330
The SPR nm
analyzer to (USB2000+VIS-NIR
curves 1100alongnm. with
The spectrometer
the minimum is finally
spectrometer, connected
Ocean
of the SPROptics, to aDunedin,
resonance computer for the
FL,
wavelength USA) datawas
were processing
from about
displayed and online
analysis.
330 nm The
to 1100 SPR nm. curves
The along with the
spectrometer is minimum
finally of the SPR
connected to aresonance
computer wavelength
for the data were displayed
processing and
on the computer screen and saved with the help of advanced software provided by Ocean Optics.
online
analysis. onThethe SPR
computer
curvesscreen
along withand saved with theofhelp
the minimum of advanced
the SPR resonancesoftwarewavelength provided by Ocean
were displayed
The processing of the measured data was carried out by Matlab software. The SPR transmission spectra
Optics.
online on Thetheprocessing
computer screen of the and measured
saved with datathe was helpcarried out by software
of advanced Matlab software.
provided by TheOcean
SPR
were transmission
obtained by measuring
spectra were
Optics. The processing
thethetransmission
of obtained measured
spectra normalized
by measuring
data was the transmission
carried
to the spectra
out by Matlab
spectrum achieved
normalized
software. The
with
to SPR
the
air as
the surrounding
spectrum
transmission medium.
achieved
spectra with air as
were the surrounding
obtained by measuring medium. the transmission spectra normalized to the
spectrum achieved with air as the surrounding medium.

Figure 1. Picture of the POF-SPR sensing platform.

Figure 1. Picture of the POF-SPR sensing platform.
Figure 1. Picture of the POF-SPR sensing platform.
2.3. Flow Cell System
2.3. Flow Cell Cell
2.3. Flow System
System
The flow-cell is schematically depicted in Figure 2. It is composed of two parts: (1) the
The flow-cell
aluminum is schematically
bottom
The flow-cell part depicted
containing in Figure
thedepicted
is schematically housing of
in the 2. It is
sensor
Figure 2. composed
frame, of twoof
the groove
It is composed parts:
housing (1) the(1)
of the
two parts: aluminum
plastic
the
bottom partfiber
optical
aluminum containing the
and thepart
bottom holehousing
housing of
containing the
the
the sensorofframe,
thermistor.
housing the groove
the sensor housing
frame, the grooveof the plastic
housing of theoptical
plastic fiber
optical
and the holefiber and the
housing of hole housing of the thermistor.
the thermistor.

Figure 2. On the left: The flow cell aluminum bottom part with the sensor frame housing for the
plastic
Figure 2. Onoptical
Figure 2.the fiber,
Onleft:
the left: and
The The the
flow cellthermistor
flow housing.
cell aluminum
aluminum On part
bottom
bottom part the right:
with
with thetheThe PDMS
sensor
sensor top
frame
frame part, for
housing
housing withthethe
for plastic
flow-channel
plastic (9.5
optical mm
fiber, long,
and 2
the mm wide, 1
thermistor mm deep)
housing. and
On the
the holes for
right: the
The fluid
PDMS inlet
top
optical fiber, and the thermistor housing. On the right: The PDMS top part, with the flow-channel and outlet.
part, with the
flow-channel (9.5 mm long, 2 mm wide, 1 mm deep) and the holes for the fluid inlet and outlet.
(9.5 mm long, 2 mm wide, 1 mm deep) and the holes for the fluid inlet and outlet.
Sensors 2016, 16, 196 4 of 9
Sensors 2016, 16, 196 4 of 9

The
The dimensions
dimensions of of the
the bottom
bottom partpart are:
are: 30
30 mm
mmˆ  30
30 mmmm ˆ 15 mm.
 15 mm. (2) (2) The
The PDMS
PDMS top top part
part with
with the
the
flow-channel and the inlet and outlet. The dimensions of the flow channel are:
flow-channel and the inlet and outlet. The dimensions of the flow channel are: 9.5 mm  2 mm  1 mm 9.5 mm ˆ 2 mm ˆ 1 mm
and
and the
the volume
volume is is about
about 20 µL. The
20 µL. The total
total dimensions
dimensions of of the
thePDMS
PDMSpart partare:
are:2020mmmmˆ  20
20 mm
mm ˆ 10 mm.
 10 mm.
The optical fiber sensor, mounted on its plastic frame (see Figure 1), fits perfectly
optical fiber sensor, mounted on its plastic frame (see Figure 1), fits perfectly into the sensor into the sensor frame
housing obtained
frame housing into theinto
obtained aluminum
the aluminumbottombottom
part so part
that,so whenthat,the
whenPDMS the part
PDMS is placed
part ison the top,
placed on
the flow-channel fits only fits
top, the flow-channel in the
onlyarea
in where
the area the sensing
where theregion
sensingofregion
the optical
of thefiber.
optical fiber.
A picture of the experimental setup with the thermo-stabilized flow cell system is shown in
Figure 3. TheThealuminum
aluminumbottom bottompart partofofthethe flow
flow cell
cell is mounted
is mounted in thermal
in thermal contact
contact withwith a Peltier
a Peltier cell
cell
(20 (20
mmmm  ˆ2020mm)mm) andand aa thermistor
thermistoris is inserted
insertedintointo
the lateral hole (see
the lateral holeFigure
(see 2). A Thermo-
Figure 2). A
Electric-Cooling (TEC) driver
Thermo-Electric-Cooling (TEC) (ILX
driverLIGHTWAVE
(ILX LIGHTWAVE LDC-3722B,
LDC-3722B,Bozeman,Bozeman, MT, USA)
MT, USA)acquires the
acquires
temperature
the temperature fromfrom
the thermistor
the thermistor and and
this this
value is used
value as feedback
is used as feedbackto drive current
to drive to the
current to Peltier cell
the Peltier
in order to lock the temperature at 23 ˝ C (˘0.1 ˝ C). The Peltier cell, in its bottom part, is in contact
cell in order to lock the temperature at 23 °C (±0.1 °C). The Peltier cell, in its bottom part, is in
with a heat-sink
heat-sink totoincrease
increaseits itsthermal
thermalefficiency.
efficiency.The The PDMS
PDMS toptop
partpart is stuck
is stuck to bottom
to the the bottom
part part
and
and
to theto sensor
the sensor cylindrical
cylindrical plastic
plastic frame
frame by by means
means of of a PMMA
a PMMA slideand
slide andfourfourscrews.
screws.In Inthis
this way,
way,
thanks to tothe
themechanical
mechanical characteristics
characteristics of the PDMS,
of the PDMS,it is possible to completely
it is possible seal theseal
to completely flowthe
channel.
flow
The transparency
channel. of the top part
The transparency of the allows checking
top part allowsthechecking
filling of the flow fillingchannel
of theand,
floweventually,
channel and, the
formation
eventually,oftheairformation
bubbles. of air bubbles.

Figure 3.
Figure 3. Picture of the
Picture of the experimental
experimental setup.
setup.

2.4. Sensor
2.4. Sensor Functionalization
Functionalization
The functionalization
The functionalization of of the sensor gold
the sensor gold layer
layer was
was achieved
achieved by by thethe deposition
deposition of of
11-mercaptoundecanoic acid 0.5 mmol/L in a H 2O/ethanol solution (10% ethanol). The thiol solution
11-mercaptoundecanoic acid 0.5 mmol/L in a H2 O/ethanol solution (10% ethanol). The thiol solution
was left
was left in
in contact
contact with
with the
the gold
gold layer
layer for
for 1212 h.
h. Once
Once the
the sensor
sensor surface
surface waswas functionalized,
functionalized, thethe fiber
fiber
was placed inside the thermo-stabilized microfluidic system. All the steps for the
was placed inside the thermo-stabilized microfluidic system. All the steps for the implementation of implementation of
the immunoassay were performed using the flow cell connected to a peristaltic
the immunoassay were performed using the flow cell connected to a peristaltic pump and keeping the pump and keeping
the temperature
temperature offlow
of the the flow
cell atcell
(23at˘(23
0.1)± ˝0.1) °C. preparation
C. The The preparation
of theofbiolayer
the biolayer (receptor)
(receptor) consisted
consisted of the
of the following steps: activation of –COOH (carboxylic) groups by
following steps: activation of –COOH (carboxylic) groups by cross-linking chemistry (200 mmol/L cross-linking chemistry
(200 mmol/L
EDC EDC andNHS),
and 50 mmol/L 50 mmol/L NHS),
covalent covalent immobilization
immobilization of mouse IgG of mouse IgG (1000
(1000 mg/L mg/Lwashing
in PBS), in PBS),
washing with PBS for removing the un-reacted antibodies, and surface passivation
with PBS for removing the un-reacted antibodies, and surface passivation with BSA (1% in PBS) in
with BSA (1% in
PBS) in order to block the remaining activated carboxylic groups and to prevent non-specific
order to block the remaining activated carboxylic groups and to prevent non-specific adsorption onto
adsorption onto the surface.
the surface.

2.5. Protocol
2.5. of the
Protocol of the Measurements
Measurements in
in Buffer
Buffer and
and Human
Human Serum
Serum
In this
In this work,
work, wewe have
have used
used aa standard
standard protocol
protocol to
to always
always measure
measure the the changes
changes inin refractive
refractive
index due to the bio-interaction under the same conditions. In particular, a washing
index due to the bio-interaction under the same conditions. In particular, a washing step with step with PBS
PBS
(buffer) after
(buffer) after the
the bio-interaction
bio-interaction was
was carried
carried out
out and
and the
the spectrum
spectrum was
was recorded
recorded after
after stopping
stopping thethe
flow thus with stable thermal and hydrodynamic conditions. Although the measurement
flow thus with stable thermal and hydrodynamic conditions. Although the measurement under under flow
conditions
flow couldcould
conditions be used to exploit
be used thethe
to exploit biosensor interactions,
biosensor it itshould
interactions, shouldbebepointed
pointedoutout that the
that the
important measurand
important measurandisisconstituted
constitutedbybythe
theRIRIchanges
changesinduced
inducedalong
alongthe the functionalized
functionalized surface
surface duedue
to
to the specific interaction between the immobilized antibody and the antigen, and not
the specific interaction between the immobilized antibody and the antigen, and not by bulk RI changes, by bulk RI
changes, simply related to the refractive index of the solutions which are pumped inside the flow
cell. This means that a washing step with PBS is necessary after each addition of the sample in order
Sensors 2016, 16, 196 5 of 9

simply related to the refractive index of the solutions which are pumped inside the flow cell. This
means that a washing step with PBS is necessary after each addition of the sample in order to measure
the real shift of the resonance determined by the antibody/antigen interaction on the fiber surface.
The antigen binding steps, with increasing concentrations of goat anti-mouse IgG ranging from
10 µg/L up to 500 mg/L in PBS, were performed by flowing the antigen solution onto the modified
Sensors 2016, 16, 196 5 of 9
sensor for 15 min followed by a washing step with PBS (buffer) of 5 min. The same assay was also
performed by spiking
to measure the realhuman
shift ofserum (diluteddetermined
the resonance 1:10 (V/V)byinthe
PBS) with goat anti-mouse
antibody/antigen interactionIgG
on at
theincreasing
fiber surface.
concentrations (from 10 µg/L up to 10 mg/L). Also in this case, the measurement of the wavelength
The antigen
shift has been bindingafter
performed steps,the
with increasing
washing ofconcentrations
the sensor by of means
goat anti-mouse IgG ranging
of PBS buffer, from to detect
in order
10 μg/L up to 500 mg/L in PBS, were performed by flowing the antigen solution onto the modified
the RI change induced only by the bound analyte, without any possible perturbation induced by bulk
sensor for 15 min followed by a washing step with PBS (buffer) of 5 min. The same assay was also
refractive indexby
performed differences.
spiking human serum (diluted 1:10 (V/V) in PBS) with goat anti-mouse IgG at
increasing concentrations (from 10 μg/L up to 10 mg/L). Also in this case, the measurement of the
3. Results and Discussion
wavelength shift has been performed after the washing of the sensor by means of PBS buffer, in
order to detect the RI change induced only by the bound analyte, without any possible perturbation
The feasibility and integration of the POF-SPR sensor platform with the ad hoc developed
induced by bulk refractive index differences.
microfluidic system were evaluated performing the model IgG/anti-IgG assay both in buffer solution
(PBS) 3.and under
Results andmore realistic and effective conditions, i.e., in human serum. Two different plasmonic
Discussion
resonances Thewere observed
feasibility in the considered
and integration wavelength
of the POF-SPR sensor range
platform (300–1100
with the adnm)hocofdeveloped
the transmission
spectra [25], whensystem
microfluidic the binding interaction
were evaluated occurs between
performing the model bio-receptor
IgG/anti-IgG andassay
analyte
bothunder investigation.
in buffer
In particular, the resonance
solution (PBS) and underdisplaying a and
more realistic blue-shift was
effective at shorter
conditions, i.e., wavelengths
in human serum. with
Tworespect to the other
different
plasmonic resonances were observed in the considered wavelength range (300–1100
one that displayed a red-shift of the SPR resonance [25]. Since the minimum at shorter wavelengths is nm) of the
muchtransmission
better defined spectra
than[25], when and
the other the binding interactionresonance
the blue-shifted occurs between bio-receptor by
is characterized and analyte response
a greater
under investigation. In particular, the resonance displaying a blue-shift was at shorter wavelengths
to the analyte concentrations, only this resonance will be taken into account hereafter. Two POF-SPR
with respect to the other one that displayed a red-shift of the SPR resonance [25]. Since the minimum
sensors
at were
shorterused to carry is
wavelengths out the two
much betterexperiments
defined thaninthe buffer
otherand
and in theserum. The two
blue-shifted sensors
resonance is exhibited
the main
characterized by a greater response to the analyte concentrations, only this resonance will be taken response
SPR resonance placed at two different starting wavelengths (even if the device
showedintothe same
account trend):Two
hereafter. 576POF-SPR
nm for sensors
the devicewere used
used toincarry
buffer, whereas
out the 626 nm for
two experiments that one used
in buffer
and
in serum. in serum. The two sensors exhibited the main SPR resonance placed at two different starting
wavelengths (even if the device response showed the same trend): 576 nm for the device used in
buffer,
3.1. Assay inwhereas
Buffer 626 nm for that one used in serum.
Figure 4 inshows
3.1. Assay Buffer the shift of the SPR resonance at around 576 nm, obtained for different
concentrations
Figure of the analyte
4 shows (from
the shift 0 toSPR
of the 500resonance
mg/L) inatbuffer
aroundsolution: the resonance
576 nm, obtained wavelength is
for different
shifted to lower values
concentrations of thewhen the(from
analyte analyte
0 to concentration increases,
500 mg/L) in buffer indicating
solution: that wavelength
the resonance the analyteiseffectively
shifted
interacts withtothe
lower values when
immobilized the analyte
receptors fromconcentration increases,
the buffer matrix hereindicating
considered.thatThe
the increase
analyte in depth
effectively interacts with the immobilized receptors from the buffer matrix here considered.
of the resonance peak exhibiting the blue-shift is a direct consequence of the stronger excitation The of the
increase in depth of the resonance peak exhibiting the blue-shift is a direct consequence of the
surface plasmon, which depends on the effective RI of the layer.
stronger excitation of the surface plasmon, which depends on the effective RI of the layer.

Figure 4. Normalized SPR spectra acquired by the POF-SPR biosensor for different analyte
Figure 4. Normalized SPR spectra acquired by the POF-SPR biosensor for different analyte
concentrations in buffer.
concentrations in buffer.
Sensors 2016, 16, 196 6 of 9
Sensors 2016, 16, 196 6 of 9

Since the parameter of interest is the measurement of the resonance wavelength shift, any
changes Since
in thetheresonance
parameter depth doisnot
of interest affect the results,
the measurement as far aswavelength
of the resonance the peak shift,
remains clearly
any changes
distinguishable.
in the resonanceBy extracting
depth do the
notminimum of the SPR
affect the results, resonance
as far for each
as the peak remainsanalyte concentration,
clearly it is
distinguishable.
possible to draw the
By extracting the calibration
minimum ofcurve of the
the SPR sensor.for
resonance Theeach
absolute
analytevalue of the wavelength
concentration, shift
it is possible toof the
draw
the calibration
transmission minimumcurve(Δλ)
of the sensor.
versus theThe absolute value
concentration of the
of the wavelength
analyte is shownshift
inof the transmission
Figure 5, together
withminimum (∆λ) versus
the Hill fitting the concentration
of experimental of the analyte
values [18,25]. is shown
The results in Figure
depicted 5, together
in Figure with the that
5, demonstrate Hill
fitting ofisexperimental
the system able to detectvalues [18,25]. The results
concentrations of thedepicted
analyteinunder
Figureinvestigation
5, demonstrate(IgG)that the system
lower than is
able
0.01 mg/L. to detect concentrations of the analyte under investigation (IgG) lower than 0.01 mg/L.

Figure 5. Absolute values of resonance wavelength shift versus the concentration of analyte and the
Figure 5. Absolute values of resonance wavelength shift versus the concentration of analyte and the
fitting of the data by Hill equation. Inset: Hill parameters.
fitting of the data by Hill equation. Inset: Hill parameters.

3.2. Assay in Human Serum

3.2. Assay in Human Serum
In order
In order to toprove
prove that thenew
that the new complete
complete optical
optical sensorsensor
systemsystem can be in
can be exploited exploited in real
real biochemical
biochemical
applications,applications,
which require which require a thermo-stabilized
a thermo-stabilized microfluidic system, microfluidic system, the
the same IgG/anti-IgG assaysame
was
IgG/anti-IgG assay was performed under more realistic
performed under more realistic and effective conditions, i.e., in serum. and effective conditions, i.e., in serum.
The Thefirst first
SPRSPR spectra were
spectra wererecorded
recorded in in
order
order totoexamine
examinethe theinfluence
influenceof of the
the sensor
sensor surface
surface
modification
modification on the resonances. Figure 6 shows the SPR resonances obtained after each step
on the resonances. Figure 6 shows the SPR resonances obtained after each step of the
of the
functionalization
functionalization process, as described
process, as described in Section
in Section 2.4. The
2.4. Thewavelength
wavelengthofofthe theSPR
SPRresonance
resonance is is shifted
shifted
to lower values
to lower (blue-shift)
values afterafter
(blue-shift) the receptor (IgG)
the receptor is immobilized
(IgG) is immobilized on the surface.
on the TheThe
surface. blocking
blockingof the
of
surface
the with
surfaceBSA didBSA
with not lead to alead
did not change
to ain resonance
change wavelength,
in resonance demonstrating
wavelength, the goodthe
demonstrating surface
good
surface
coverage. coverage.the
Moreover, Moreover,
absencethe of absence of a detectable
a detectable wavelength wavelength
shift aftershift
the after the first injection
first injection of human of
human
serum, which serum, whichother
contains contains other
kinds ofkinds
IgG of IgGnot
(but (butthe
not specific
the specific one),
one), demonstratedthe
demonstrated the good
specificity of the sensor that should result in a signal only in
specificity of the sensor that should result in a signal only in presence of anti-mouse presence of anti-mouse IgG (specific
IgG
analyte).
(specific The preliminary
analyte). The preliminaryresultsresults
on the on
assay
theinassay
human in serum
humanare shown
serum in shown
are Figure 7inthat details
Figure the
7 that
shift of the SPR resonance at around 626 nm, obtained for the different concentrations
details the shift of the SPR resonance at around 626 nm, obtained for the different concentrations of of analyte (from
0 to(from
analyte 10 mg/L) 0 to spiked
10 mg/L)inhuman
spikedserum.
inhumanEvenserum.
in this case,
Eventhein resonance
this case, wavelength
the resonance is shifted to lower
wavelength is
values when the analyte concentration increases.
shifted to lower values when the analyte concentration increases.
Sensors 2016,16,
Sensors2016, 16,196
196 77 of
of 99
Sensors 2016, 16, 196 7 of 9

0.9545
0.9545
Normalized Transmitted Light Intensity
Normalized Transmitted Light Intensity

0.954
0.954
Buffer
Buffer
Buffer post IgG
Buffer
Bufferpost IgG
post BSA
0.9535 Buffer
Bufferpost BSA
post serum
0.9535 Buffer post serum

0.953
0.953

0.9525
0.9525
610
610 615
615 620
620 625
625 630
630 635
635 640
640
Wavelength [nm]
Wavelength [nm]

Figure
Figure 6.6.6.
Figure Normalized
Normalized SPR
NormalizedSPR spectra
SPRspectra acquired
spectraacquired
acquired by
by the POF-SPRsensor
the POF-SPR
POF-SPR sensor
sensorinin buffer
inbuffer for
bufferfor the
forthe different
different
the steps
steps
different ofof
steps of
the
thesurface
surface functionalization
functionalization process
process and for the first
first injection
injection of
of serum
serum (not
(not spiked).
spiked).
the surface functionalization process and for the first injection of serum (not spiked).

The
Thetotal
totalshift
shift of
of the
the SPR
SPR resonance
resonance detailed in
in Figure
Figure 77 isis roughly
roughly2.22.2nm,
nm,which
whichisisslightly
slightly
The total shift of the SPR resonance detailed in Figure 7 is roughly 2.2 nm, which is slightly lower
lower
lowerthan
thanthat
thatobtained
obtainedwhenwhen thethe assay
assay was performed
performed ininbuffer
bufferfor forthe
thesame
sameanalyte
analyteconcentration
concentration
than that obtained when the assay was performed in buffer for the same analyte concentration of
ofof1010mg/L
mg/L(2.7
(2.7nm
nm from
from Figure
Figure 5).
5). This could be ascribed
ascribed toto the
the higher
highercomplexity
complexityofofthe
theserum
serum
10 matrix
mg/L (2.7 nm fromtoFigure
withrespect
respect simple5).
PBS This could be ascribed
buffer, to the higher complexity of the serum matrix
matrix with to simple PBS buffer, which implies
implies aa decrease
decreaseof ofthe
theantigen
antigenmobility
mobility[26].
[26].
with respect to simple PBS buffer, which implies a decrease of the antigen mobility [26].

Figure 7. Normalized SPR spectra acquired by the POF-SPR biosensor for different analyte
Figure
Figure 7.7. Normalized
Normalized SPR spectra acquired by the POF-SPR biosensor for different analyte
concentrations in serum.SPR spectra acquired by the POF-SPR biosensor for different analyte
concentrations
concentrationsininserum.
serum.
These preliminary results represent a new paradigm for biochemical sensing applications. In
These
These
fact, preliminary
the preliminary
new resultsrepresent
results
sensor system represent
simplifies a new
athe
new paradigm
paradigm
use of forfor
the POF-SPR biochemical
biochemical sensing
sensing
sensor platform andapplications.
applications.
increasesInitsIn
fact,
fact,
the new thesensor
new
reliability sensor
for system system
simplifies
biochemical simplifies
sensing use ofthe
theuse
theapplications. of use
POF-SPR
The the sensor
POF-SPR
of sensorand
platform
the integrated platform
increases
system could andits increases
also allow theits
reliability for
reliability
biochemical for
implementation biochemical
sensing an assaysensing
ofapplications. The
protocolapplications.
use The use ofsystem
of the integrated
including a regenerationthe integrated
could
step system
to also allow
obtain thecould
the also biolayer
allow the
implementation
specific
ofimplementation
available for a of
an assay protocol an
new assay protocol
interaction
including including
in consecutive
a regeneration stepa assay
regeneration
to cycles.
obtain step to biolayer
Several
the specific obtain
examples the specific
of biolayer
regeneration
available for a new
available
procedures
interaction for a new interaction
can be foundassay
in consecutive in
in thecycles. consecutive
literature [27] and
Several assay
represent
examples cycles. Several examples
a future issue,procedures
of regeneration which willcan of regeneration
be taken into in
be found
procedures
theaccount
literature can
with[27] be
the found
and in the
exploitation
represent ofaliterature
the [27] POF-SPR
proposed
future issue, and represent
which system
will a future
to a into
be taken issue,
real which
application.
account with willthebeexploitation
taken into
account with the exploitation of the proposed
of the proposed POF-SPR system to a real application. POF-SPR system to a real application.
Sensors 2016, 16, 196 8 of 9

The other kind of regeneration which could be considered in this type of biosensors, is the
complete restoration of the gold sensor surface by removing not only the bound analyte but also the
immobilized bioreceptor and the functional layer (thiols in most cases when using gold). This step
is rarely performed in SPR biosensors because of the difficulty in removing the thiols from the
gold surface [28]. This last kind of regeneration requires strong treatments, such as electrochemical
cleaning [29], ultraviolet/ozone exposure [30], and ammonia-hydrogen peroxide mixture (APM)
solution stripping [31], which are often performed out of the integrated fluidic system. Being the
aim of this work the demonstration of the feasibility of the integration of a POF-SPR sensor into a
thermo-stabilized flow cell, this kind of regeneration was beyond the scope of the work.

4. Conclusions
In this work, the first experimental measurements obtained with a POF-SPR sensor integrated with
a thermo-stabilized flow cell have been presented. In particular, the effectiveness of the new complete
optical sensing platform has been demonstrated thanks to the development of the microfluidic system,
which has been used for both the functionalization of the sensor surface and the antibody-antigen
binding experiments obtained in buffer solution (PBS) and in human serum. These experimental
results show that the POF-SPR sensor platform is appropriate for biochemical applications based on
fluidics, even in a real and complex environment such as serum.

Acknowledgments: Francesco Chiavaioli wishes to thank the Italian Minister of University and Research
(MIUR) under the grant N. RBFR122KL1. S. Tombelli wishes to thank the European Community for the project
Hemospec (FP7-611682).
Author Contributions: Francesco Baldini, Francesco Chiavaioli and Cosimo Trono of the Institute of Applied
Physics “Nello Carrara” (CNR) realized the thermo-stabilized flow cell for biochemical sensing applications.
Luigi Zeni and Nunzio Cennamo of the Department of Industrial and Information Engineering (Second University
of Naples) realized the optical sensor platform. Sara Tombelli and Ambra Giannetti of the Institute of Applied
Physics “Nello Carrara” (CNR) realized the functionalization process on gold film. All authors realized the
experimental measurements.
Conflicts of Interest: The authors declare no conflict of interest.

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