Real-time detection of DNA interactions with long-
period fiber-grating-based biosensor
Xianfeng Chen,
1,
*
Lin Zhang,
1
Kaiming Zhou,
1
Edward Davies,
1
Kate Sugden,
1
Ian Bennion,
1
Marcus Hughes,
2
and Anna Hine
2
1
School of Engineering and Applied Science, Aston University, Birmingham, B4 7ET, UK
2
School of Life and Health Sciences, Aston University, Birmingham, B4 7ET, UK
*
Corresponding author: chenx2@aston.ac.uk
Received May 17, 2007; revised July 13, 2007; accepted July 14, 2007;
posted July 24, 2007 (Doc. ID 83181); published August 20, 2007
Using an optical biosensor based on a dual-peak long-period fiber grating, we have demonstrated the detec-
tion of interactions between biomolecules in real time. Silanization of the grating surface was successfully
realized for the covalent immobilization of probe DNA, which was subsequently hybridized with the comple-
mentary target DNA sequence. It is interesting to note that the DNA biosensor was reusable after being
stripped off the hybridized target DNA from the grating surface, demonstrating a function of multiple
usability.
© 2007 Optical Society of America
OCIS codes: 060.2370, 050.2770
.
During the past decade, optical biosensors capable of
detecting biomolecular interactions have become
valuable tools for use in medical diagnosis and life
sciences as well as environmental monitoring and
other applications. The high-efficiency immobiliza-
tion technique has been developed to enable the func-
tionalization of silica glass support [1]. Several opti-
cal biosensors have been presented that use DNA
microchips, a microcavity, an optical ring resonator, a
planar waveguide, and optical fiber gratings [2–8].
However, some of these demonstrated optical biosen-
sors have limitations for real-time hybridization
studies and monitoring hybridization kinetics. Here
we implement an optical biosensor based on a dual-
peak long-period fiber grating (LPFG) for detecting
biomolecular interactions at a silica–liquid interface
with the advantages of high sensitivity, real-time
monitoring, and reusability.
To achieve high-sensitivity detection of the de-
signed biomolecular interaction, the most sensitive
LPFG structures should be used. It has been re-
ported that the coupling condition of LPFGs with
relatively short periods are close to the dispersion
turning points, resulting in conjugate dual-peak clad-
ding modes that are extremely sensitive to external
perturbations [9,10]. Several dual-peak LPFGs with
a relatively small period 共⬃160
m兲 were UV in-
scribed in H
2
-loaded SMF-28 fibers employing the
point-by-point method and a frequency-doubled Ar
laser. All the gratings were annealed at 80°C for 48 h
to stabilize their optical properties. Figure 1 shows
the spectral evolution of a 30 mm long LPFG with a
period of 161
m during the UV inscription process.
It is clear that with increasing UV exposure the con-
jugate dual peaks were increasing in strength and
moving close to each other as the coupling condition
approached the dispersion turning point.
The ability of LPFGs to couple light from the fiber
core mode to cladding modes allows optically detect-
ing the change in refractive index at the grating sur-
face. This thus provides an optical detection method
to monitor biochemical and biomolecular interac-
tions. Figure 2 shows the basic scheme of the func-
tionalization of a LPFG as a DNA-array biosensor.
Silanization is a process for modification of the glass
surface to adsorb biomolecules. The LPFG surface is
first silanized, followed by the activation of cross
linkers to facilitate the immobilization of probe DNA
and then to be used to monitor in situ the hybridiza-
tion of targeted DNA. The DNA hybridization process
modifies the refractive index of the LPFG surface,
thus resulting in its spectral shift. By demodulating
the spectral shift, the designed DNA hybridization
can be monitored with high sensitivity.
All the biochemical experiments were performed in
a fume cupboard. To minimize the bend cross sensi-
tivity, the dual-peak LPFG with a 161
m period was
placed straight in a V-groove container on a Teflon
plate, and all the chemicals and solvents were added
and withdrawn from the container by carefully pipet-
ting.
Prior to silanization, the LPFG was cleaned by im-
mersion in 5 M hydrochloric acid (HCl) for 30 min at
room temperature, followed by rinsing in deionized
Fig. 1. (Color online) Spectral evolution of a dual-peak
LPFG with a period of 161
m under increased (arrow di-
rection) UV exposure.
September 1, 2007 / Vol. 32, No. 17 / OPTICS LETTERS 2541
0146-9592/07/172541-3/$15.00 © 2007 Optical Society of America
(DI) water three times and drying in the air. Silaniza-
tion of the LPFG surface was performed by immers-
ing the cleaned grating sample in fresh 10%
3-aminopropyl-triethoxysilane (APTS) (Sigma-
Aldrich Company Ltd.) for 30 min, also at room tem-
perature.
To immobilize biomolecules covalently to the glass
surface, a chemical bond has to be formed between a
functional group of biomolecules and the amino
group of the linker [2]. As it is well known in biocon-
jugate chemistry, dimethyl suberimidate [DMS, the
molecular structure shown in Fig. 3(b)] is water
soluble and membrane permeable and is one of the
best cross-linking agents to convert the amino groups
into reactive imidoester cross-linkers [11]. The imi-
doester functional group is one of the most specific
acylating groups available for the modification of pri-
mary amines and has minimal cross reactivity to-
ward other nucleophilic groups in proteins [12]. For
activation of the fiber surface, the silanized LPFGs
were immersed in 25 mM DMS in phosphate-
buffered saline (PBS) solution for 35 min at room
temperature. The activated sensors were rinsed by
DI water three times, dried, and stored in a vacuum
desiccator for immobilization of either protein or
probe DNA.
To provide a simple method to determine whether
biomolecules are immobilized on the fiber glass sur-
face, green fluorescent protein (GFP), which is an in-
trinsically fluorescent protein that has been used ex-
tensively as a tool in biology to enable imaging [13],
was employed to detect the attachment of protein
onto the fiber surface. The DMS-activated LPFG fi-
ber, as described above, was incubated in 1 mg/ml
GFP in PBS for 16 h at room temperature. For GFP
fluorescence detection, the GFP-deposited fiber was
observed under a Zeiss Axioskop microscope with a
UV light source by using appropriate filters (Filter
Set 9, with excitation wavelength between 450 and
490 nm, and the emission filter LP515, which allows
wavelengths above 515 nm to be viewed). The image
was captured and is shown in Fig. 3(a), exhibiting
successful protein immobilization. For comparison,
an untreated fiber was also observed under the mi-
croscope; no fluorescence was observed (not shown).
The immobilization of probe DNA process was car-
ried out by incubation of the activated LPFG in 1
M
probe DNA (as shown in Table 1) in PBS for 16 h at
room temperature. The LPFG was measured at the
beginning and end of the immobilization process, and
Fig. 4(a) shows the measured spectra of the red peak
of its dual peaks (the peak at the longer wavelength
side). Note that an offset wavelength of ⬃70 nm from
the original position (Fig. 1) of the red peak was due
to the thermal annealing and the immersion in PBS
solution. By defining the resonance wavelength using
the centroid calculation method, we noticed that
there was a blueshift in wavelength of 254 pm after
16 h of deposition, indicating that the grating surface
has been modified. We also noticed that the transmis-
sion loss of this peak has increased by ⬃1.5 dB. This
could be caused by some roughness of the immobi-
lized grating surface.
Hybridization was executed with the target DNA
listed in Table 1. After cleaning with DI water, the
grating sensor was rinsed in 6 ⫻ SSPE (0.9 M NaCl,
0.06 M NaH
2
PO
4
, and 0.006 M EDTA), then im-
mersed in fresh 1
M target DNA in 6⫻ SSPE buffer
for 60 min at room temperature. The grating wave-
length was monitored in situ throughout the hybrid-
ization process, and the hybridization-induced wave-
length shifts against time are plotted in Fig. 4(b),
showing a nonlinear characteristic. We may regard
that there are two stages associated with the hybrid-
ization reaction process in 60 min. The rapid reaction
occurred in the first 3 min, showing a wavelength
shift rate of 86 pm, followed by a much slower reac-
tion process with a rate of 9 pm/min from
3 to 60 min. The overall wavelength shift induced by
the hybridization reaction in 60 min is ⬃715 pm. In
comparison with a previously reported biosensor
based on a core-etched fiber Bragg grating [8], our
dual-peak LPFG biosensor has not just achieved a
three times higher reaction rate but also maintained
the fiber robustness.
For realizing a practical optical biosensor, reusabil-
ity is an important and must-have function. To this
end, we have assessed the reusability of our LPFG
sensor. The above DNA-hybridized LPFG sensor was
washed three times in a freshly prepared stripping
buffer of 5 mM Na
2
HPO
4
and 0.1% (w/v) sodium
dodecyl sulfate (SDS) at 95° C for 30 s [2], then
Fig. 3. (Color online) (a) Fluorescence on the fiber surface
indicates (b) successful DMS activation.
Fig. 2. Basic scheme of the functionalization of LPFG fiber surface for the generation of biosensors.
2542 OPTICS LETTERS / Vol. 32, No. 17 / September 1, 2007
rinsed with DI water and dried for the rehybridiza-
tion. The grating spectra, as shown in Fig. 5(a), were
obtained before and after the stripping procedure. A
blueshift of 1257 pm was observed after the strip-
ping, indicating that the hybridized target DNA was
being released back into solution by denaturation. Af-
ter stripping, the sensor was rehybridized by immer-
sion in 2
M target DNA (a doubled concentration of
DNA was used to enable detecting a larger spectral
change) in 6⫻ SSPE buffer for 60 min at room tem-
perature. A 1165 pm wavelength increase was mea-
sured over 60 min [Fig. 5(b)], showing a wavelength
shift rate of 119 pm/min in the first 3 min followed
by 16 pm/min to 60 min. Although the enhanced sen-
sitivity showing in this reuse hybridization is only re-
sponding to the higher concentration of target DNA,
it clearly demonstrates the success of the sensor re-
usability.
In conclusion, a novel optical biosensor based on a
dual-peak LPFG has been successfully demonstrated
and used for detection of the DNA interactions in real
time with high sensitivity. Our experiment clearly
shows that the covalent linkage between the probe
DNA and silanized LPFG surface has not been af-
fected by the repeated heating and cooling stripping
procedure. The effective noncovalent probe–target
DNA bond, along with its resistance to breakdown,
makes the LPFG an ideal reusable biosensor for bio-
molecular interaction monitoring. It may be possible
to further enhance the biosensing sensitivity by em-
ploying lightly etched LPFG structures as we demon-
strated before [10]. It will also be interesting to look
at the advantage of this biosensor in further biomo-
lecular interaction, for example, in developing a
probe to discriminate between single nucleotide poly-
morphisms [14].
This work was supported by the UK Engineering
and Physical Science Research Council (EP/D500427/
1).
References
1. H. R. Luckarift, J. C. Spain, R. R. Naik, and M. O.
Stone, Nat. Biotechnol. 22, 211 (2004).
2. M. Beier and J. D. Hoheisel, Nucleic Acids Res. 27,
1970 (1999).
3. F. Vollmer, D. Braun, and A. Libchaber, Appl. Phys.
Lett. 80, 4057 (1995).
4. A. Ksendzov and Y. Lin, Opt. Lett. 30, 3344 (2005).
5. J. L. DeRisi, V. R. Iver, and P. O. Brown, Science 278,
680 (1997).
6. G. Boisde and A. Harmer, Chemical and Biochemical
Sensing with Optical Fibers and Waveguides (Artech
House, 1996).
7. M. P. DeLisa, Z. Zhang, M. Shiloach, S. Pilevar, C. C.
Davis, J. S. Sirkis, and W. E. Bentley, Anal. Chem. 72,
2895 (2000).
8. A. N. Chryssis, S. S. Saini, S. M. Lee, H. Yi, W. E.
Bentley, and M. Dagenais, IEEE J. Sel. Top. Quantum
Electron. 11, 864 (2005).
9. X. Shu, L. Zhang, and I. Bennion, J. Lightwave
Technol. 20, 255 (2002).
10. X. Chen, K. Zhou, L. Zhang, and I. Bennion, Appl. Opt.
46, 451 (2007).
11. G. T. Hermanson, Bioconjugate Techniques (Academic,
1996).
12. G. Mattson, E. Conklin, S. Desai, G. Nielander, D.
Savage, and S. Morgensen, Mol. Biol. Rep. 17, 167
(1993).
13. S. L. Rea, D. Wu, J. R. Cypser, J. W. Vaupel, and T. E.
Johnson, Nat. Genet. 37, 894 (2005).
14. M. D. Hughes, Z. R. Zhang, A. J. Sutherland, A. F.
Santos, and A. V. Hine, Nucleic Acids Res. 33, e32
(2005).
Fig. 4. (Color online) (a) LPFG’s spectra monitored at the
beginning and end of probe DNA immobilization process.
(b) Wavelength evolution of grating sensor against time
during hybridization of target DNA.
Fig. 5. (Color online) (a) Spectra of the LPFG before and
after the stripping procedure. (b) Wavelength shift against
time during the rehybridization process.
Table 1. Sequences and Modifications of the Probe and Target Oligonucleotides
Oligonucleotide 5
⬘
End Modification Sequence 3
⬘
End Modification
Probe none GCA CAG TCA GTC GCC NH
2
Target none GGC GAC TGA CTG TGC none
September 1, 2007 / Vol. 32, No. 17 / OPTICS LETTERS 2543
