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Fabry-Pérot Immunosensor with UV Crosslinking Surface Immobilization
Elaine Silvana Vejar
Department of Biomedical Engineering and Biotechnology
University of Massachusetts, Lowell, Lowell, MA 01854
Abstract: This project consists of the fabrication of a Fabry-Pérot sensor which uses a
novel RNA immobilization technique onto an optical fiber tip using UV cross-linking.
Three sets of cleaved optical fiber were treated with pure RNA solution, diluted RNA
solution, and DI water, respectively. They were then put into the UV cross-linker for 2
minutes. Scanning Electron Microscopy (SEM) photos of the treated fiber end indicate
that RNA has been successfully immobilized onto the fiber surface. This may lead to an
efficient immobilization method for preparation of various optical fiber biosensors.
I. Introduction
Optical sensors have been widely used in a broad array of applications in chemical1
and biological sensing and portray a promising outcome in the medical field. Fiber optic
sensors are able to operate under high temperatures, are immune to electromagnetic
interference and display multiplexing ability2. Biosensors are composed of the following
elements; biocatalyst, transducer and output. The optimal goal of an immunosensor is to
detect, test, and transmit information about a specific biological target in a fast, accurate
and inexpensive approach.
Fabry- Pérot (FP) sensors are desirable because of their small size and high sensitivity
attributes3. However, current immobilization techniques onto optical sensors involve
cumbersome process that may increase the risk of contamination and even damage of the
biocomponent4 (RNA, DNA, antigen/antibody). The most common methods for
immunosensor analysis are enzyme-linked immunosorbent assays (ELISA). ELISA is
used to detect and amplify an antigen-antibody reaction; the amount of enzyme-linked
antigen bound to the immobilized antibody being determined by the relative
concentration of the free and conjugated antigen and quantified by the rate of enzymic
reaction5. ELISA analysis is based on chromogenic or fluorogenic substrates to produce a
signal.
In an effort to enhance optic fiber signal a study used gold-nanoparticles. The
immunosensing process takes about 9 minutes from the start of the antibody binding to
the surface regeneration process. However it is very lengthy to prepare the gold surface.
Two gold films are used and bonded to the surface by e-beam evaporation. The process
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for the first gold film takes about ten minutes and approximately two hours for the second
film6.
Fluorescent labeling methods have also commonly used by many studies to study
antigen/antibody bonding. Fluorescence preparation can also be a lengthy procedure
where a risk for human error is vulnerable and often produce large numbers of false
positives7. In this project, a simple immobilization method was demonstrated by using a
UV cross-linking technique. The immobilization preparation time can be drastically
reduced. The orientation of the bio-sample must be determined to create effective
annealing to the complementary target.
II. Principle
A FP sensor consists of two closely spaced parallel internal mirrors m1 and m2 of
single mode fiber8 separated by a length l and refractive index n. Incident light (I0) is
continuously transmitted every time it comes into contact with a mirror wall resulting in
multiple reflectivities r1 and r2 beams interfering with each other and thus creating high
resolution interferometer9. Considering only the first two reflections Ir1 and Ir2 the
highest intensity can be obtained in Eq. 1 where θ is the face difference of the
interference and Ir is the intensity if the reflected light.
The multiple internal reflections that occur in FP interferometer can be seen in
Fig. 1.
Fig. 1. Principle of Fabry-Pérot sensor
lResonant cavity
Fiber core
I0It1
Ir1
Ir2
2
II. Experimental Procedure
Sensor FabricationThe Fabry-Pérot Sensor fabrication consists of the following components: light
source, three-port optical circulator, Micron Optics SI720 optical spectrum analyzer
(OSA) and of the sensing fiber. A schematic diagram is presented in Fig.2.
Fig. 2. Fabry-Pérot sensor Schematic Diagram
Fiber PreparationSingle mode fiber is used throughout the experiments. All fibers undergo the
following procedure:
- Strip the outer jacket using a fiber-optic stripping tool
- Cleave each fiber using a high precision cleaver
- Wipe each fiber after cleaving with lint free swabs dipped in alcohol
- Perform ultrasound cleaning on bundled fiber
To test for high-quality cleaving, the OSA was attached to the three-port circulator
using a long fiber. Port 1is connected to the OSA laser, port 2 is connected to the fiber to
be cleaved, and port three is connected to the OSA laser detecting channel. Using the
OSA, the loss can be set to 0 prior to the connection of Port 2 with the fiber, then control
the loss under 0.5dB while cleaving. A small loss indicates that the fiber end face is of
good quality.
Hydrofluoric (HF) Acid Fiber EtchingThe FP sensor reflective cavities are formed in a very meticulous process of dipping
the fibers into Hydrofluoric Acid (HF) of 48% concentration for 20 to 25 minutes. Fused
Light source Circulator
Sensing fiber
Optical spectrum analyzer
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silica fibers have an approximate etch rate of 1µm per minute at room temperature, the
germanium doped core has a higher etching rate10. A schematic diagram of the fiber
etching procedure is seen in Fig. 3. Once the fibers have been removed from the HF they
are rinsed twice in de-ionized for three minutes each time.
Fig. 3. Hydrofluoric acid fiber etching setup
1 Zhu, Y. et al, “Miniature Fiber Optic Pressure Sensor for Medical Applications”, Photonics Technology Letters, IEEE., vol. 17, no. 2, pp. 447-449, Feb. 2005. 2 Fang, J. et al, “A new Processing Technique for Interferometric Fiber-Optic Sensors”, Optical Fiber Communication Conference, 1999, and the International Conference on Integrated Optics and Optical Fiber Communication. OFC/IOOC apos; 99. Technical Digest., vol. 2, pp. 223 – 225, 19993 Tseng, Y.T. et al, “ Gold-Nanoparticle Enhanced In-Situ Immunosensor Based on Fiber-Optical Fabry-Perot Interferometry”, Proceedings IEEE Conference on Nanotechnology, Japan, July 20054 Tseng Y., Wu Y., Yang C., Wang M., Tseng F., “Gold-nanoparticle Enhanced in-situ Immunosensor based on fiber-optical Fabry-Perot Interferometry”, 2005 5th IEEE Conference on Nanotechnology, v 2, 2005 5th IEEE Conference on Nanotechnology, 2005, p 117-120.5 http://www.lsbu.ac.uk/biology/enztech/immuno.html6 Tsenga, Y.T., Wu, Y.C., Yang C.S., Wan, M.C., Tseng, F.G., “Gold-nanoparticle enhanced in-situ immunosensor based on fiber-optical Fabry-Perot Interferometry”. Nanotechnology, 2005. July 2005: 845 - 848 vol.27 “Immunosensors”, http://www.cpeo.org/techtree/ttdescript/imusens.htm8 Taylor, H. “Principles and Applications of Fiber-Optic Fabry-Perot sensors”, Summaries of papers presented at the Conference on Lasers and Electro-Optics, 1998. CLEO 98. Technical Digest.9 Nave, R., “Fabry-Perot Interferometer”, http://hyperphysics.phy-astr.gsu.edu/hbase/phyopt/fabry.html#c110 Wang, X., “Label-free DNA Sequence Detection Using Oligonucleotide Functionalized Fiber Probe with a Miniature Protrusion." http://scholar.lib.vt.edu/theses/available/etd-08142006-211154/unrestricted/final.pdf
HF
DI 1Water
FUME HOODDI 2Water
4
Fiber Surface Immobilization Using a UV CrosslinkerTo immobilize the RNA onto the optical tip previously cleaved and clean fibers were
used. The benefits of using a crosslinker are various. A crosslinker has been useful for the
crosslinking of DNA or RNA onto coated glass slides, nylon, nitrocellulose or nylon-
reinforced nitrocellulose membranes. In addition, it dramatically decreases baking time to
just mere seconds11. Our goal is to prove that a crosslinker will also be useful with optic
fibers. Optic fibers prove to be an excellent candidate since it is composed of glass.
The initial step for the experiment was to denatured the dsRNA ladder by placing it in a
micro centrifuge tube and then in a water bath at a temperature of 97º C for 35 minutes.
Once out of the water bath the tube was immediately placed in an ice bucket to prevent
re-naturing of the RNA. There were three types of samples of 20 micro litters each made:
a control (just water), a diluted solution 1:10 with water, and dsRNA. In each solution 5
fibers tips were clipped and enclosed in a centrifuge tube. The tubes were incubated for
24 hours and then crosslinked twice in an Xl-1000 UV Crosslinker. The samples were
then placed in a refrigerator.
Fabry-Pérot Reflective CavitiesThe reflective cavities are made by splicing two previously HF etched fibers using a
Fujikura FSM-20CSII splicer. In Fig. 4 a schematic diagram of a FP sensor air cavity is
presented.
In Fig.5 images of etched fibers during the splicing process are presented.
11 UVP, Inc., “Uses of the UV C-100 Crosslinker in the Laboratory”, http://www.uvp.com/pdf/ab-114.pdf
Etched Fiber 2 Etched Fiber 2
FP air cavity
5
Fig. 5. Etched Fiber Splicing
IV. Results and Discussion
The digital images in Fig.6 show fibers etched in HF for 20 and 25 minutes. The air
cavity formed after 25 minutes is more defined that the etched fiber in 20 minutes. HF is
an efficient and inexpensive method for creating FP cavities since it is useful for
repeatability of similar samples. The use of HF is simple and it requires no expensive
equipment for the fiber modification.
a. 20 Minutes b. 25 Minutes c. 20 and 25 minutes
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Fig. 6. Fabry-Pérot cavities formed by HF etching. Fibers etched in HF for 20 minutes (a). Fibers etched in HF for 25 minutes (b) and same images as (a) and (b) with different resolution (c).
Scanning Electron Microscope (SEM) ImagingSEM imaging was chosen as a method to explore immobilization of the RNA
onto the optical fiber tip. The scanning electron microscope is promising because is able
to produce high-resolution images of a sample surface. SEM images have a characteristic
three-dimensional appearance and are useful for judging the surface structure of the
sample. SEM analysis requires that the fibers are very small samples and that they are
bound with a thin copper sheet to prevent scattering and distortion of the image. The
three different sets of optical-fibers were studied.
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The images obtained in the SEM provided very positive results. As expected, the
images gathered from the control fibers showed a relatively smooth surface with minor
alterations to the surface as seen in Fig. 7. When the set of fibers treated with RNA were
compared it was concluded that the RNA fibers treated in the diluted solution show some
activity on the surface as seen in Fig. 8, but not as much as the fibers treated in the
original RNA concentration solution. The fibers treated in the concentrated RNA solution
show high activity on the surface as presented if Fig. 9.
To further investigate and conclude the orientation of the immobilized RNA on
the fiber, the experimentation of immobilizing a strain of RNA with a specific size and
sequence need to be performed. The experiment will serve to confirm the hybridization of
the immobilzed RNA strain to a tagged RNA chain. A simple tagging method using
ethidium bromide may yield good results.
Fig. 7. Fibers treated in De-ionized water
Fig. 8. Fibers treated in diluted RNA solution
8
Fig. 9. Fibers treated in pure RNA solution
V. ConclusionOur experiment results show that UV cross-linking is an efficient immobilization
method for RNA onto optical fiber tip. This may be very beneficial for the fabrication of
optical fiber biosensors for RNA detection.
VI. AcknowledgementsThis project could have been possible with out the extensive collaboration of many
individuals. Dr. Ada from the Campus Materials Characterization Laboratory provided
valuable technical help with the SEM imaging. Dr. Bruce Jackson and Dr. Jamie Wilson
made available their biotechnology lab at Mass Bay Community College and also
provided valuable insight. Dr. Gu made his Bioprocessing Lab available for the etching
component of the projects and last but not least to Prof. Wang for providing guidance
throughout the whole process.
VII. References
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