Fabry-Pérot Immunosensor with UV Crosslinking Surface Immobilization

Elaine Silvana Vejar

Department of Biomedical Engineering and Biotechnology

University of Massachusetts, Lowell, Lowell, MA 01854

Elaine_Vejar@student.uml.edu

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

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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

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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

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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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