Sensors and Materials, Vol. 14, No. 6 (2002) 339-345
MYUTokyo

S &M 0493

Fiber Optic Couplers for Protein Detection
Michael Henning
Veridian Corporation, 1115 5th St., SW; Charlottesville, VA 22902, U.S.A.
(Received March 26, 2002; accepted April 10, 2002)

Key words:

fiber optic, coupler, biosensor, assay

Numerous applications in areas such as medical diagnostics, environmental monitor­
ing, and food safety require selective protein detection. A variety of optical detection
methods have been developed, but many of them require the use of a fluorescent labelingdye. Label-free optical techniques such as surface plasmon resonance (SPR), use ofresonant mirrors, and interferometry have also been developed. Here we report the use ofa fiber optic coupler as a platform for bioassays/biosensing. The coupler fabrication anddesign elements crucial to the sensor performance are described, and protein detection isdemonstrated.

1.

Introduction

I. I Refractive index-based detection

The response of many biosensors and assays currently in use is based on absorbance or
fluorescence of the analyte molecule or of the dye used as a tag. Fluorescence-based
techniques are much more sensitive than absorbance-based ones and, therefore, provide
lower limits of detection. Unfortunately, most common analyte molecules are not naturally
fluorescent. Therefore, a fluorescent dye molecule or a tag, such as a quantum dot, must be
incorporated in the analysis. Methodologies include linking the dye/tag to the analytemolecule or the receptor, performing a "sandwich" assay with a secondary-labeled mo­
lecu1ar recognition element, or displacing fluorescently labeled molecules with the unlabelledanalyte molecules in a competitive format. Such techniques complicate detection byadding steps and reagents. In addition, the attachment of fluorescent tags can interfere withthe molecular binding event. The typical protein limit of detection for fluorescence-based
techniques is 1 ng/mL (pM for 150 KDa protein).0-3l
Label-free detection methods simplify detection by reducing sample preparation andeliminating reagents. Many state-of-the-art, label-free optical detection methods measure

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Sensors and Materials, Vol. 14, No. 6 (2002)

refractive index changes induced by analyte binding. Such techniques include surface
plasmon resonance (SPR), use of resonant mirrors, and interferometry. Table 1 provides
a list of many state-of-the-art bio-detection systems, the corresponding limits of detection,and select representative publications.

l .2 Fiber optic couplers

A variety of biosensors that utilize optical fibers have been developed_(l4,i6-l9l Here we
describe the use of fiber optic couplers as a bioassay/biosensor platform. These couplers
are fabricated by simultaneously heating and pulling two single-mode fibers to create a"waist" region where the two fibers are thermally fused and the fiber diameter is reduced
from an initial value of 125 µm per fiber to 5-15 µm per fiber. After this fusion, lightpropagating in a single input fiber is split at the "waist" region and then propagates down
the two fibers. In this "waist" region, the fiber cores become too small to contain the
propagating mode, and therefore light propagates through the surrounding cladding. Thus,in the coupled region, the light is guided through a "core" composed primarily of theoriginal fiber cladding material, and whatever surrounds the fused region, such as the
sample solution, becomes the new cladding. As a result, the division of power between thetwo fibers becomes a strong function of the index of refraction in the small volume
surrounding the fiber surfaces in the coupled or "waist" region. Theoretical approxima­
tions of the response of a simplified version of such a coupler have been described
previously.<20,21 >The coupler can be used as a biosensor if receptor molecules are attached to the couplersurface. Binding of the analyte molecule to the receptor molecule changes the refractive
index and hence the critical angle at the coupler surface, thereby inducing a shift in the
coupling ratio. Therefore, the change in the ratio of the output couplers can then be used todetect a binding event. A simplified diagram depicting the coupler response is shown in
Fig. 1. The application of fiber optic couplers as an immunoassay detection platform has
been suggested previously,CZ2l and here we report experimental results and design modifi­
cations made to improve the reproducibility and sensitivity of the coupler response to
analyte binding.

Table 1A lisr of many state-of-the-art bio-detection systems, the corresponding limits of detection, and selectrepresentative publications.
Limit of Detection*(analyte = protein)
Sensor Type
Fluorescence
ng/mL
SPR
ng/mL
ng/mL
Resonance mirrorµglmL
Grating couplers
Bidiffractive grating couplers/
ng/mL
Multilayer gratings
ng/mL
Hartman interferometerng/mL
Mach-Zehnder interferometer
Optical difference interferometerµg/mL* For a 150 KDa protein, such as IgG, ng/mL = pM = ppb.

Select References(1-3)(4, 5)(5, 6)(7)(8, 9)(10-12)(13, 14)
(15)

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Sensors and Materials, Vol. 14, No. 6(2002)

Receptors on Coupler Surface
tput 1 = 50%

l"P"la1)

Analyte Binds to Receptor

9utput 1 = 20%

�tput 2 = 80%

Fig. 1. Schematic depicting the light output change induced in a fiber optic coupler due to analytebinding to a receptor immobilized on the coupler surface. Not to scale.

2.

Materials and Methods

Couplers were either purchased (Empirical Technologies Corporation, ETC) or fabri­cated in-house. The Veridian fabrication involves an automated coupler pulling station_C23>
Two single-mode fibers (Corning SMF-28) are held at a fixed position at one end, and the
opposite ends are run to a moveable stage through a tensometer. The pull length ismeasured with encoder motors and the entire pulling process is monitored by computer
software. The central region of the fibers is positioned in a 23OO °C furnace (ETC) that
automatically moves during the drawing process in order to keep the furnace positioned in
the critical "waist" region of the fibers. If the furnace is not moved during the pulling
process, the couplers produced are skewed in the taper because heat was not being appliedconsistently to the coupled region. The ETC couplers used in this study were produced
using such a system and, thus, had a skewed coupling region.
During the Veridian pulling process, light is launched into one fiber input and theoutput intensity of the two fibers is monitored. Collecting this data in real time and using
the software-controlled pulling station allow a coupler to be drawn to a specific coupling
ratio, or length, which can be monitored with the pulling software. The station C23> wasdesigned to pull couplers with only small variations coupler to coupler in order to reduce
the number of variables affecting the coupling ratio.c20-2n
A crossover occurs as a coupler is pulled through the 50:50 split in the light output.
With the current automated station, couplers have been pulled up to 7 crossovers. Model­
ing of the single-mode-fiber coupler showed that the sensitivity of the coupler was
substantially affected by the number of crossovers during the pulling process.< 24J Experi­mentsC25l performed using a series of couplers pulled to the first through the fifth crossover342

Sensors and Materials, Vol. 14, No. 6 (2002)

also indicated that properties such as sensitivity and signal-to-noise ratio were affected by
the pull length. Couplers pulled to the third crossover provided optimal performance;
therefore Veridian couplers pulled to the third crossover (Fig. 2) were chosen for this study,
and were compared to ETC couplers which were pulled to the first crossover. The Verdian
couplers tested have a "waist" diameter of approximately 18±1 µm, and the ETC couplers
had a waist diameter of approximately 30±2 µm. The waist sizes of both ETC and Veridian
couplers were measured using an optical microscope.
The Veridian couplers and the purchased couplers tested were fabricated using a single­
mode fiber, in which theoretically there is only a single core mode propagating in the fiber.
However, even in a single-mode fiber there can be multiple core modes and, additionally,
as light is launched into a fiber through a connector, higher order and cladding modes areoften generated that can interfere with the dominant mode.C26l These modes are introduced
at the connection from the light source to the coupler and are generated as light is coupledback into the output fiber cores from the waist region. Due to the short length of the fiber
optic coupler, these modes will not be attenuated simply by the fiber length, as is common
in communications applications. Therefore, standard cladding/higher order mode reduc­tion techniques were used to eliminate or significantly reduce these modes on both the
input and output ends of the couplers. The response of the modified versions of both the
Veridian couplers and the ETC couplers was compared with that of unmodified couplers.
Both the Veridian couplers and the ETC couplers were mounted in a quartz half shellmeasuring 3.5 inches in length and 0.25 inches in diameter. In order to prepare the coupler
for use as a biological sensor, receptor molecules were immobilized on the coupler surface,

Coupler 3 Pulled to Third Crossover
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Fig. 2. Real-time monitoring of coupler fabrication. The two coupler output powers (arbitrary
units), Pl and P2, are plotted as functions of the coupler pull length (mm). The intersections of thetwo lines indicate crossovers where the power distribution between the two fibers is 50:50.

Sensors and Materials, Vol. 14, No. 6(2002)343

as described below. Because both the coupler holder and the coupler are made of quartz,
the receptor molecules will be immobilized on both surfaces and, therefore, analytebinding will also occur on both the coupler and the holder. Loss of analyte to the holder
surface may reduce the sensitivity of the system because the area of the quartz half is manyorders of magnitude larger than the area of the fiber optic sensing region. Therefore, theVeridian coupler holder was passivated to minimize the adsorbance of biological mol­ecules to the coupler frame.
The couplers were treated to immobilize receptor molecules on the fiber surface. The
cells were prepared in sets of five couplers that were mounted in a holder and treatedsimultaneously. The receptor molecule was an antibody, rabbit anti-goat immunoglobulin
G (IgG), and the analyte molecule was goat IgG. All couplers sets (Veridian andpurchased) were prepared using the following procedure: 1) 30-min soaking in a 1:1
solution of methanol and hydrochloric acid, 2) rinsing in water, 3) 30-min soaking in 50%
H2SO4 solution, 4) water rinse, 5) rinsing in phosphate buffer saline (PBS), 6) incubation
with rabbit anti-goat IgG solution, 7) incubation in a non-protein blocker. Colorimetricand fluorescence techniques were used to verify the immobilization of the rabbit anti-goat
IgG on the coupler surface.c27J

3.

Results and Discussion

Experiments were run to show the capability of fiber optic couplers to function as
bioassays/biosensors and the efficacy of the various design enhancements describedabove. The receptor-modified couplers were interfaced with an input light source and theoutput light from each fiber was monitored and collected a rate of 5 points/s. The couplers
were tested in a continuous fluid flow system. To measure the response to the analyte, thefollowing solutions were introduced into the continuous flow system: 1) buffer containing
the non-protein blocker, 2) 27 µglmL whole-molecule goat IgG solution, and 3) buffer
containing the non-protein blocker. Due to the high binding constant of antibody-antigen
interactions, a final buffer rinse (step 3) should not cause a significant change in the coupler
output if the analyte (goat IgG) binds to the receptor (rabbit anti-goat IgG).
The results achieved with the Veridian and the ETC couplers that were not modified toreduce higher order/cladding modes were inconsistent (data not shown). Therefore, themode reduction modification appears crucial to the coupler operation as a biosensor, andboth Veridian and the ETC couplers were modified for further testing. Prior to mode
reduction modification, the average percent light ratio for the two output fibers was
between 50:50 and 25:75 for both the Veridian and the ETC couplers. When the cladding/higher order modes were suppressed in the Veridian couplers, the output light ratios
remained similar. However, when these modes were suppressed in the ETC couplers, thelight output from one lead was almost completely eliminated and the average percent lightratio for the two output fibers became 95:5 to 99: 1, indicating that the light in one output
was primarily made up of cladding and higher order modes.
Figure 3 shows typical results achieved using the mode-suppressed ETC couplers andVeridian couplers. All couplers were tested in sets of five, but the response of only one
coupler from each set is shown to avoid redundancy. As described above, the couplers

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Sensors and Materials, Vol. 14, No. 6 (2002)

Modified Purchased Coupler

Veridian Coupler

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Fig. 3. Typical coupler response to analyte binding. Left - Purchased coupler (ETC) modified toreduce higher order/cladding modes. Right - Veridian coupler pulled to the third crossover andmodified to reduce higher order/cladding modes. Fiber output 1 (black) - left axis. Fiber output 2(grey) - right axis. Data shown are responses to the following solutions: 1) buffer, 2) 27 µg/mL goat
IgG, 3) buffer, 4) 227 µg/mL rabbit anti-goat IgG.

were exposed to a buffer, then to a solution of goat IgG and then, again, to a buffer. Bothcoupler types responded irreversibly to the goat IgG. In order to verify that the goat IgG
was bound, a sandwich assay was performed by introducing rabbit anti-goat antibody into
the flow system. If the goat IgG was bound to the couplers, it would bind its conjugate, therabbit anti-goat IgG, and there would be an additional change in the index of refraction
surrounding the sensing region of the coupler. As shown in Fig. 3, both coupler types also
responded irreversibly to the rabbit anti-goat IgG, as expected. The signal-to-noise ratio
achieved is much higher for the Veridian couplers, indicating that the Veridian designmodifications used may produce a more sensitive biosensor/bioassay platform.
Experimentation has shown that the fiber optic coupler does provide a viable platform
as a biological/chemical sensor. However, the original coupler designc22l must be modified
to enhance the signal output, increase the signal-to-noise ratio, and isolate the detection
area on the coupler. Veridian Corporation accomplished these goals and showed signifi­
cant improvement in signal output, and reproducibility by suppressing higher order modes,
pulling the couplers beyond the first crossover, and passivating the quartz half.

Acknowledgements
Partial funding for this research was provided by the United States Marine Corps. The
author gratefully acknowledges the hours spent in basic research by the entire MOBAD
team and particularly Susan Barker who graciously and with expert knowledge made thispaper readable. David Angeley also provided the core optics knowledge that led to the
reduction of the higher order and cladding modes which provided the breakthroughtechniques to produce a working single-mode fiber optic bioassay device.

Sensors and Materials, Vol. 14, No. 6(2002)

345

References

2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27

C. A. Rowe, L. M. Tender, M. J. Feldstein, J.P. Golden, S. B. Scruggs, B. D. MacCraith, J. J.
Cras and F. S. Ligler: Analytical Chemistry 71 (1999) 3846.
C. A. Rowe, S. B. Scruggs, M. J. Feldstein, J.P. Golden and F. S. Ligler: Analytical Chemistry71 (1999) 433.
K. Schult, A. Katerkamp, D. Trau, F. Grawe, K. Camm.an and M. Meusel: Analytical ChemistJ.y
71 (1999) 5430.
Biacore: Product information, www.biacore.com (2001)
D. Hall: Analytical Biochemistry 288 (2001) 109.
AffinitySensors: Iasys product information, www.affinity-sensors.com (2001)
M. P. DeLisa, z. Zhang, M. Shiloach, S. Pilevar, C. C. Davis, J. S. Sirkis and W. E. Bentley:
Analytical Chemistry 72 (2000) 2895.
T. O'Brien, L. H. I. Johnson, J. L. Aldrich, S. G. Allen, L.-T. Liang, A. L.Plummer, S. J. Krakand A. A. Boiarski: Biosensors & Bioelectronics 14 (2000) 815.
W. A. Challener, J. D. Edwards, R. W. McGowan, J. Skorjanec and Z. Yang: Sensors andActuators, B 71 (2000) 42.
N. F. Hartman: U.S. Patent 4,940,328 (1990)
N. F. Hartman: U.S. Patent 5,623,561 (1997)
B. H. Schneider, J. G. Edwards and N. F. Hartman: Clinical Chemistry 43 (1997) 1757.
L. M. Johnson: U.S. Patent 4,515,430 (1985)
R. A.Potyrailo, S. E. Hobbs, G. Hieftje and M. J. Fresenius: Analytical Chemistry 362 (1998)
349.
C. Stamm, R. Dangel and W. Lukosz: Optics Communications 153 (1998) 347.
0. Wolfbeis: Analytical Chemistry 72 (2000) 81R.
M. Mehrvar, C. Bis, J. Scharer, M. Moo-Young and J. Luong: Analytical Sciences (2000) 677.
W. Tan, R. Kopelman, S. L. R. Barker and M. T. Miller: Analytical Chemistry 71 (1999) 606A.
J. Janata: Analytical Chemistry 73 (2001) 150A.
J. Bures, S. Lacroix and J. Lapierre: Applied Optics 22 (1983) 1918.
F. Gonthier, S. Lacroix and J. Bures: Optical and Quantum Electronics 26 (1994) S l35.
D. Gerdt and J. C. Hen-: U.S. Patent 5,494,798 (1996)
M. Henning, R. Reynolds, K. Sellers and S. Barker: Patent Pending Serial No. 10/117,655
D. Angeley: unpublished results (2001)
M. Henning: unpublished results (2001)
C. DeCusatis and G. Li: "Fiber-Optic Communication Links" in Fiber Optics Handbook. M.
Bass and E. W. Stryland, Eds. (2002) 6.17
C. Earle and M. L. Wagner: unpublished results (2001)