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Design of Capillary Waveguide Biosensors
Harbans Dhadwal1, Paul Kemp2, Josephine Aller2 and Megan Dantzler2
State University of New York at Stony Brook
of Electrical and Computer Engineering
2Marine Sciences Research Center
1Department
Excitation through the
capillary wall
DESIGN CONSIDERATIONS
AIM
To develop design rules for capillary waveguide biosensors, based
based on
evanescent wave excitation and detection of freely propagating fluorescence.
fluorescence.
BACKGROUND
A typical capillary is a series of cylindrical shells with different refractive indices. The interior space provides a channel for transport of the target molecules in a fluid stream. Next is a thin oligonucleotide
probe coating on the order of 10-15 nm in thickness. A typical method for binding probes involves a streptavidin molecule covalently bonded to the silica surface and linked to a biotinylated probe molecule lying
orthogonal to the capillary surface. The effective thickness of the coating layer is determined by the total length of the surface bound probe, including streptavidin (ca 5nm), biotin (3 nm), the probe molecule itself
(ca 7 nm for a 20-mer), and a fluorochrome molecule. The physical dimensions of the fluorochrome we use, Alexa 532, have not been determined but are likely to be on the order of several nm in diameter
(Molecular Probes, Inc.). The practical upper limit to probe length is determined by the penetration depth of the evanescent wave, which is a less than one wavelength (e.g. 532 nm for the laser we used). The next
shell consists of the capillary wall, which is made from pure silica and has a refractive index close to 1.5. Finally, the outer surface of the capillary can be coated or immersed in another fluid. For this discussion, we
consider an optically transparent capillary wall surrounded by air.
Various optical configurations for excitation and collection of emission
from molecules bound to the interior surface of a capillary have been
used. EWFE – evanescent wave field excitation, FETGM – fluorescent
emission tunneled onto the guided modes, DEX – direct excitation, FPFE
– free propagating fluorescence,
The optimal design problem is primarily concerned with determining
the inner and outer radii of a silica capillary such that the optical
sensor will provide the highest transmission of free fluorescence
radiated through the capillary wall and into the surrounding medium.
We are not optimizing for transfer of fluorescence into the guided
modes of the capillary wall. For the CWS described here, the
excitation and emission optical geometries can be optimized
independently.
X
X
transmitted
transmitted
trapped
Fluorescent
molecule
o
Y
Z
capillary wall
coating layer
Refractive index
n3
n2
n1
d1
d2
(d)
(a)
t
n4
Y
Fiber
EW FE
FETGM
FETGM
Capillary
DEX
(c)
(e )
FP FE
DEX
Surface rendering of the efficiency η(Rd, d1), of the
free propagating fluorescence transmitted from the
outer capillary surface. The grid points are
computed using 20,000 rays from dye molecules
immobilized in a thin coating layer (t/λ<0.2) on the
interior capillary surface. ηmax = 79% for Rd > 1.34.
Calculation of fluorescence emission.
Typical ray propagation from
fluorescence point sources located on
the coating/fluid interface.
FP FE
FP FE
EW FE
Geometries that keep the excitation and emissions paths separated have
are preferred because of the reduced in-band background signal.
CONCLUSION
We have outlined a design procedure for CWS and demonstrated one
implementation of a capillary waveguide biosensor. A sensitivity of 30 pg.mlof DNA probe was obtained using a 1 mm fiber to collect free propagating
fluorescence emanating from the capillary surface at right angles to the
evanescent wave excitation. The size of the portable capillary waveguide
biosensor can be considerably reduced as the fluorescence signal is collected
from only a 1 mm length of capillary. Although not reported here, the sensor
can be reused over several tens of cycles. Additionally, the limit of detection
is inversely proportional to the extinction ratio and directly proportional to
the square root of the measurement interval. Assuming that signal and
background counts increase proportionally in response to excitation power,
then the limit of detection can be extended by reducing the in-band
fluorescence background, through the use of an improved detection geometry
or through use of hybridizing solutions with lower autofluorescence. The
usefulness of this biosensor design for study microbial processes in natural
environments will guide future refinements and design options.
Detection Limit
EXPERIMENTAL SETUP
A schematic of a capillary waveguide biosensor. LD – excitation source, PD – photodetector, MO – microscope objective, BS –
beam splitter, OPF1 – transmitting optical fiber , OFC – opto/fluid connector, HE – heating element, OPF2 – optical fiber (or
fiber array) collector, BCL – biconvex lens, HNPF – holographic notch-plus filter, BPF – band pass filter, OPF3 – optical fiber
relay, PMT – photomultiplier, PC – computer based data acquisition, S – syringe containing sample, V – stop valve, CAP –
capillary, CL – coating layer, FA – fiber array, FPFE – free propagating fluorescence emission, FETGM - fluorescent emission
tunneled onto the guided modes of the capillary wall.
PD
MO
BCL
LD
HNPF
BS
BPF
BCL
OPF2
OPF1
OPF3
HE
FETGM
S
PC
V
PMT
Fiber array
(17) 0.33 mm
10 |2
10 |1
10 |0
10 |-1
10 |-2
10 |-5
10 |-4
10 |-3
10 |-2
10 |-1
Concentration (æg/ml)
Response of the CWS at a
concentration of 30.63 pg.ml-1 of
probe.
multiple receiving optical fibers
fluid orrifce
Silica thin wall capillary
Coating of probe molecules
flow out
2.00
30
20
Dark
10
Denature
0
sample injection port
Hybridize <n S >
40
3
Fluorescence emission collected by
a linaer array of 17 optical fibers
Photon counts (x10 3)
50
fluid port
opto/fluid connector
Support from NSF under contracts OCE-0083193 and OCE-9907983 is appreciated.
1 mm fiber
10 |3
FPFE
Optical fiber delivers excitation
laser light at 532 nm to capillary
Acknowledgements
Normalized photon counts [ (<nS>/<nB>) - 1]
1
Most commonly, detection limit (DL) is expressed in terms of the lowest
concentration giving rise to a measurable signal that is three standard deviations above
the background signal. In this paper, we are going to use the terminology of a modern
digital communication system to define the sensitivity in a more quantitative measure.
The reliability of a digital communication channel is expressed in terms of a bit-errorrate (BER) which measures the number of times a digital signal (one or zero) is decoded
incorrectly from a train of pulses comprising ones and zeros. Borrowing these ideas, we
can express the background signal (absence of target species) as being equivalent to a
zero and the detection of the target species equivalent to a digital one. In terms of
photon detection, <nS >, <nB> represent the average counts per sample interval T, of the
target and background signals, respectively. We define the limit of detecting target
species in term of a target-detection-error-rate (TDER). One common approach is to
express TDER as a ratio of the number of errors Ne to the total number NT of
measurements, that is, TDER=Ne/Nt. For example, a TDER = 10-3 implies that the
target was misidentified once only in one thousand measurements, that is, a false
detection probability of 0.001.
0
300
600
<n B >
900
Time in seconds
Fiber optic connectors
Cylindrical housing
Photon counts (x10 )
DEX
(b)
Excitation through the
capillary core
1200
1500
1.60
1.20
<n B >
<n S >
0.80
0.40
0.00
0
45
90
135
180
225
270
Time in seconds
A Workshop on The Next Generation of in situ Biological and Chemical Sensors in the Ocean – Woods Hole Oceanographic Institution, July 13-16, 2003
315
360
405
450
From the figure we note that nS, σS, nB, σB are
1.06 kcps, 25 cps, 1.00 kcps, and 38 cps,
respectively. For a one second measurement
interval these translate into an extinction
ratio of ε=0.94, Q=1.2 and a TDER=0.115.
Howver, if we extend the measurement
interval to ten seconds the TDER is reduced
to 1.35 x 10-3 . In our measurements we have
opted to use a sixty second measurement
interval, which gives a TDER = 10-15 ,
corresponding to a negligible false detection
probability. In practice, the upper limit of
measurement interval is determined by the
desired response time for the sensor.