probabilistic estimation of detection characteristics for surface...
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Probabilistic estimation of detection characteristics for surface-enhanced localized
surface plasmon resonance biosensing
Heejin Yang
The Graduate School
Yonsei University
Department of Electrical and Electronic Engineering
Probabilistic estimation of detection characteristics for surface-enhanced localized
surface plasmon resonance biosensing
A Master’s Thesis
Submitted to the Department of Electrical and Electronic Engineering
and the Graduate School of Yonsei University
in partial fulfillment of the
requirements for the degree of
Master of Science
Heejin Yang
December 2014
This certifies that the master’s thesis of
Heejin Yang is approved.
____________________________________ Thesis Supervisor: Donghyun Kim ___________________________________ Thesis Committee Member: Taewon Hwang ___________________________________ Thesis Committee Member: Dong Ha Kim
The Graduate School
Yonsei University
December 2014
i
Acknowledgments
First and foremost, I would like to express my deepest appreciation to my advisor,
Professor Donghyun Kim, who has supported me throughout my Master’s course.
Without his devoted and constant support, it would have been impossible for me
to complete my Master’s course. In spite of busy works of his own, he was
willing to take time to listen to my thoughts, help me on a better path of life, give
advice on my research, and provide the best environment for my research.
I would also like to express my warm thanks to my thesis committee.
Professor Taewon Hwang helped me find the basic concept of my study. With
constructive questions, Professor Dong Ha Kim helped me see the things I would
have otherwise overlooked. This dissertation would have never been
accomplished without their support.
I am also thankful for the past two years that I have spent with my colleagues
in the Biophotonics Engineering Laboratory. Even though I have not had an
opportunity to work with Dr. Jongryul Choi, Dr. Yeonsoo Ryu, Mr. Yonghwi Kim,
and Taewoong Lee, my research has been largely inspired by their research. Dr.
Youngjin Oh has helped me with the fabrication of nanostructure using e-beam
lithography and advised me, from research to my religious faith. I have to mention
Wonju Lee for giving me great help in RCWA calculation, illustration of
detection models, and my life in the laboratory. My time working with Taehwang
Son and Hongki Lee was an extraordinary experience. Also, I am grateful to
Haena Kim, Kiheung Kim, Changheon Lee, and Hyunwoong Lee.
ii
I thank Kangsoo Kim, Minki Kim, and Hojung Jin as well for their
encouragement and advice.
I would like to express my deepest thanks to my family for always staying
on my side.
Lastly, I would like to thank God for being my savior..
iii
Table of Contents
Acknowledgments
List of tables
List of figures
List of acronyms
Abstract
Chapter 1 Introduction
1. Biosensors
2. Surface plasmon resonance biosensors
2.1 Theoretical background
2.2 Detection characteristics
2.3 Enhancement of detection characteristics and colocalization
3. Overlap integral
Chapter 2 Probabilistic estimation of detection characteristics
1. Method and model
1.1 Electromagnetic field amplitude: nanoisland and its nearfield
1.2 Permittivity modeling: Poisson distribution
1.3 Three detection models
2. Probabilistic interpretation of detection characteristics
2.1 Non-specific detection
2.2 Non-colocalized detection
2.3 Colocalized detection
iv
2.4 Noise characteristics of nanoplasmonic detection
3. Results and discussion
3.1 Detection characteristics of three detection models
3.4 Wavelength dependence
3.5 Discussion
Chapter 3 Conclusion
References
국문요약
v
List of tables
Table 1.1 Historical landmarks in the biosensor and its development
Table 1.2 Type of transducers with property
Table 2.1 RCI at 95% CL for concentration and 2.03E11 selected as the
target concentration that produces normalized OI = 0.001 in non-
specific detection with target size = 25 nm
vi
List of figures
Figure 1.2.1 Two semi-infinite dielectric media with ε1 and ε2 separated by a
interface plane at z = 0.
Figure 1.2.2 Dispersion curve for SP.
Figure 1.2.3 Prism configuration with dielectric constants ε1 < ε2 (a) Otto
configuration (b) Kretschmann configuration.
Figure 1.2.4 Concept of a SPR biosensor.
Figure 2.1.1 Comparison of fabrication process: (a) e-beam lithography for
customized nanostructure. (b) Thermal annealing for nanoisland.
Figure 2.1.2 SEM images of nanoisland with varied thickness of evaporated
film.
Figure 2.1.3 Correlation length: (a) correlation coefficient of nanoisland (2 2
m2) (b) profile of correlation coefficient along the horizontally
dashed axis, and (c) profile of correlation coefficient along the
vertically dashed line.
Figure 2.1.4 Schematic process of near field calculation.
Figure 2.1.5 Schematic illustration of probability density function given by
Poisson distribution when concentration C (same as χS) = 1 in
arbitrary unit (histogram in blue) and 10 (in red).
Figure 2.1.6 Schematics of three different detection models.
Figure 2.3.1 (a) Normalized OI for non-specific detection of targets of varying
size. A color band is CI with 95% CL. (b) Magnified image of a
rectangle in Figure 2.1.6a. The vertical arrow is the range of
normalized OI to be possible at the concentration producing
normalized OI =1. The horizontal arrow is the range of producible
concentration of normalized OI = 1.
vii
Figure 2.3.2 (a) Normalized OI for non-colocalized detection of targets of
varying size. A color band is CI with 95% CL. (b) Magnified
image of a rectangle in (a).
Figure 2.3.3 Normalized OI for colocalized specific detection. Although there is
a CI with 95% CL, the CI appears as a line and thus is hard to
distinguish between the CL and the mean.
Figure 2.3.4 Comparison of normalized OIs generated by three models with
varying concentrations with target size at 25 nm.
Figure 2.3.5 RCI curves of varying concentration with target size = 25 nm in the
three models.
Figure 2.3.6 RCI curves of varying concentration with target size = 25 nm in the
three models.
Figure 2.3.7 Near field patterns of same nanoisland at various wavelengths. (a)
Incident wavelength λ = 488 nm, (b) 633 nm, and (c) 760 nm.
Figure 2.3.8 Histograms of normalized fields of nanoislands at various
wavelengths.
Figure 2.3.9 Normalized OI of the colocalized detection at different
wavelengths λ = 488 nm, 633 nm, and 760 nm of varying
concentration with target size 25 nm.
Figure 2.3.10 (a) RCI at different wavelengths λ = 488 nm, 633 nm, and 760 nm.
(b) Magnified RCI at undistinguishable wavelengths arget size 25
nm.
viii
Figure 2.3.11 List of abbreviations
CI CL CLT EMT LOD OI PR RCI RCWA SAM SEM SP SPP SPR
confidence interval confidence level central limit theorem effective medium theorem limit of detection overlap integral photo resist relative confidence interval rigorous coupled-wave analysis self-assembled monolayer scanning electron microscope surface plasmon surface plasmon polariton surface plasmon resonance
ix
Abstract
The present study is on detection characteristics of surface-enhanced surface
plasmon resonance biosensors. The random nature of near-field generated by
nanoisland brings the need for probabilistic modeling. Detection characteristics,
represented by overlap integral, of three detection models - non-specific, non-
colocalized, and colocalized detection models - were calculated based on the
probability theory. The calculated overlap integral is proportional to the target
concentration. The effect of target size on the reliability of normalized overlap
integral is limited compared to that of target concentration. Overlap integral is the
highest and confidence interval is the lowest in colocalized detection. Compared
to the non-specific detection model, the colocalized detection model got the
detection limit enhanced by four more orders.
Keywords: biosensor, surface plasmon resonance (SPR), SPR biosensor, semicontinuous nanostructure, random nano island, probability theory
1
Chapter 1
Introduction
1. Biosensors
A sensitive and selective recognition of materials has always been an important
issue in scientific research.
This is an important research topic also in the fields of biomedical
engineering, biochemistry, and biophysics, because of the need regarding drug
development, medical diagnostics, environmental issues, and more. Recent
demands in many fields have required biosensing for detecting biomolecules,
microorganisms, and macromolecular interactions.
High sensitivity and selectivity have usually been achieved by high-liquid
chormatography, gas chromatography, and mass spectrometry, and high cost and
preparation of samples for these useful techniques have accelerated development
of economical methods that are easy to use. Biosensors may be one such solution
[1,2].
The term "Biosensor" was introduced by Karl Cammmann in 1977 [3]. He
defined a biosensor as a chemical sensor where the detecting system uses a
biochemical mechanism. Leland C. Clark is known to be the inventor of the first
biosensor [4]. Clark published his paper on glucose biosensor in 1956. The device
was made up of an oxygen electrode coated with glucose oxidase. The electrode
2
measures the oxygen concentration. The reduced amount of oxygen concentration
is converted into the amount of glucose.
Table 1.1 Historical landmarks of biosensor and its development.
Year Event References
1962 First glucose enzyme electrode Clark, L. C. & Lyons, C. [1]
1973 Glucose enzyme electrode based on
peroxide detection Guilbault, G. G. & Lubrano, G. J. [2]
1975 Launch of the first commercial glucose
sensor system
Yellow Springs Instruments glucose
biosensor
1977 Karl Cammann introduced the term
“ biosensor ” Cammann, K. [3]
1980 First fibre optic pH sensor for in vivo
blood gases Peterson. [5]
1982 First fibre optic-based biosensor for glucose : Demonstration of in vivo
glucose monitoring
Shichiri, M. et al. [6]
1983 First surface plasmon resonance (SPR)
immunosensor Liedberg, [7]
1990 SPR based biosensor by Pharmacia
BIACore Jonsson
2001 Ellipsometric Biosensors Arwin [8]
Present Quantum dots, nanoparticles,
nanowires, nanotubes, etc Ghoshal [9-10]
A biosensor is generally defined as a device to detect chemical or biological
molecules, or microorganisms. A biosensor consists of a biological active
substance and a transducer. The biological active substance, called a receptor, is
used to selectively bind the analyte or functional group of interest, which may be
either organic or inorganic. Antibody, enzyme, and DNA are widely used as
receptors in biosensors [11-15]. A transducer is a platform to transform chemical
3
or physical responses of a biological recognition event into measureable
parameters. Optical biosensors are biosensors with transduction based on optical
principles. They are classified not only by the basis but also by whether the label
association attaches to the target or not.
Table 1.2 Type of transducers with properties Type Property
Optics
Luminescence Fluorescence Adsorption
Phosphorescence Surface enhanced Raman scattering
Dispersion Refraction spectroscopy
Electrochemistry Impedimetry Amperometry Voltammetry
Thermodynamics Heat of reaction
Adsorption
4
2. Surface plasmon resonance biosensors
2.1 Theoretical background
2.1.1 Excitation
Free electrons excited by the electromagnetic field at a metal/dielectric interface
collectively and coherently oscillate; such oscillations, are called SP oscillations
[16,17]. Consider two semi-infinite nonmagnetic media with dielectric functions
ε1 and ε2 are separated by a planar interface at z = 0. Maxwell’s equations without
external sources can be expressed as follows [18]:
Figure 1.2.1 Two semi-infinite dielectric media with ε1 and ε2 separated by an
interface plane at z = 0.
(1.2.1)
(1.2.2)
∙ ∙ 0 (1.2.3)
and
ε2ε1
z = 0
5
∙ 0 (1.2.4)
where i is i-th media. Media 1 is below z = 0 and media 2 is above z = 0.
Solutions of Eqs. (1.2.1) ~ (1.2.4) can be analyzed in the s-polarized and p-
polarized electromagnetic modes. For an ideal surface, if waves propagate along
the interface, there must be a component of the electric field normal to the surface.
Thus, the p-polarized wave is considered. The conditions in which a travelling
wave with the magnetic field H parallel to the interface (p-polarized wave) may
propagate along the surface (z = 0), with the fields declining in the positive (z > 0)
and negative (z < 0) directions are caculated.
, 0, | | (1.2.5)
and
0, , 0 | | (1.2.6)
qi represents the magnitude of a wave vector parallel to the surface.
When Eqs. (1.2.1) and (1.2.6) are substituted into Eqs. (1.2.1) ~ (1.2.4), the results
are as follows:
1 1 1 1 , (1.2.7)
2 2 2 2 , (1.2.8)
and
2 2
2 . (1.2.9)
In the boundary conditions, the component of the electric and magnetic fields
parallel to the surface must be continuous. From Eqs. (1.2.7) and (1.2.8),
6
1
11
2
22 0 (1.2.10)
and
1 2 0 (1.2.11)
are derived. For Eqs. (1.2.10) and (1.2.11), a solution exists only when the
determinant is zero, i.e.
1
1
2
20 (1.2.12)
This is the SP condition.
The 2D wave vector q from the boundary conditions is entered into Eq.
(1.2.9), i.e. q1=q2=q. Thus, Eq. (1.2.12) can be expressed as follows [19]
, (1.2.13)
w/c represents the magnitude of the light wave vector.
7
2.1.2 Dispersion relation
Consider a Drude semi-infinite metal in vacuum. Dielectric functions of medium
is
1 12
(1.2.14)
and ε2 = 1 [18], where η represents a positive infinitesimal. Thus, Eq. (1.2.13) can
be expressed as follows:
(1.2.15)
The blue line shows the light line ω = cq in free space and the red solid line
shows the SPP in the dispersion relation of Eq. (1.2.15). There is no intersection
between the red line and the blue line at low frequency. Thus, the incident light
cannot directly excite the SPP in free space. The red solid line approaches the
surface plasma frequency line. Because the dispersion curve of photons in metal
can lie across the red line at the point where the momenta match, SPPs can be
excited in metal.
8
Figure 1.2.2 Dispersion curve for SP.
ω=ckx
kx=
1
0.6
0.8
0.4
0.2
0
ωsp=ωp/
0 0.5 1 1.5 2 2.5 3
ω/ωp
Kx (arbitrary units)
momentum matching
9
2.1.3 Penetration depth
From Eqs. (1.2.13) and (1.2.9) (with q1 = q2 = q), the SP decay constant ki normal
to the interface is derived as follows
2
1 2. (1.2.16)
Eq (1.2.16) can define a penetration depth or attenuation length L = 1/ki where the
electromagnetic field becomes 1/e.
10
2.2 Surface plasmon resonance
To demonstrate SP excitation, Otto and Kretschmann independently propose
prism configuration [21,22]. The difference between the two proposals is the
location of low refractive index media between a metal film and the prism. In
Otto’s configuration which is for SP of solid phase media, low refractive index
media are inserted under the metal, whereas in Kretschmann’s configuration, the
metal adheres to the surface of the prism without low refractive index media. The
latter configuration is a more efficient structure for the creation of SP. There is
also a grating configuration, but its inaccuracy places the grating-configuration-
based sensor at a disadvantage; because the incident light passes through the
sample, it may have an accuracy issue when the sample is absorptive.
Figure 1.2.3 Prism configurations with dielectric constants ε1 < ε2 (a)
Otto configuration and (b) Kretschmann configuration.
As previously stated, the momentum of incoming photons and the SP can be
matched. Such matching is called SPR, and the incident angle of photons is called
θε0
ε1
ε2
prism
SP
θε0
ε1
ε2
prism
SP
(a) (b)
re
an
w
fu
ph
re
pr
2.
Th
se
co
esonance an
nd plasmon
where sinθR
unction of
hotons decr
esonance an
roperty, SPR
3 Detection
he perform
ensitivity,
ommonly de
ngle. Resona
happens, an
represents
additional
reases when
ngle is a sen
R can be the
Figu
n characteris
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LOD, and
efined as fo
ance condit
nd it is deriv
s the reson
dielectric l
n the incide
nsitive value
e basis of a
ure 1.2.4 Co
stics
PR biosens
d cost-effic
llows:
11
tion occurs
ved as follo
0
nance angl
layer such
ent angle ap
e of the diel
biosensor.
oncept of a
sor is eval
ciency. Th
when the e
ows
1 2
1 2
e and ε0 r
as air. Th
pproaches t
lectric funct
SPR biose
luated by v
e sensitivit
exact matchi
represents t
he intensity
he resonanc
tions, and b
nsor.
various crit
ty, S, of b
hing of phot
(1.2.1
the dielect
y of reflect
ce angle. T
because of th
teria such
biosensors
ton
17)
ric
ted
The
his
as
is
12
(1.2.18)
where P is the measured output of the sensor; I is the sensor input; n is the
refractive index of the sensor platform; SRI is the sensitivity on refractive index
change; and ESF is the efficiency of the surface functionalization. Thus, the S is
the ratio of the change in the sensor output (e.g., wavelength or resonance angle)
to the change in the input (e.g., concentration of analyte). The S can be divided
into SRI and ESF. SRI is related to a method of modulation and excitation of SP, but
not ESF [23].
LOD is a metric with respect to the resolution of a biosensor and indicates
the minimum concentration of analyte that can be detected from the absence of
analyte [23]. LOD is calculated from the measured output of the sensor and the
standard deviation of a blank sample. The concentration of analyte that generates
the sensor output of three standard deviations is the LOD [24].
13
2.4 Enhancement of detection characteristics and colocalization
SPR biosensors are advantageous in being real-time and label-free. However, they
are not sensitive enough to detect a single molecule because of its label-free
nature. Several techniques have been proposed to improve the sensitivity, such as
conjugation with gold, silver, magnetic, or carbon-based nanoparticles, surface
modification with nanostructure and metamaterials; and colocalization of the
biointeractions of interest [11,25-28].
Colocalization is a technique to make an overlap between biointeraction and
a field localized by nanostructure. Dielectric or metal is evaporated at an angle to
coat the nanostructure except the regions very nearby the nanostructure. It is
similar as the situation where a shadow is formed right near the building which
receives the sunlight. The exposed regions of the surface are used as the binding
regions to capture the functionalized targets, allowing colocalization. Experiments
have discovered that the sensitivity increases by 1000 times compared to the
traditional detection [11,29-33].
Despite the sensitivity enhancement by colocalization is experimentally
proven the sensitivity characteristics is generally vague to understand [26]. Due to
the difficulty of alignment and the limitation of the prediction on the localized
near-field position, exact colocalization is difficult to achieve.
14
3. Overlap integral
SPR biosensors are founded on an enhanced evanescent field in the
metal/dielectric interface. The field distribution in the region where the enhanced
evanescent field exists determines the sensor sensitivity for a perturbation in the
analyte. Shalabney and Abdulhalim demonstrate that a shift in the wavevector k
corresponds to the OI, which corresponds to the interaction volume Vin [34,35]
δ∙∗∙ ∙
∙∗∙ ∙
(1.3.1)
where Ei and ki represent the electrical field and its wave vector before the
refractive index variation from perturbation in analyte; Ef is the changed electrical
field after the perturbation.; and δk is the change in the wave vector because of the
change of the dielectric constant from ε to ε + δε. Since δk represents the change
in the incident angle or the wavelength change, δk/δε indicates the sensitivity of
the sensor corresponding to the OI normalized by the total energy. An
enhancement of the sensitivity can be mathematically accomplished by enlarging
the interaction volume to increase the evanescence depth, improving the field
intensity, and using a material with a high electric constant.
Based on Eq. (1.3.1), the OI can be simplified as follows [34]:
OI | | (1.3.2)
Et is the normalized tangential electromagnetic field amplitude and ε is the
permittivity. Therefore, Eq. (1.3.2) has the unit of energy. The total energy on the
surface with the localized target can be related to the radiative energy which can
15
be measured with the SPR signal. Hence, the near-field quantities in the
interaction volume may be transduced into the far-field characteristics. For
example, the LOD can be molecularly interpreted as the number of target
molecules (N) generating a reference change in the reflectance (R) in the SPR
biosensor. Based on the OI, the redefined LOD is closely related to the
perturbation in the OI from the biointeraction of interest. In addition, the detection
sensitivity can be redefined as the ratio of the change in the OI to the number of
target molecules.
Thus, OIs help understand the relation between the near-field and the far-
field. In previous research, OIs have been used to optimize the nanostructure in
different features and structural parameters and predict the sensitivity of the
experimental model [26,36].
OIs have been deterministically treated, but the far-field characteristics of a
SPR biosensor can calculated with the simplified OI Eq. (1.3.2) in the manner of
the probabilistic approach. If the sampling function for the numerical calculation
is larger than one target molecule, the target permittivity may be approximately
derived as follows:
ε ∑ (1.3.3)
ε and ε each represent the permittivity of the target and the buffer
(ε2 2 and ε2 2 ). represents the locations of Nt target
molecules. When Eq. (1.3.3) is put into Eq. (1.3.2), the result is as follows:
OI ∑ | | | | (1.3.4)
16
From Eq. (1.3.4), the sensitivity is derived as follows:
∆
∆∑ | | ∑ | | ⁄ (1.3.5)
This is because the right hand side of Eq. (1.3.4) is a constant. Although the OI
becomes a simple integral form, two components, the permittivity and the field,
remain to analytically evaluate the OI. Due to the complex action of targets such
as contact inhibition or aggregation, it is complicated to precisely model the target
and its distribution. Also, the field is largely unknown.
The permittivity of the target and the buffer solution is treated as a
permittivity of a layer derived from the EMT [26,36]. Not considering the nature
of a random variable, however, the EMT-based evaluation is deterministic. In this
thesis, the probabilistic aspect of OI is focused on the colocalization. The
reliability in estimating the concentration of a target is also discussed.
17
Chapter 2
Probabilistic estimation of detection
characteristics
1. Method and Model
1.1 Electromagnetic field amplitude: nanoisland and its nearfield
To produce highly localized SP, the nanostrucutures on the metal surface are
fabricated by Focused-ion beam, e-beam lithography, or others. For example, e-
beam lithography is the practice of SEM by focusing a beam of electrons to draw
customized shapes on a surface coated with an electron-sensitive resist which can
be either positive or negative. Exposed to the e-beam, the electron resistor is
transformed into either more or less soluble structure. Due to the difference in the
solubility, either the exposed or non-exposed regions of the resistor are selectively
removed, with the surface immersed in the developing solution. After the
developer is rinsed on the surface, the vapor metal or dielectric covers the opening
region, and the residual resistor is removed. Although this practice is powerful in
that it can draw the patterns of nanostructure without mask, it has low throughput,
is not cost-effective, and is time-consuming. Also, the SEM, the evaporator for
vapor deposition, and various chemicals are required for the e-beam lithography.
18
Figure 2.1.1 Comparison of fabrication process: (a) e-beam lithography for
customized nanostructure. (b) Thermal annealing for nanoisland.
Random nanoisland, which is a semi-continuous structure, can be simply
synthesized by annealing the surface covered with the metal. It increases the cost-
effectiveness and reduces the synthesizing time [15]. Also, the excitation of the
highly localized SPs on the random nanoisland is experimentally observed [22]. It
is applied in various ways such as resonance energy transfer [37,38], surface-
enhanced Raman spectroscopy [39-42], enhancement and quenching of
fluorescence and photoluminescence [43-48], highly luminescent light emitting
diodes [49], solar cells [50,51], spontaneous light emission [52], and far-field
super-resolution microscopy [53].
Evaporation
PR spincoating
e-beam lithography
Development
Evaporation
Removal
Customized nanostructure
Thermal annealing
(a) (b)
Semicontinuousnanoisland
be
ch
is
Fig
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19
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20
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21
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22
1.2 Permittivity modeling: Poisson distribution
The distribution of target molecules at a certain concentration in space can be
analyzed with the stochastic geometry which is for random spatial patterns. In
enhancing sensing characteristics, where the target molecules are exactly located
is less important than whether they exist inside the highly localized near-field; one
target molecule in the highly localized near-field increases the sensing
characteristics more than a target molecule located outside does. In the stochastic
geometry, the number of points at a given rate is a random variable and is known
to follow the Poisson distribution [59]. The Poisson distribution is a discrete
probability distribution that demonstrates the probability of a given number of
events occurring in a fixed interval or space when these events occur with a
known average rate and independently of the time since the last event [60].
If the target molecules show no preference for any point and the total
volume of the target molecules is small that it is negligible compared to that of the
highly localized near-field, the random variable of target molecules can be
modeled according to the Poisson point distribution. In this case, the probability
mass function is
P!
. (2.1.1)
χ is the known average number of target molecules per unit volume, which is the
F
P
Figure 2.1.
oisson distr
.5 Schemat
ribution wh
(h
ic illustrati
hen concen
histogram i
23
ion of prob
ntration C (
in blue) and
bability den
(same as χ
d 10 (in red
nsity functio
χS) = 1 in ar
d).
on given by
rbitrary un
y
nit
24
1.3 Three detection models
Non-specific and specific detection have been considered. Randomness in the
non-specific detection arises from the distribution of target objects and the
intrinsic nature of nanoislands. Randomness in the specific detection is related to
the probability of binding target molecules to probe layer on the surface. The
binding process in the specific detection is stochastic and is considered as a
negligible factor in this study [61,62].
Figure 2.1.6 shows a simple model used to calculate the characteristics of
SPR detection in the non-specific and the specific models on nanoislands. The
models have a 20-nm thin underlying silver film. The dielectric permittivity and
the size of target biomolecules have been treated the same as those of adenovirus
particles (dielectric, nav = 1.366). The notation of target concentration has
followed En (n: integer) represents 10n /L = 10n-9 /m3 which is normally used
for the virus concentration. The non-specific detection model in Figure 2.1.6a has the probabilistically
distributed target in 3D. In the specific detection model illustrated in Figure 2.1.6b,
the distribution of target molecules is planar on the recognition layer with
antibodies that selectively capture the target. The binding position on the layer
follows the probabilistic distribution. In the colocalized detection model shown in
Figure 2.1.6c, the target molecules are bound to the localized field inside the
recognition layer and the binding position is probabilistically distributed. For
th
na
in
us
hese three m
anoislands.
F
We obs
ncident light
sed for SP
models, the
Figure 2.1.
served dete
t at = 48
PR sensing.
e randomne
6 Schemati
ction chara
8, 633, and
. The refle
25
ess of near
ics of three
acteristics a
d 760 nm. T
ectance cha
r-field distr
e different d
and localiza
These three
ange occurr
ibutions fo
detection m
ation on th
wavelength
ring as the
ollows that
models
he p-polariz
ths are wide
e result of
of
zed
ely
f a
26
biointeraction was calculated at the fixed incident angle of 60. It is assumed that
a 2.3-nm chromium layer adhered to an SF10 substrate (refractive index ns =
1.723) in the ambient buffer solution (nbuffer = 1.33). Also, we assumed that
nanoislands were made of a thermally annealing 5-nm silver film on top of the
chromium layer. Optical constants of silver and chrome at = 488, 633, and 760
nm were taken [63].
27
2 Probabilistic interpretation of detection characteristics
2.1 Non-specific detection
To make the analysis simple, it is assumed that is divided into four levels,
while ε is divided into two levels depending on the location , i.e.,
, ∈ |0.75 max min max, ∈ |0.50 max min 0.75 max min, ∈ |0.25 max min 0.50 max min
, ∈ |min 0.25 max min
(2.2.1)
and
ε ∈
(2.2.2)
Hence, Eq. (21) can be expressed as follows:
{ } { }∑∑ ≈-3
0= 12
023
0= 12
02 ++)(= i itiiii itiitii KAεESεEKAεEKASεEOI (2.2.3)
represents the number of target molecules in the i-th region (i = 0, 1, 2, 3)
which is an area corresponding to one of the four levels. At is the volume of one
target, and S represents the total volume of the i-th region. In this case, S and is
disjoint and independent of each other. Therefore, can be treated as the
independent Poisson random variable with parameter Si. Eq. (2.2.3) is
approximated based on the same assumption in Eq. (2.1.1) that S ≫ Eq.
(2.1.1) can be converted into
P!
. (2.2.4)
28
The OI is normalized by the optical signature of ambient buffer solution
without any target molecules. Normalized optical signature OIis interpreted as
follows:
OI∑ 2
0S21
30
OI , (2.2.5)
where OI ∑ 230 0Si. OI is a new Poisson random variable with a
parameter ∑ S30 , where 2
0S OI and
21A OI . Because each is independent of each other, OIcan be
modeled as a Gaussian random variable by the CLT [64]. The CLT is a theory in
which a Gaussian random variable is approximated from the summation of
sufficient independent random variables. As the number of independent random
variables becomes sufficient, their sum approaches a Gaussian random variable.
Inversely speaking, it is hard to model the sum of insufficient random variables
into a Gaussian random variable. In this case, the number of random variables is
close to insufficient. Nonetheless, OI can be converted into a Gaussian random
variable because 0 and 1 are Poisson random variables, and a Poisson
random variable is close to a Gaussian random variable. The mean and the
variance of OI are given by
OI2 ∑ ∑3 0
30 (2.2.6)
and
OI2 ∑ 23
0 (2.2.7)
From Eqs. (2.2.6) and (2.2.7), the area concentration of target, χ, is derived as follows:
29
OI ∑3 0∑ S3
0 (2.2.8)
Because of the Gaussian random variable, OI, also is a Gaussian random
variable with expectation and variance
(2.2.9)
and
σ 2 ∑2S3
0
∑ S30
2 (2.2.10)
Thus, the reliability of estimating the target concentration can be calculated in the
given CL [64]. The 95% CI of χ is
1.960 χ 1.960 . (2.2.11)
The 99% CI of χ is
2.575 χ 2.575 . (2.2.12)
Corresponding to a target concentration with 95% CL, the CI of the estimated
≡ is given by
1.960 OI 1.960 . (2.2.13)
The CI of the estimated ≡ with 99% is given by
2.575 OI 2.575 . (2.2.14)
The averages derived from the probabilistic model are consistent with those
from the deterministic model described in Eqs. (1.3.3) - (1.3.5) with the limited
ranges of target size up to 25 nm. If the target size exceeds 25 nm, the correlation
between the probabilistic and the deterministic models decreases. This is because
the assumption that the target size is small enough to be neglected compared to
30
the size of the localized field is repudiated above 25 nm. Thus, this study
performed the estimation for the target size 1 to 25 nm.
31
2.2 Non-colocalized detection
Non-colocalized detection is achieved by a probe layer on the metal surface
capturing target molecules. The influence of a probe layer on the near-field
distribution is imperceptibly small. Hence, in non-colocalized detection is
approximately identical to that in non-specific detection. The permittivity of the
outside volume to capture target molecules is treated to be ε0. The OI outside the
binding layer is constant at 20S . Eq. (2.2.3) in non-specific detection is given
by
OI ∑ S , A ∑ S , (2.2.15)
Si,inside is the volume of the i-th region in the binding layer, and Si, outside is that of
the outside. Si in non-specific detection is the sum of Si, inside and Si, outside in this
case. Furthermore, Eqs. (2.2.4) ~ (2.2.8) can be explained as follows:
P ,
!, (2.2.16)
OI∑ 2
0S ,21
30 ∑ 2
0S ,30
OI (2.2.17)
OI2 ∑ , ∑3 0
30 ∑ 2
0S ,30 OI (2.2.18)
OI2 ∑ 2
,30 (2.2.19)
OI ∑3 0 ∑ 20S , OI
30
∑ S ,30
(2.2.20)
OI ∑ S , 20S , OI , and 2
1A OI . Finally,
the CI with CLs can be obtained through Eqs. (2.2.9) ~ (2.2.14).
32
2.3 Colocalized detection
In colocalized detection, the effect of the probe layer on the approximation of
is negligible. Thus, remains identical. S2 and S3 are the regions
covered with the probe layer. The OI is expressed as
OI ∑ S , A ∑ S , ∑ S , .
(2.2.21)
Also, Eqs. (29) ~ (32) are changed to
OI∑ , ∑ , ∑ ,
(2.2.22)
, S , S , OI
(2.2.23)
∑ , (2.2.24)
∑ ∑ , ∑ ,
∑ , (2.2.25)
Then, the CI can be obtained through Eqs. (2.2.9) ~ (2.2.14).
33
2.4 Noise characteristics of nanoplasmonic detection
There are two factors that can bring noise to SPR biosensors. One such factor is
related to the technical performance, such as instrumentation from vibration or
stray light, for example. The other noise factor is related to the intrinsic noise
associated with the fundamental phenomena such as thermodynamic refractive
index fluctuation. If the noise sources are assumed to be probabilistically
independent of each other, the noise sources affect perturbations of the target
permittivity and the field amplitude that, consequently, bring noise to the OI.
Thus,
OI OI O (2.2.26)
Due to CLT, the noise term on the right hand side which is the sum of
various independent noise sources is treated as a Gaussian random variable with
expectation μ 0 and variance σ2 2 [65]. Because OI and
O can be considered independent and OI as the sum of the two
independent Gaussian random variables, OI becomes another Gaussian
random variable with mean μ Exp OI and variance σ2 2OI2. The
probability density function p is written as
ε, . (2.2.27)
and represent the statistical deviation in the tangential field amplitude
and target permittivity. The variance of OI is larger than that of OI without
O . The variance is the key factor for CI; an increase in the variance
34
increases the CI. The effects of noise factors are inversely proportional to the
LOD and, thus, the lower noise factor increases the LOD. Because the present
study demonstrates a particular model of detector, the LOD should be considered
as a relative scale rather than an absolute scale. Then, it is meaningful to relatively
compare the LODs of detection scenarios because noise factors in the scenarios
are independent of each other. Therefore, the LOD is evaluated with the relative
magnitude of optical signature generated in each of the detection scenarios.
35
3 Results and discussion
3.1 Detection characteristics of three detection model
3.1.1 Non-specific detection model
Consider a rectangular parallelepiped and assume that its base is a lateral plane of
2 2 m2 and the height is one penetration depth from the surface. If the
penetration depth is 100 nm, four target molecules exist inside the box at E10.
Figure 2.3.1 (a) describes the detection sensitivity as the optical signatures that
cause non-specific detection at a specific concentration evaluated for nanoislands.
The optical signatures are obtained by dividing the calculated OI into that of
ambient buffer without target molecules.
It is found that optical signatures monotonically increase with the size and
the target concentration. Optical signatures significantly changes with the target
size and are almost proportional to the target concentration. Because OI and
ΔOI/ΔN may be associated with the LOD and the detection sensitivity, the results
in Figure 2.3.1 imply that the LOD and the sensitivity for detecting targets of
larger size are enhanced. Such enhancement is related to the growth of effective
volume which is exposed to the near-field and occupied by the target.
Figure 2.3.1 shows the average characteristics using effective medium and
probabilistic characteristic as CI in the non-specific detection. An increase of CI
with target size shows that a larger target causes higher uncertainty at the same
concentration. For example, the CI with 95% CL for targets of ϕ = 25 nm at a
concentration of 2.03E11 is from 0.00075195 to 0.001248 with an average of
36
0.001, i.e., the relative confidence interval (RCI) defined by dividing the CI into
an average to be 0.5. The target concentration producing OI = 0.001 ranges from
1.594E11 to 2.631E11 with an average of 2.03E11, i.e., the RCI in the target
concentration of0.49. An identical concentration (2.03E11) produces the almost
constant RCI of 0.5 with target size of ϕ = 10, 15, and 20 nm although the CI at
the concentration increases with target size. This is because target size increases to
the average optical signature at the concentration. However, CI remains the same
as the target concentration changes. Hence, the RCI is inversely proportional to
the target concentration.
r
ho
Figur
varying s
rectangle in
to be po
orizontal a
e 2.3.1 (a) N
size. A color
n Figure 2.
ossible at th
arrow is the
Normalized
r band is C
1.6a. The v
he concentr
e range of p
37
d OI for no
CI with 95%
vertical arr
ration prod
producible
1.
on-specific
% CL. (b) M
ow is the ra
ducing norm
concentrat
detection o
Magnified i
ange of nor
malized OI
tion of norm
of targets of
image of a
rmalized O
I =1. The
malized OI
f
OI
I =
38
3.1.2 Specific detection model
The probe layer for the target recognition is assumed to uniformly cover the
surface in the specific detection model. The detection is limited to the target
molecules, although they are randomly captured by the layer. In other words, the
number of bindings can be identical to the target concentration. Computationally,
the target molecules are laterally distributed beneath the surface on top of the
binding layer. For simplicity, the specific detection model is estimated with
different scenarios, the non-colocalized and the colocalized detection models.
In the specific detection model, it is assumed that target molecules do not
prefer the surface as described in Figure 2.3.2. Figure 2.3.2 shows the calculated
optical signature with various target concentrations. The tendency in the results of
the non-colocalized detection is similar to the case of non-specific detection. On
the other hand, the magnitude of optical signature in non-colocalized detection is
much larger than that of optical signature in non-specific detection, which is due
to the nature of the surface. Following the evanescent nature, the near-field
exponentially decays of the surface. Thus, the optical signature is enhanced
compared to that of non-specific detection. Also, the number of target molecules
captured by the probe layer is generally larger in specific detection; as the target
concentration increases, the effective are is enlarged at a faster pace. Hence, the
stronger near-field and the wider effective area contribute to amplify the optical
signature in specific detection.
de
sp
0.
co
Figure 2
varying s
Compar
etection is s
patial rando
046857 to
oncentration
2.3.2 (a) No
size. A color
red to non-
smaller than
omness. On
0.050386
n of targets
ormalized O
r band is C
re
-specific de
n that in non
n the 95%
with an a
are ϕ = 25
39
OI for non-
CI with 95%
ectangle in
etection, th
n-specific de
CL, an op
average of
5 nm and E
-colocalized
% CL. (b) M
(a)
he variance
etection bec
ptical signa
0.048622
11. Also, th
d detection
Magnified i
e in no
cause of a d
ature range
when the
he uncertain
n of targets
image of a
on-colocaliz
decrease in t
es from OI
size and t
nty is 7.3%
of
zed
the
I =
the
in
te
In
sp
t
m
w
op
si
lo
to
erms of the R
n other wor
pecific detec
Figure
there is the
Colocali
molecules ex
would be ha
ptical trapp
gnificant b
ocalization w
The opt
o the target
RCI, compa
rds, the cert
ction.
e 2.3.3 Norm
e CI with 9
ized detect
xclusively b
rd to exper
ing in the c
because the
with an arbi
ical signatu
concentratio
ared with R
tainty in de
malized OI
5% CL, it
between
tion can be
bind in the l
rimentally p
calculation.
probabilist
itrary structu
ure in coloc
on. Because
40
RCI = 71%
etection is d
I for coloca
appears as
n the CL an
e probabili
localized fi
perform col
. Neverthele
tic interpre
ure.
calized detec
e the target
in the non-s
drastically i
lized specif
s a line so it
nd mean.
istically tre
elds as desc
localization
ess, the res
etation can
ction can be
molecules
specific det
improved b
fic detectio
t is hard to
eated in th
cribed in Fi
n on nanoisl
ults are pro
be applied
e calculated
bind to th
tection mod
by conducti
on. Althoug
distinguish
hat the targ
igure 2.3.3.
lands witho
obabilistica
d to the fie
d with respe
he probe lay
del.
ing
gh
h
get
. It
out
lly
eld
ect
yer
in
nu
de
ca
ca
th
ca
fo
op
be
de
n the locali
umber of ta
etection. Ca
an achieve t
alculation fo
he trend of
alculated op
ollows that o
ptical signa
ecause the d
etection than
Figure
w
zed field, t
arget molec
alculated t
the highest
or average b
f average o
ptical signa
of non-colo
ature is the
distribution
n in the oth
e 2.3.4 Com
with varyin
the target c
cules to the
this way, t
estimation
based on an
optical sign
atures. The
ocalized and
e highest a
n of the nea
er two scen
mparison of
ng concentr
41
concentratio
e volume o
the optical
with nanois
n effective m
natures. Fig
e overall tr
d non-specif
among the
ar-field has
narios.
f normalize
rations wit
on correspo
of the locali
signature f
slands. The
medium are
gure 2.3.3
rend of av
fic detection
three dete
a stronger
ed OIs gene
h target siz
onds to the
ized field i
for colocali
results of t
meaningfu
shows the
erage optic
ns; however
ction scena
influence i
erated by th
ze at 25 nm
e ratio of t
in colocaliz
ized detecti
the tradition
ul in followi
trend of t
cal signatur
r, the value
arios. This
in colocaliz
hree model
m.
the
zed
ion
nal
ing
the
res
of
is
zed
ls
42
Figure 2.3.3 illustrates that CI’s end-point and start-point are hard to
distinguish and that the CI is narrower than in non-specific and non-colocalized
detection. For the case where the targets are ϕ = 25 nm and the concentration is
E11, an optical signature ranges from OI = 1.5206 to 1.537 with an average of
1.5288 and the uncertainty is 1.1% in RCI. The RCI for this case is shown in
Table 2.1 for the detection models. It is confirmed RCI decreases with the order
from non-specific, non-colocalized, and to colocalized detection models. The
lowest uncertainty in the colocalized detection is achieved by limiting the freedom
of where target molecules are located. In other words, the distributable dimension
is relatively reduced from 3-dimension in non-specific detection and 2-dimension
in non-colocalized detection to 1-dimension in colocalized detection. Thus, the CI
is minimum and the value of optical signatures is maximum in colocalized
detection. Also, RCI slightly changes with target size.
Table 2.1 RCI at 95% CL for concentration and 2.03E11 selected as the
target concentration that produces normalized OI = 0.001 in non-specific
detection with target size of 25nm
Target concentration
Non-specific Non-colocalized Colocalized
9E 7.5023 0.7706 0.1170 10E 2.3723 0.2436 0.03695 11E 0.7118 0.07259 0.01078
2.03 x 11E 0.4963 0.05061 0.007514 12E 0.2372 0.02443 0.003717
ta
is
w
sc
in
re
ge
fo
de
an
The RC
argets of ϕ =
monotonic
while the opt
cale are alm
nterpretation
elated to th
enerating R
or non-spec
etection am
nd non-spec
Figure
CIs of the th
= 25 nm at
cally reduce
tical signatu
most identi
n based on
he resolutio
RCI = 1. The
ific, non-co
mplifies LOD
cific detectio
e 2.3.5 RCI
hree detect
the 95% CL
ed with targ
ure largely d
cal for the
the Poisson
on of biose
e LOD beco
olocalized,
D by 120 a
on.
I curves of
nm in
43
tion models
L by varyin
get concentr
decreases. T
e three dete
n process. I
ensors is d
omes 7.0E1
and coloca
and 11700
varying co
the three m
s in Figure
ng the target
ration becau
The slopes o
ection mod
In the prese
defined as t
10, 7.2E8, a
alized detec
times comp
oncentration
models.
2.3.5 are o
t concentrat
use CI sligh
of the RCI l
dels due to
ent study, L
the target
and 0.60E7,
ctions. Then
pared to no
n with targ
obtained w
tion. The R
htly increas
lines on a lo
o probabilis
LOD which
concentrati
, respective
n, colocaliz
on-colocaliz
get size = 25
ith
RCI
es,
og-
stic
h is
ion
ly,
zed
zed
5
44
In summary, colocalized detection enhances the optical signatures,
detection uncertainty related sensitivity, and LOD, compared to other detection
models, based on the probabilistic interpretation which are results obtained by the
traditional methods.
3.
A
at
sh
at
co
be
Th
or
ta
F
4 Waveleng
According to
t longer wav
horter wave
t an adva
olocalized d
enefit at a
hus, SPR se
rder to inve
arget size of
Figure 2.3.6
(a) In
gth depende
o previous re
velengths [5
elength. Alth
antage in
detection m
longer wav
ensing usin
estigate this
f 25 nm at li
6 Near field
ncident wav
ence
esearch, con
53-55]. How
hough non-
SPR sensi
may decrease
velength is
g colocalize
nature, opt
ight wavelen
d patterns
velength a =
45
nventional
wever, near
-specific an
ing at a lo
e at the sam
offset by
ed detection
tical signatu
ngth λ = 48
of same na
= 488 nm, (
SPR detecti
r-fields are
d non-coloc
onger wav
me wavelen
the weaker
n may have
ures from co
88, 633, and
anoisland a
(b) 633 nm,
ion has high
efficiently
calized dete
elength, th
ngth. This i
r near-field
e the best w
olocalized d
760 nm we
t various w
, and (c) 76
her sensitiv
localized a
ection may
he benefit
is because t
d localizatio
wavelength.
detection w
ere compare
wavelengths
60 nm.
ity
t a
be
of
the
on.
In
ith
ed.
s.
id
at
Fi
co
de
at
th
Figure
Figure 2
dentical in sp
t three diffe
igure 2.3.6
oncentration
ecreases as
t λ = 633 an
he effect of n
e 2.3.7 Hist
wavelen
2.3.8 shows
pite of diffe
ferent wave
6. On the
n and as the
the wavelen
nd 760 nm
near-field lo
tograms of
ngths at= 4
s that the o
erent wavele
elengths dem
other han
e wavelengt
ngth further
are compar
ocalization
46
normalized
488 nm, 633
overall tren
engths. The
monstrate n
nd, optical
th shifts fro
r shifts to 7
red, the diff
on optical s
d fields of n
3 nm, and 7
nds of optic
e distributio
noticeable d
l signature
m 488 to 6
760 nm. Wh
fferent shifts
signature is
nanoisland
760 nm.
cal signature
ns of near-f
differences
increases
33 nm. Opt
hen the opti
s in the grap
dominant.
ds at variou
es are almo
fields induc
as shown
with targ
tical signatu
ical signatur
aph imply th
us
ost
ced
in
get
ure
res
hat
=
nm
0.
O
stu
si
pr
ra
Figu
wavelengt
For the
25 nm, the
m)/OI(760
0108, and 0
I is highest
udy, the d
gnatures. T
robability th
atio of the
ure 2.3.8 No
ths aveleng
optical sign
e calculated
nm) = 1.67
0.0133 resp
t with the lo
distribution
Then, RCI
hat target m
volume of
ormalized O
gths ifferen
vary
natures at λ
d ratios ar
7. The RCI
ectively for
owest RCI a
of near-fie
decreases
molecules e
f target mo
47
OI of the co
nt waveleng
ying concen
= 488, 633
re OI(633 n
Is at E11 s
r λ = 488, 6
at λ = 633 n
eld strongl
in more a
exist in a sp
olecule to
olocalized d
gths λ = 488
ntrati
3 and 760 n
nm)/OI(488
hown in Fi
633 and 760
nm. Accordi
ly influenc
a localized
pecific regi
the volum
detection at
8, 633, and
m with th
nm) = 1.74
igure 2.3.9
0 nm. It is in
ing to the an
es the tren
field. The
ion S is
me of the r
t different
760 nm of
he targets of
4 and OI(6
are 0.013
nteresting th
nalysis in th
nd of optic
e geometric
defined as
region, whi
f ϕ
33
35,
hat
his
cal
cal
s a
ich
de
th
ne
ecreases and
he field cha
ear-field are
Figure
760 nm. (b
d reduces th
aracteristics
e closely rel
e 2.3.9 (a) R
b) Magnifie
he uncertain
such as in
lated to OI a
RCI at diff
d RCI at u
48
nty at a mor
ntensity and
and RCI.
ferent wave
undistinguis
760 nm.
re localized
d the total
elengths =
shable wav
d field. This
volume of
= 488, 633,
velengths λ
s suggests th
the localiz
and 760 nd
λ = 488 and
hat
zed
d
d
49
3.5 Discussion
Researchers in the field of biosensors are interested in whether a single molecule
may be detected by their systems. In this chapter, plasmonic detection has been
discussed with the probabilistic approach. Whether a nanoisland-based SPR
sensor is able to detect a single molecule using colocalized detection was studied;
the results indicate that the capacity of detectable binding is lower than fg/mm2. If
a molecule weights 100 kDa and the size of the localized field is 1 mm2, the LOD
would be a few hundred molecules under the best conditions. This implies that it
is quite challenging to detect a single molecule with nanoisland based on
colocalized detection. Nevertheless, OI conceptually signifies that colocalization
is able to further enhance LOD by optimizing the size of the target molecule and
localizing the strong near-field over the total field of detection.
Colocalized detection produces the highest optical signature and the lowest
uncertainty compared to the other detection models. However, there is a tradeoff
because, despite the advantages of colocalized detection, it takes an additional
experimental process to demonstrate colocalization. For sensing that requires
moderate detection characteristics, non-specific and non-colocalization can be an
alternative solution.
50
Chapter 3
Conclusions
Surface-enhanced SPR biosensors’ detection characteristics based on a
probabilistic model are researched. The randomness arising from near field
distribution of being produced by nanoisland increases to the need for
probabilistic approach. Non-specific, non-colocalized and colocalized detection
models were considered and calculated their detection characteristics. The
tendency of calculated OI shows proportional increase with target concentration.
It follows the previous research based on EMT. However the effect of target
concentration on uncertainty of SPR signal represented by normalized OI is
limited than that of target size. In other words, the CI is less affected by target
concentration than target size. Detection characteristics represented by the CI and
the amplitude of normalized OI were the optimum in colocalized detection. Based
on the probabilistic models, LOD of colocalized detection model was enhanced by
more 4 orders than that of non-specific detection model.
51
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국문요약
표면 플라즈몬 공명 바이오 센서의 검출 특성에 관한 확률적
추정과 플라즈몬 식각을 이용한 동시국소화 구현
표면 향상 표면 플라즈몬 공명 바이오 센서의 검출 특성이
연구되었다. 나노섬 구조에서 만들어지는 근접장 패턴은 무작위적으로
분포하기 때문에 확률적 해석의 접근을 요한다. 검출 특성 분석을 위해
확률적으로 해석된 비선택적, 비동시국소화 그리고 동시국소화 검출
모형에서 중첩 적분을 계산하였다. 공통적으로 나타난 특징으로, 중첩
적분 크기는 표적 분자 농도에 비례하며 증가하는 경향을 보였고 표적
분자 크기의 영향도 컸다. 한편, 센싱 신호의 신뢰성은 표적 분자
크기의 영향보다 표적 분자 농도의 영향을 받았다. 동시국소화 모형은
가장 큰 값의 중첩 적분과 가장 신뢰할 만한 신뢰성을 보여주었다.
비선택적 모형과 비교 시, 동시국소화 모형은 검출 한계를 10000 배
향상 시킬 수 있는 것으로 계산되었다.
.
핵심 용어: 바이오 센서, 표면 플라즈몬 공명, 표면 플라즈몬 공명
바이오 센서, 준연속 나노구조, 나노섬 구조, 확률 이론