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Taking whispering gallery-mode single virus detection and sizing to the limitV. R. Dantham, S. Holler, V. Kolchenko, Z. Wan, and S. Arnold Citation: Appl. Phys. Lett. 101, 043704 (2012); doi: 10.1063/1.4739473 View online: http://dx.doi.org/10.1063/1.4739473 View Table of Contents: http://apl.aip.org/resource/1/APPLAB/v101/i4 Published by the American Institute of Physics. Related ArticlesSingle particle demultiplexer based on domain wall conduits Appl. Phys. Lett. 101, 142405 (2012) Studies on CdS nanoparticles prepared in DNA and bovine serum albumin based biotemplates J. Appl. Phys. 112, 064704 (2012) Effect of molecule-particle binding on the reduction in the mixed-frequency alternating current magneticsusceptibility of magnetic bio-reagents J. Appl. Phys. 112, 024704 (2012) Field dependent transition to the non-linear regime in magnetic hyperthermia experiments: Comparison betweenmaghemite, copper, zinc, nickel and cobalt ferrite nanoparticles of similar sizes AIP Advances 2, 032120 (2012) Optofluidics incorporating actively controlled micro- and nano-particles Biomicrofluidics 6, 031501 (2012) Additional information on Appl. Phys. Lett.Journal Homepage: http://apl.aip.org/ Journal Information: http://apl.aip.org/about/about_the_journal Top downloads: http://apl.aip.org/features/most_downloaded Information for Authors: http://apl.aip.org/authors
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Taking whispering gallery-mode single virus detection and sizing to the limit
V. R. Dantham,1 S. Holler,1,2 V. Kolchenko,3 Z. Wan,4 and S. Arnold1,a)
1Microparticle Photophysics Lab, Polytechnic Institute of NYU, Brooklyn, New York 11201, USA2Department of Physics, Fordham University, Bronx, New York 10458, USA3Department of Biological Sciences, NYC College of Technology, Brooklyn, New York 11201, USA4Department of Physics, Hunter College of CUNY, New York, New York 10065, USA
(Received 17 May 2012; accepted 12 July 2012; published online 27 July 2012)
We report the label-free detection and sizing by a microcavity of the smallest individual RNA virus,
MS2, with a mass only �1% of InfluenzaA (6 vs. 512 ag). Although detection of such a small
bio-nano-particle has been beyond the reach of a bare spherical microcavity, it was accomplished
with ease (S/N¼ 8, Q¼ 4� 105) using a single dipole stimulated plasmonic-nanoshell as a
microcavity wavelength shift enhancer, providing an enhancement of �70�, in agreement with
theory. Unique wavelength shift statistics are recorded consistent with an ultra-uniform genetically
programmed substance that is drawn to the plasmonic hot spots by light-forces. VC 2012 AmericanInstitute of Physics. [http://dx.doi.org/10.1063/1.4739473]
Early detection of virus at ultra-low concentration is im-
portant in identification and elimination of pathogens.
Recently, spherical optical microcavities have succeeded in
detecting individual InfluenzaA virions and quantifying their
size and mass [512 ag]. The signal/noise (S/N) ratio in those
experiments would have precluded the detection of single
viruses such as polio (14 ag) or the smallest RNA virus MS2
with a mass only �1% of InfluenzaA (Ref. 1) [6 vs. 512 ag].
Herein, we report the label-free detection and sizing by a
microcavity of MS2.2 Although detection of such a small
bio-nano-particle was beyond the reach of a bare micro-
spherical whispering gallery mode (WGM) biosensor,3 it is
accomplished with ease (S/N¼ 8) using a WGM-nanoplas-
monic-hybrid (WGM-h) composed of a spherical dielectric
microcavity with a nanoplasmonic receptor at the equator
(i.e., center of the WGM ring of light).4 Unlike our earlier
work on plasmonic enhancement that used the quadrupole
mode of a nanoshell at 633 nm to sense a polystyrene (PS)
nanoparticle, and only demonstrated a wavelength shift
enhancement of �4�, the current experiments excite the
plasmonic dipole by moving the excitation into the near
infrared (780 nm), and demonstrate a much larger overall
enhancement of �70�. This allows the MS2 virus to be
detected with a modest hybrid mode Q of only 4� 105. In
what follows we will (1) describe an analytical theory for the
dipole plasmonic enhancement by a nanoshell, (2) present
our experimental results, and (3) interpret these results.
The resonance wavelength shift of a dielectric WGM
resonator due to nanoparticle entering the evanescent field
Dkr was described earlier.5 It comes down to a simple result
of 1st order perturbation theory—the fractional wavelength
shift is equal to the cycle-averaged work required for the
local field to polarize the nanoparticle Wp divided by the
cycle averaged energy within the cavity Wc; Dkr=kr
¼ Wp=Wc. This so called “reactive sensing principle (RSP)”
has been tested extensively and correctly predicts the wave-
length shift for a nanoparticle of a given polarizability
adsorbing to a microcavity’s equator. For a Rayleigh particle,
Wp may be written as Wp ¼ ð1=4ÞRe½aex� jE0ðrvÞj2, where
aex is the polarizability in excess of the surrounding medium,
and E0ðrvÞ is the amplitude of the evanescent field at the posi-
tion of the analyte. The wavelength shift generated by the
RSP is proportional to polarizability which in turn is propor-
tional to mass. Our interest is principally in detecting individ-
ual virus with masses so small that a bare cavity produces a
wavelength shift below the noise.3 For a microspherical cav-
ity, the smallest virus currently detected is InfluenzaA with a
mass of 512 ag.6 The S/N ratio for these experiments was 3,
implying that the limit of detection would be �170 ag. The
detection of MS2 with a mass of only 6 ag will require an
enhancement of >30�. The RSP indicates that enhancing the
intensity jE0ðrvÞj2 at the adsorption site relative to overall
energy in the cavity should generate a proportional enhance-
ment in wavelength shift. A dipolar plasmonic mode of a
nanoshell receptor with an inner silica core (radius r1 and
dielectric constant Es) and an outer shell of gold extending to
radius r2 has a peculiar but useful properties in this regard.
These properties are most easily revealed by using Rayleigh
theory, where simple analytical results point to the spectral
region in which the enhancement is optimized.
A Rayleigh sized gold nanoshell excited into a dipole
plasmon resonance develops an intensity at its hot spots
jEhsj2 in excess of the intensity jE0j2 that stimulates it. For
the lowest order transverse electric (TE), WGM interacting
with a Rayleigh sized nanoshell on the equator the evanes-
cent wave polarizes the shell allowing the field at the hotspot
to be written as the sum of the driving field E0 (modeled as a
plane wave) and an induced field Eind from the nanoshell.
The maximum intensity enhancement on one of the hotspots
north or south of the equator RE;max is easily evaluated from
a quasi-electrostatic model7
RE;max ¼jEhsj2
E20
¼ E0 þ EindðrhsÞE0
��������2
¼ 3 Eg
Eg þ 2Eeg
��������2
; (1)
where Eg and Ee are the dielectric constants of the gold shell
and environment (i.e., water), respectively. The easiest way
a)Author to whom correspondence should be addressed. Electronic mail:
sarnold935@aol.com.
0003-6951/2012/101(4)/043704/4/$30.00 VC 2012 American Institute of Physics101, 043704-1
APPLIED PHYSICS LETTERS 101, 043704 (2012)
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to understand this enhancement is to examine the denomina-
tor in the rightmost expression of Eq. (1). The plasmonic
dipole resonance occurs when the real part of this denomina-
tor goes to zero
Re½Eg� ¼ �2Ee Re½g�; (2)
where
Re½g� ¼ ReEsPþ Egð3� PÞ
Esð3� 2PÞ þ 2EgP
� �with P ¼ 1� r1
r2
� �3
:
For a solid gold particle for which r1¼ 0, Re[g]¼ 1, provid-
ing the well known resonance condition Re½Eg� ¼ �2Ee for
which resonance occurs in water at �540 nm and the
enhancement is j3Eg=Im½Eg�j2 �37, limited by the Im½Eg�.4At longer wavelengths such as 770 nm, Im[Eg] is much
smaller, but unfortunately the solid gold nanosphere’s reso-
nance cannot reach this wavelength. This is where a core
shell structure “out shines” the solid gold nanosphere. With
ðr1=r2Þ¼ 0.85 and for a silica core, Eq. (2) gives
Re[g]¼ 4.9, for which the dipole resonance shifts to 770 nm,
and enhancement grows to�340. Fig. 1 shows enhancement
spectra calculated from Eqs. (1) and (2) for a 50 nm diameter
core shell structures having a variety of shell thicknesses.
Although the wavelength shift enhancement of the hybrid
mode will be depleted somewhat by spatial decay of the
local field across the adsorbed virus, the interaction with a
dipole mode minimizes this effect by having an intensity
which drops off considerably slower than all other plasmonic
multipoles. As a result, the wavelength shift enhancement
for MS2 is found to be diminished by about a factor of 3
from the surface intensity enhancement, which easily
exceeds our goal of 30�.
All experiments were carried out in a poly-dimethyl silox-
ane (PDMS)-glass microfluidic cell3 held at 25.00 6 0.01 �C.
Contained within were a silica microsphere (radius R� 45 lm)
and a tapered fiber for evanescently coupling light into a
WGM of the microsphere as shown in Fig. 2. The laser driving
the guided wave in the fiber was an isolated distributed feed-
back laser (DFB) oscillating near 780 nm and tuned in wave-
length using a saw tooth current supply. The wavelength of a
WGM signature dip in transmission through the fiber at kr was
detected by a photodiode and measured using a LabVIEW run
multi-point parabolic fit. In what follows we describe the
attachment of the plasmonic structure to the microsphere reso-
nator, and the preparation of the MS2 virus.
Assembling the WGM-h was accomplished by using
carousel light forces8 generated by the WGM of the bare
microsphere (inset in Fig. 2).4 In particular, the gradient
force draws a gold nanoshell (r1� 60 nm, t� 12 nm from
Nanospectra Biosciences) to the equator of a silica micro-
sphere.4 Since both the silica microsphere and gold nano-
shells have negative charge on their surfaces, nanoshells are
repelled from the microsphere’s surface unless there are
strong WGM gradient forces.8 To aid these forces salt is
added (NaCl at 20 mM) to increase the conductivity of the
solution and thereby reduce the range of the electrostatic
fields emanating from the silica-water and gold-water inter-
faces. Attachment of a single nanoshell, likely at a silica
defect site,9 was verified for all of the WGM-h assemblies
through light scattering and a simultaneous shift in resonance
wavelength. This was followed within seconds by washing
for 5 min with DI water to eliminate all suspended nanoshells
from solution. During this period, the laser was turned off.
Attachment of only a single shell was facilitated by working
with a small shell concentration of 1.3 fM and carefully tim-
ing the washing. In each assembly, the nanoshell was veri-
fied to remain attached in the same apparent place without
the aid of the optical gradient force. Next, we will describe
the preparation of the MS2 virus and its injection into the
microfluidic cell.
MS2 was prepared by inoculating E. coli bacteria with
viable virus stock [American Type Culture Collection
(ATCC), Manassas, VA], harvesting the amplified number
of viruses, and dispersing them in saline solution. The result-
ing solution was tested by dynamic light scattering and found
to show no evidence of clustering. Since MS2 is genetically
programmed, it is perfectly uniform in size compared with
artificial nanoparticles such as PS or gold.
After identifying a mode of the WGM-h from a dip in
transmission through the fiber, MS2 viruses were then
injected so that their resulting concentration in the cell was
330 fM, with the cell brought to a salt concentration of
60 mM; again to aid adsorption.
FIG. 1. Enhancement in the WGM-hybrid wavelength shift calculated
quasi-statically for an infinitesimally small particle binding at T on a gold
nanoshell 50 nm in diameter having thicknesses ranging from the full radius
(i.e., solid gold) down to 3.75 nm.
FIG. 2. Microfludic WGM biosensor with the image of an assembled
WGM-h resonator.
043704-2 Dantham et al. Appl. Phys. Lett. 101, 043704 (2012)
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Before recording dip trace characteristics of the WGM-h,
we adsorbed virus onto a bare resonator. A wavelength shift
binding curve was recorded with characteristic of nonspecific
adsorption with no detectable steps.3 Next, we carried out the
same experiment with the bare resonator replaced by a WGM-
h. A portion of a typical dip trace is displayed by the upper
trace (black) in Fig. 3(a). In this figure, we can see clear steps
due to adsorption of individual MS2 virus on the nanoshell.
The underlying slope matched that of adsorption by a bare
silica resonator at the same time following injection within
the experimental noise. This clearly shows the ease with
which one can distinguish adsorption on plasmonic particle
from its silica substrate. The plasmonic enhancement is
entirely responsible for this contrast; adsorption of individual
MS2 virus at the equator of the bare resonator of the same
radius produces a theoretical shift �0.25 fm, which is well
below the r.m.s. background noise of 2 fm (lower trace). The
16 fm wavelength shift step shown near the center of the upper
trace represents an enhancement of �64�. In this particular
experiment, 28 total steps were recorded after the first over an
interval of 3200 s. The largest of these was 17 fm for an
enhancement of 68�. The experiment was repeated with dif-
ferent WGM-h assemblies with similar results.
In Fig. 3(b), the 28 events are represented by a histo-
gram. This histogram bares little resemblance to experiments
with artificial nanoparticles or InfluenzaA virus. When light
forces draw nanoparticles to the equator of a bare cavity we
expect the distribution of wavelength shifts to reflect the dis-
tribution of mass, which is typically Gaussian (i.e., bell
shaped) for artificial nanoparticles. This is also the case for
InfluenzaA, since the cell membrane coat on the virus’ cap-
sid can vary from one virus to another. The histogram in Fig.
3(b) simply shows more events as the wavelength shift is
reduced followed by non-symmetric termination for smaller
resonance shifts. We believe this is because MS2 is pro-
grammed by its viral genome to be essentially uniform, and
because all of the 28 events are on the same nanoshell. The
increase in the number of events having smaller shifts occurs
because only one virus can occupy each of the two hot spot
centers (e.g., T in Fig. 1), and as the polar angle h increases
along the plasmonic sphere the geodesic parallels increase in
circumference as the sinðhÞ, thereby enabling more particles
to fit at larger angles.
In addition, increasing polar angle for a dipole mode
leads to a lower surface intensity and therefore a smaller
shift. Where the light force becomes too small to compete
for adsorption events the distribution terminates. The differ-
ence between this behavior and a Gaussian is clear, distinct,
and associated with light induced adsorption10 of an ultra-
uniform virus on a single spherical nanoshell.
A key question is whether the size of the virus can be
extracted from the measured signal. The answer will be af-
firmative for the largest wavelength shift. However, there are
some limitations to obtaining a simple analytic expression
imposed by using commercial nanoshells. Unfortunately, a
shell having an outer circumference to wavelength ratio of
2pr2=k ¼ 2pð71:5 nmÞ=780 nm ¼ 0:58 can be treated as a
Rayleigh particle only in a gross approximation. This led us
to simulate the intensity using the finite element method
(FEM, COMSOL). Fig. 4(a) shows the result at 780 nm for a
shell with a core radius of 60 nm and a shell thickness of
11.5 nm having a virus (refractive index¼ 1.5, typical of vi-
rus) of radius a¼ 12.5 nm on its point of highest intensity.
The dipole pattern is apparent along with some distortion
caused by the electromagnetic wave changing phase across
the structure. This causes the highest intensity point to be
slightly in the forward direction. The enhancement we are
interested in is the wavelength shift for the virus seated on
the gold hot spot to that on the silica equator Dkg=Dks which
from the RSP is the polarization energy of the virus in con-
tact with the nanoshell in comparison with that on the silica
equator. This ratio requires integration over the body of the
virus.
We have modeled this enhancement as a function of the
virus radius a using FEM and found an analytical expression
using the shell parameters in Fig. 4(a)
FIG. 3. (a) Shift of resonance wavelength above 780.674 nm of a WGM res-
onator R¼ 45 lm having a gold nanoshell attached at its equator due to the
adsorption of MS2 viruses (upper trace). The lower trace shows the back-
ground without MS2 or the gold nanoshell (r.m.s. noise 2 fm). Insets show
the recorded spectrum SD for the hybrid resonator (Q� 4� 105) and an illus-
tration of MS2 virus (radius a� 13.6 nm). (b) Step number statistics for all
of the steps recorded over 3000 s.
043704-3 Dantham et al. Appl. Phys. Lett. 101, 043704 (2012)
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Dkg=Dks ¼ A½r2=ðr2 þ faÞ�6; (3)
with A¼ 155.56 and f¼ 0.76. The wavelength shift ratio
reaches 155.56 for an infinitesimally small adsorbate and
falls rapidly as the adsorbate increases in size [Fig. 4(b)] due
to the attenuation of the near field with range. Eq. (3) is the
key to finding the size of the virus from the experimental
wavelength shift Dkg but requires working out Dks. Fortu-
nately, the latter has been worked out earlier and is propor-
tional to the adsorbate volume; Dks / a3.6 With the
appropriate expression from Ref. 6 incorporated into Eq. (3),
we arrived at a solution for the virus size
a � G
1� 2Gfr2
� � ; where G ¼ R5=6
D1=3A1=3k1=6r
ðDkgÞ1=3; (4)
R is the microsphere radius, and the dimensionless parameter
D which includes the dielectric properties of the virus,
microsphere and water has a value6 of 1.50. For the largest
measured shift of 17 fm, we obtain a viral radius of 13.3 nm
which is in excellent agreement with a neutron diffraction
analysis of MS2 for which the reported outer radius is
13.6 6 1.0 nm.2
In this paper, we have demonstrated single particle
detection of the smallest RNA virus by using a microcavity-
hybrid having a modest Q factor (4� 105), and used the
RSP to determine its size from the largest wavelength shift
signal. Detection was possible because of an amplified wave-
length shift signal (17 fm) due to a nanoshell plasmonic
dipole enhancement of �70�. This amplification allowed
detection in the presence of r.m.s. noise of 2 fm. As a conse-
quence of this noise level, our limit of detection from Eq. (4)
is a � 5:7 nm (0.4 ag), below the size of all known viruses.
By contrast with our wavelength shift of 17 fm for MS2,
the detection of higher refractive index polystyrene particles
of comparable size on a ultra-high Q microtoroid (�108)
only produced a signal of 0.4 fm, and required a noise reduc-
ing reference interferometer to lower the r.m.s. noise to
0.2 fm.11 Combining the two advances of plasmon enhance-
ment and reference interferometry should prove very power-
ful for the next challenge, label-free single protein detection.
We have estimated the limit of detection using Eq. (4) for
our experimental setup by adding a reference interferometer
of our own and it is found to be a� 2 nm, a dimension below
that of a typical protein.
Further sensitivity can be gained by using plasmonic
structures with additional lightning rod enhancements, such
as nano-rods.12 We have merely scratched the surface of
what is likely to be possible.
The authors thank the NSF for supporting this work
(Grant No. CBET 0933531). We also wish to thank N. L.
Goddard of Hunter College for making her student Z. Wan
available for preparing the MS2 virus.
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FIG. 4. (a) FEM simulation of the park-
ing of MS2 virus at one of the dipole
lobes of a plasmonic nanoshell with
60 nm inner core radius of silica and
11.5 nm shell thickness of Au. The field
intensity at the point of contact grows to
just over 252� the field of the 780 nm
TE mode as indicated by the rainbow
scale on the left. (b) shows the enhance-
ment in the wavelength shift calculated
from FEM for different a values, fitted
with an analytical expression (Eq. (3)).
043704-4 Dantham et al. Appl. Phys. Lett. 101, 043704 (2012)
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