Peer Review File
Reviewers' comments first round:
Reviewer #1 (Remarks to the Author):
The manuscript by Kumar et al entitled “Overcoming Evanescent Field Decay Using 3D-Tapered
Nanocavities for On-Chip Targeted Molecular Analysis” discusses how using an “open,” tapered,
metal-insulator-metal waveguide enables the formation of a rather uniform, strongly enhance
electric field. Due to the “openness” of the cavity the enhanced electric field is accessible to e.g.
analyte molecules and can therefore be used as a sensing platform. According to the Authors, the
key breakthrough reported in the submitted manuscript is the creation of an enhanced electric field
which is very uniform. Hence, biosensing with this device is not limited by the inhomogeneous field
and the device works, according to the Authors, just as well for a short fluorescent molecule (tag) or
a long one. The Authors first present a numerical analysis of the field enhancement in the device and
then use fluorescent labels of different length/height to demonstrate uniform fluorescence
enhancement within the whole MIM waveguide.
In general, I find little that is wrong with this manuscript/work. It is pretty well written and easy to
follow/understand, however, certain statements are a little confusing and/or lack precision. In my
opinion the only drawback of this manuscript is that the concepts which are its basis are already very
well studied. There are two key parts to making this device – the energy concentrator (taper) and an
appropriately sized final MIM waveguide – are not unique. Perhaps the combination of the above
mentioned elements in this particular realization is unique, but there are no new physical concepts
involved in this work.
The “overcoming of the evanescent field decay” is done using a well-studied MIM waveguide. Its
“[non]evanescent field” in the final, narrow part is the same practically regardless of what how the
energy is coupled into it, neglecting any transient effects. It is simply given by the geometry. Such
MIM waveguides have been presented as biosensors before by, e.g. Dell’Olio et al (Design of a New
Ultracompact Resonant Plasmonic Multi-Analyte Label-Free Biosensing Platform, Sensors 17, 1810
(2017)), as well as others.
I am puzzled by the “tip-length effect” phrase. The “cavity” (and here, in contrast to the Authors, I
use “cavity” to refer only to the narrow, final part) is merely a MIM waveguide with a finite length so
that a Fabry-Perot resonance can be used to modulate the field intensity in the hot spot (note, that
the hot spot becomes confined in the third dimension).
Also, while I understand the reason behind using the 3D taper, I do not think that the tapered part is
all that important in achieving “3 to 4 times greater u<sub>A</sub> (field uniformity)” (as stated on
page 4). The uniformity is ensured by the MIM waveguide, while the role of the 3D taper is to
compress the incident field (from the large MIM waveguide).
Reviewer #2 (Remarks to the Author):
Reviewer Blind Comments to Author
Manuscript Number:
Title: Overcoming Evanescent Field Decay Using 3D-Tapered Nanocavities for On-Chip Targeted
Molecular Analysis
Recommendation: Reject (Publish in a different journal)
This paper presents a 3D tapered nanocavity which provides the enhancement factor of 1100 and
the 40-fold fluorescence enhancement uniformity compared to a conventional bowtie nanoantenna.
They fabricated the 3D tapered nanocavity device by using focused ion beam (FIB) milling and gold
deposition on the SiO2/Si substrate. They also measured fluorescence signal (AF-750) from the
biotin-streptavidin binding using the 3D tapered nanocavity under the near-infrared LED excitation.
While the paper contains some of the necessary data, it is missing experimental results and
discussion for why a 3D tapered nanocavity was made and how it is substantially better than all
previous works.
1. The novelty of a 3D tapered nanocavity is not clear. The 3D tapered nanocavity structure is
basically same as the previously reported 3D tapered metal-insulator-metal waveguide (Choo et al.
Nature Photonics, 2012). Please clearly deliver the novelty of this structure (3D tapered nanocavity)
compared to previous SPP or LSPR sensors in terms of device and performance (E-field
enhancement, fluorescence signal enhancement, etc.).
2. There are parameter studies using the FDTD calculation in the supporting information. However,
overall experimental studies are insufficient (Fig. 2 and Fig. 3). They only show simple experimental
results for the 3D tapered nanocavity, the tip only structure, and the conventional bowtie
nanoantenna. It would be better to show and discuss experimental results for the 3D tapered
nanocavities with different physical dimensions as shown in the numerical results.
3. The device coated with biotin can capture the streptavidin high concentration. What is the limit of
low concentration?
4. They demonstrated specific and single molecular capture using the nanocavity which has similar
dimensions with the target molecule. In this paper, they capture the IgG antibody (length 15~20 nm)
using the nanocavity with 20 nm x 20 nm size. If this single molecule capture depends on the size of
nanocavity and the target molecule, additional experimental results for different sizes (at least two
or three) can support the principle of single molecule capture.
5. In addition, the authors should carefully proofread the manuscript. For example,
- Please insert scale bars for all figures of 2D E-field intensity profiles.
- In Figure 3, the alphabets (numbers) for sub-figures and descriptions don’t match. In addition, it
would be better to effectively display their results to place the results under the corresponding
schematic illustrations for effective delivery of results.
- On page 4, Finite-Domain Time-Difference is incorrect terminology for FDTD. Finite Difference Time
Domain (FDTD) is correct terminology.
- In Figure S4b, the direction of arrow is strange. It should be in the opposite direction.
The authors present an interesting concept to improve the fluorescent enhancement intensity using a
plasmonic nanotapered cavity.
The concept is novel,
and will
be of interest to the biosensing research
community, and also to
other br
anch
es
of photonics
reserach
.
The work is very thorough, and the results
are convincing. However, the
manuscript
is not written very well, and
a more
balanced view of the
importance of the work should be presented
.
Comments regarding writing and presentation of results
Some of the examples
about the writing and organization are described below
a) In page 3 of the manuscript the authors mentioned
“This
engineered hotspot within the device tip was used for highly efficient and uniform fluorescent
enhancement
–
the enhancement factor up to 1100 times and 40x improvement in uniformity compared to
a bowtie antenna
-
....”
Reading this, my impression was the 110
0 enhancement was compared to the bowtie antenna. But from
page 6
of the Supplementary Materials, I understand that the enhancement is compared to a flat silica
surface. Such ambiguity about one of the central claim
s
of the manuscript should be avoided.
b
)
In Figure 1 the authors show the schematic of a three dimensional structure and in Figure 2 they the
discussed the field distribution over
its
cross section. But since the coordinate system was not clearly
defined in these figures, one has to go through
the text and caption trying to confirm if x and y are
in the
lateral and vertical direction
s
.
c) According to the caption
s
,
Fig. 2(c) and (d) shows
E
2
enhancement
profiles
. I believe these are plots
of magnitude of
E
2
from simulation, and not enha
ncement with respect to some other structures.
If it was
really enhancement (ratio of field for structure under consideration with respect to field of some other
structure) the quantity on the color bar of Fig. 2(c) and y axis of Fig. 2(d) should be number
s
and not
E
2
.
Presentation of data in this way may mislead the reader.
d) In page 7
of the manuscript
the authors mention
“The net enhancement experienced by a
fluorophore
is defined by the EM field intensity and the quantum
efficiency enhancement th
at are discussed in Section S4, Figures S8
-
1 and S8
-
2
.
”.
However, in S4 the authors use the term “quantum yield gain” instead of “quantum efficiency
enhancement”. Why not use the term “quantum yield gain” in the manuscript too?
Section S4 has the followi
ng description regarding quantum yield gain
“
A dipole source was placed at varied locations along the x or y axis to monitor the radiation from the
dipole and the tip structure.
r
was obtained by measuring the transmission through a closed box around a
dipole source (Eq S4
-
1).
0
0
P
P
struture
r
“
There was no discussion at all about what
r
,
0
,
P
structure
,
P
0
stand for.
The entire manuscript should be reviewed carefully to i
mprove clarity
.
Comments about claims made in the manuscript
I agree with the authors that nonuniformity of field in a bowtie antenna is a fundamental limitation, and it
is commendable that the authors attempted to address this issue. However, the solutio
n they proposed comes
with many other challenges.
In a real life application, one would need to have an array of these devices to
do analysis of a finite volume of sample. Hand
l
ing such a case with bowtie antennas are relatively straight
forward.
For examp
le, it is relatively easy to have
collection of many
bowtie
antennas on a substrate which
will provide many hot spots. The
entire
array of bowtie antenna
s
can be illuminated with a very simple
scheme
. The scheme the authors pro
posed on the other hand,
will
have
a much large
r
footprint for the same
number of hotspots and
will
need a more complicated tapered structure
s
for light coupling to the tip.
This does not mean the contributions the authors made are not useful. But they
should recognize and clearly
di
scuss the limitations of their design
, and provide some suggestions about how future work may address
some of these limitations
.
Response to the reviewers:
We indicate the reviewers’ comments in bold, text
from the manuscript in italics, and edited text in
underlined italics.
Reviewers' comments: Reviewer #1 (Remarks to the Author):
The manuscript by Kumar et al entitled “Ov
ercoming Evanescent Field Decay Using 3DTapered
Nanocavities for On-Chip Targeted
Molecular Analysis” discusses how using an “open,” tapered,
metal-insulator-metal waveguide enables the fo
rmation of a rather uniform, strongly enhance
electric field. Due to the “openness” of the cavity
the enhanced electric field is accessible to e.g.
analyte molecules and can therefore be used as a sensing platform.
According to the Authors, the key breakthrough
reported in the submitted manuscript is the
creation of an enhanced electric field which is very
uniform. Hence, biosensing with this device is
not limited by the inhomogeneous field and the devi
ce works, according to the Authors, just as well
for a short fluorescent molecule (tag) or a long one. The Authors first present a numerical analysis
of the field enhancement in the device and then
use fluorescent labels of different length/height to
demonstrate uniform fluorescence enhancement with
in the whole MIM waveguide. In general, I
find little that is wrong with this manuscript/work. It is pretty well written and easy to
follow/understand, however, certain statements are
a little confusing and/or lack precision. In my
opinion the only drawback of this manuscript is
that the concepts which are its basis are already
very well studied.
Response:
We thank the reviewer for their constructiv
e comments and encouraging remarks about our
manuscript. We have added discussi
on and supporting evidence to further elucidate the major challenge
in the field that is solved by our device, along with
the design novelties relevant to solving this challenge.
We have also added further experi
mental data and revised some of the calculations correcting some
errors. We hope that our answers and revised
discussion satisfy the reviewer’s concerns.
While plasmon enhanced fluorescence has been discu
ssed as a potential revolutionary technique for
sensitive detection of single or few biomolecules, r
estrictions associated with size and placement of
molecules have prevented this method to be widely u
sed in the field. Various reports have outlined the
impact of changing fluorophore distance with resp
ect to efficiency of plasmonic enhancement on a
nanoplasmonic substrate (
References 8 -14
in the main manuscript). Puchkova
et al.
also discuss the main
challenge preventing the widespread application of
a dimer nanoantenna (DN) plasmonic device for
fluorescence enhancement as “positioning the biologica
l assay of interest at the DN’s hotspot”.
[Puchkova, Anastasiya, et al. Nano letters 15.12 (2015): 8354-8359.]
Herein we have demonstrated that an open linear
3D-tapered geometry can uniquely solve this long-
standing challenge associated with plasmon enhanced
fluorescence, while providing one of the highest
fluorescence enhancements reported by plasmonic nanostructures. {Table S1}.
We introduce the following c
oncepts in this manuscript:
a)
Fabrication scheme for manufacture of a linear 3D taper nanocavity accessible to fluid flow and
capture of molecules (Figure 1).
b)
Analyze the coupling of fluorophores to the confin
ed field within the narrow tip at the end of
taper. We demonstrate that the energy confinem
ent generated by 3D-tapered waveguides can be
efficiently used for enhancement of emission from molecules by analyzing the change in quantum
yield (the radiative decay rate of fluorophores) w
ithin the tip leading to further improvement in
fluorescence (Figure S8 1-3)
c)
First realization and demonstration of the hi
ghly uniform fluorescence emission enhancement
within the 3D-tapered device for a range of
molecules overcoming size and distance-dependent
restrictions (Figure 4).
The introduction and discussion section have now been modified to clarify these aspects.
Page 3: “
Herein, we demonstrate that 3D-tapered gap pl
asmon nanocavity can o
vercome a long-standing
limitation for plasmon-enhanced fluorescence de
monstrating powerful emission enhancement
independent of the size or position of the molecules within the nanocavity
. The 3D-taper results in
confinement of the electromagnetic field collected th
roughout the body of the device into a tiny cavity ~
3300× smaller in volume for an |E|
2
enhancement close to 500. Furtherm
ore, our analysis reveals that the
3D-taper geometry improves the coupling of molecula
r emitters to the electromagnetic field, delivering
up to 8-28% improvement in the radiative decay ra
tes and thus leading to even stronger enhancement of
fluorescence. We specifically trap and observe single
antibody molecules or
arrays of molecular
assemblies within the device, as well as detect low
concentration (10 pM) prot
ein molecules diffusing in
solution. Significantly, optimizing the taper angle and a
narrow final tip geometry of the device results in
generation of a highly uniform electromagnetic field
volume at the hotspot (40× improvement in
uniformity compared to a bowtie nanoantenna). Combi
nation of the strong electromagnetic confinement,
powerful coupling of the emitter to the confined field and a homogenous electromagnetic field volume
result in obtained enhancements around
2200 (enhancement figure of mer
it ~ 260) for molecular heights
ranging from few angstroms to 20 na
nometers, which can be further extended to 50 nanometers using the
current design approach. The enhancement factor (EF)
was calculated with respect to non-enhancing
silica substrate such as glass chips commonly u
sed in bioassays and enhancement figure of merit
quantifies device performance independent of the fluorophore quantum yield
18
. Overcoming the molecule
placement limitation for plasmonic enhancement of fl
uorescence such as presented in this manuscript can
finally allow this technique to be reliably and wide
ly applicable for a broad range of biological assays
including complex mo
lecular assemblies.
”
Page 9: “
We demonstrate 3D-tapered gap
plasmon nanocavities which provide one of the highest
enhancement of fluorescence obtained by plasmonic n
anostructures (EF: ~ 2200 with figure of merit ~
260), independent of the size of the molecular assemb
lies used in the assay.
Overcoming molecule size
and placement-dependent extreme variation in plas
monic fluorescence enhancement has been a major
challenge restricting widespread application of th
is method in bioassays. The nanostructure geometry
presented in this manuscript demonstrates a way
to overcome this limitation thereby improving the
consistency and range of plasmon-enhanced emi
ssion for diversely sized assembly of molecules.
Simultaneously, we also demonstrate capture and visua
lization of single antibodies at the tip as well as
sensing of proteins at low concentrations (10 pM).
These advantages can be readily transferred towards
applications such as highly sensitive biosensing us
ing molecular labels of varying sizes and analysis of
single molecules or tightly controlled arrays of molecu
les for protein orientation, protein function, and
biological polymer formation studies
32-34
.”
There are two key parts to making this devi
ce – the energy concentrator (taper) and an
appropriately sized final MIM waveguide – are not unique. Perhaps the combination of the above
mentioned elements in this particular realization
is unique, but there are no new physical concepts
involved in this work. The “overcoming of the evanescent field decay” is done using a well-studied
MIM waveguide. Its “[non]evanescent field” in th
e final, narrow part is the same practically
regardless of what how the energy is coupled into it, neglecting any transient effects. It is simply
given by the geometry.
Response
: The challenge we solve in this manuscript is
specifically a device which provides one of the
highest plasmonic enhancements obtained using plasmoni
c nanostructures (Table S1), independent of the
size of the molecules utilized
in the assay (Figure 4).
While MIM waveguides have been discussed previously
as energy concentrators, efficient fluorescence
enhancement within a plasmonic nanocavity depends on bo
th the energy concentration at the hotspot and
the coupling between the emitter and the electromagnetic field [1-3].
[1] Lakowicz, Joseph R. "Radiative decay engin
eering 5: metal-enhanced fluorescence and plasmon
emission." Analytical biochemistry 337.2 (2005): 171-194.
[2] Chikkaraddy, Rohit, et al. "Single-molecule strong coupling at room temperature in plasmonic
nanocavities." Nature 535.7610 (2016): 127.
[3] Mack, David L., et al. "Decoup
ling absorption and emission process
es in super-resolution localization
of emitters in a plasmonic hotspot."
Nature communications 8 (2017): 14513.
In addition to introducing the fabr
ication scheme for an open, linearly
3D-tapered device, which allows
molecule delivery to hotspot we have also analy
zed the coupling of emitters within the hotspot, and
realized the specific benefits provided by the tapere
d geometry. We found that the radiative decay of the
fluorophore was improved by a further 8-28% due to the 3D-taper.
The concepts that are newly realized in our manuscript are:
a)
Fabrication scheme for manufacture of a linear 3D taper cavity accessible to fluid flow and
capture of molecules (Figure 1).
b)
Analyze the coupling of fluorophores to the confin
ed field within the narrow tip at the end of
taper. We demonstrate that the energy confinem
ent generated by 3D-tapered waveguides can be
efficiently used for enhancement of emission from molecules by analyzing the change in quantum
yield (the radiative decay rate of fluorophores) w
ithin the tip leading to further improvement in
fluorescence (Figure S8 1-3)
c)
First realization and demonstration of the em
ission enhancement uniformity within the 3D-
tapered device for a range of molecules (Figure 4).
We discuss in the manuscript:
Page 2 “
While several reports have discussed
novel nanoscale geometries that improve the confinement of
EM fields leading to strong fluorescence enhancements
15-18
, engineering a hotspot that resolves the
distance challenge between the emitter and the
nanostructure surface has remained elusive
.
Powerful fluorescence enhancement within a nanostruc
ture independent of variation in molecule size and
position can be expected to rely on several important fa
ctors: a) strong confinement of electromagnetic
field b) powerful coupling of the emitter to the field for enhancement of emission and c) a hotspot
geometry which generates uniform electromagnetic field
distribution. At the same time, the hotspot
geometry needs to be large enough for commonly u
sed protein-protein binding assays (i.e. larger than
antibodies, ~ 15 nm). Metal-insulator-metal (MIM)
structures utilizing surface-plasmon-polariton (SPP)
propagation have been known to enable
efficient confinement of EM energy
19-22
. Specifically, waveguides
with a 3D taper that rely on adiabatic compression of the SPP mode inside the MIM gap have been
shown to provide extreme volumetric
nanoscale confinement of EM fields
23-27
. While extremely promising
for energy confinement and transfer, the potential
of 3D-tapered designs for coupling with fluorescent
emitters and bioassays had remained unrealized primarily
due to closed monolithic structures which
prevented molecular integration with
these devices. Furthermore, as electromagnetic field intensity on a
plasmonic surface is maximum at the metal-dielectric
interface, close packing of multiple metal-dielectric
interfaces such as in very thin
metal-insulator-metal (MIM) gaps, can result in integration of these
multiple field profiles within the gap creating a mo
re homogeneous field distribution. Therefore, a 3D-
tapered structure provides unutilized
potential towards these goals, for
enabling confinement of a large
amount of incident electromagnetic energy into a tiny MIM gap. In order to take advantage of the
previously known as well as unexplored abilities of 3D
-tapered MIM devices, we designed and fabricated
a fluidic channel-like 3D-tapered gap plasmon nanocav
ity, allowing ready access of the nanostructures
to molecules in solution (Figure 1a).”
The reviewer rightly suggests that homogeneous field di
stribution within the cavity is primarily due to the
geometry of the tip at the end of the waveguide. Ho
wever, the linear 3D-taper is necessary for efficient
volumetric confinement of electromagnetic field into
this tip, which has a volume ~3300 times smaller
than the initial device body. Light collected throughout
the body of the device is squeezed efficiently into
the hotspot as a result of the 3D taper and we obt
ain a combination of powerful electromagnetic field
confinement with uniform field distribution (both are
important for an efficient sensing device). Figure 2e
shows that the 3D taper helps improve the energy c
onfinement to about an order of magnitude higher
compared to a 2D-tapered tip.
Such MIM waveguides have been presented as biosensors before by, e.g. Dell’Olio et al (Design of a
New Ultracompact Resonant Plasmonic Multi-Anal
yte Label-Free Biosensing Platform, Sensors 17,
1810 (2017)), as well as others.
Response:
The reference indicated by the reviewer uses a plasmonic Bragg grating and uses SPR-based
refractive index sensing for detection of molecular
binding. The device geometry, analytical method and
motivation is very different compared to our manuscript.
The detection region in the reference article is compos
ed of Bragg gratings with
width 90 nm and height
100 nm. Due to the wider cavities the electromagnetic
confinement would be weaker and the hotspots
reside at the corners of the structures as seen in Fi
gure 5 (as opposed to merging of hotspots due to narrow
cavities and resultant homogeneous field distribution in our tips).
Crucially, the reference manuscript focuses on SPR-b
ased local refractive index change, where the
sensitivity is dependent on the net mass of molecule
s adhering to the surface and the problems of emitter
coupling, fluorophore distance and position are not a
pplicable. SPR-based refractive index sensing is a
commonly used method using commercial tools (Bia
core, GE Healthcare Life Sciences, USA) and
predominantly utilized for analysis of binding
kinetics coefficients between proteins.
The challenge we solve in this manuscript is a
device which provides extremely efficient fluorescence
enhancements, and even more significantly - the abilit
y to provide this enhanc
ement independent of the
size of the molecules utilized in
the assay. While various nanostructu
res and MIM waveguides have been
shown as energy concentrators and biosensors in the p
ast, none of them have demonstrated their ability to
solve this fundamental challenge
associated with plasmon-enhanced fluorescence (to the best of our
knowledge).
I am puzzled by the “tip-length effect” phrase. The
“cavity” (and here, in cont
rast to the Authors, I
use “cavity” to refer only to the narrow, final part)
is merely a MIM waveguide with a finite length
so that a Fabry-Perot resonance can be used to mo
dulate the field intensity in the hot spot (note,
that the hot spot becomes confined in the th
ird dimension). Also, while I understand the reason
behind using the 3D taper, I do not think that the ta
pered part is all that important in achieving “3
to 4 times greater u<sub>A</sub> (field uniformity)” (as stated on page 4). The uniformity is
ensured by the MIM waveguide, while the role of
the 3D taper is to compress the incident field
(from the large MIM waveguide).
Response:
We agree with the reviewer that the major role
played by the 3D-taper is to compress the field
into the 20 nm wide tip, thus providing about 7-
times higher electromagnetic field confinement as
compared to 2D-tapered cavity and approximately
50-times higher confinement compared to 20 nm tip
alone (Figure 2e). The uniformity is primarily a result of geometry of the narrow tip region, as even a tip
(with no taper) by itself provides less variation in el
ectromagnetic field within the cavity compared to a
bowtie structure (Figure 2e). However, our analysis
also shows that tapered structures do see a further
improvement (3-4×) in field uniformity compared to devices with no taper (Figure 2e).
The width of the tip (final narrow cavity) is maintain
ed at 20 nm. We modulate the length of the tip and
observe that shorter tip (20 nm being the short
est) results in the highest electromagnetic field
enhancement as well as fluorescence enhancement (Figures 2f, 2g, 3c, 3d, S3, 4e).
Reviewer #2 (Remarks to the Author):
Reviewer Blind Comments to Author
Manuscript Number: Title: Overcoming Evanescen
t Field Decay Using 3D-Tapered Nanocavities
for On-Chip Targeted Molecular Analysis
Recommendation: Reject (Publish in a different journal)
This paper presents a 3D tapered nanocavity wh
ich provides the enhancement factor of 1100 and
the 40-fold fluorescence enhancement uniformit
y compared to a conventional bowtie
nanoantenna. They fabricated the 3D tapered nano
cavity device by using focused ion beam (FIB)
milling and gold deposition on the SiO2/Si subs
trate. They also measured fluorescence signal
(AF-750) from the biotin-streptavidin binding us
ing the 3D tapered nanocavity under the near-
infrared LED excitation. While the paper contains
some of the necessary data, it is missing
experimental results and discussion for why a 3D
tapered nanocavity was made and how it is
substantially better than all previous works.
Response:
We thank the reviewer for their constructiv
e comments which have helped us improve the
depth of description and clarity in our manuscrip
t. We have added several experimental data
recommended by them as well as discussion pertaining
to the novelty and application of the 3D-tapered
cavity. We have also revised the enhancement calculati
ons adding new analysis and correcting errors. We
hope that our revisions answer their concerns satisfactorily.
1.
The novelty of a 3D tapered nanocavity is not
clear. The 3D tapered nanocavity structure is
basically same as the previously reported 3D ta
pered metal-insulator-metal waveguide (Choo et
al. Nature Photonics, 2012). Please clearly deliver
the novelty of this structure (3D tapered
nanocavity) compared to previous SPP or LSPR
sensors in terms of device and performance (E-
field enhancement, fluorescence signal enhancement, etc.).
Response
–
We have now expanded on the novelty in th
e manuscript and added comparisons with
respect to other devices presented in the past (Table S1).
The challenge we solve in this manuscript is speci
fically a device which provides one of the highest
enhancements obtained using plasmoni
c nanostructures (Table S1), and even more significantly - the
ability to provide this enhancement independent of
the size of the molecules utilized in the assay
(Figure 4).
While MIM waveguides have been discussed prev
iously as energy concentrators, efficient
fluorescence enhancement within a plasmonic nanocavit
y depends on both the energy concentration
at the hotspot and the coupling between the
emitter and the electromagnetic field [1-3].
[1] Lakowicz, Joseph R. "Radiative decay engin
eering 5: metal-enhanced fluorescence and plasmon
emission." Analytical biochemistry 337.2 (2005): 171-194.
[2] Chikkaraddy, Rohit, et al. "Single-molecule strong coupling at room temperature in plasmonic
nanocavities." Nature 535.7610 (2016): 127.
[3] Mack, David L., et al. "Decoupling absorp
tion and emission processes in super-resolution
localization of emitters in a plasmonic hots
pot." Nature communications 8 (2017): 14513.
In addition to introducing the fa
brication scheme for an open, linearly 3D-tapered device, which
allows molecule delivery to hotspot we have al
so analyzed the coupling
of emitters within the
hotspot, and realized the specific benefits provided by the tapered geometry.
The concepts that are newly realized in our manuscript are:
a)
Fabrication scheme for manufacture of a linear 3D taper cavity accessible to fluid flow and
capture of molecules (Figure 1).
b)
Analyze the coupling of fluorophores to the confin
ed field within the narrow tip at the end of
taper. We demonstrate that the energy confinem
ent generated by 3D-tapered waveguides can be
efficiently used for enhancement of emission from molecules by analyzing the change in quantum
yield (the radiative decay rate of fluorophores) w
ithin the tip leading to further improvement in
fluorescence (Figure S8 1-3)
c)
First realization and demonstration of the em
ission enhancement uniformity within the 3D-
tapered device for a range of molecules (Figure 4).
While various nanostructures and MIM waveguides
have been shown as energy concentrators and
biosensors in the past, none of them have dem
onstrated their ability to solve this fundamental
challenge associated with plasmon-enhanced
fluorescence (to the best of our knowledge).
We discuss the details in manuscript as follows:
Page 2 “
While several reports have discussed novel
nanoscale geometries that improve the
confinement of EM fields leading
to strong fluorescence enhancements
15-18
, engineering a hotspot
that resolves the distance challenge between the
emitter and the nanostructure surface has remained
elusive.
Powerful fluorescence enhancement within a nanostruc
ture independent of variation in molecule size
and position can be expected to rely on several impor
tant factors: a) strong confinement of
electromagnetic field b) powerful coupling of the em
itter to the field for enhancement of emission and
c) a hotspot geometry which generates uniform elect
romagnetic field distribution. At the same time,
the hotspot geometry needs to be large enough for co
mmonly used protein-protein binding assays (i.e.
larger than antibodies, ~ 15 nm). Metal-insulator-
metal (MIM) structures utilizing surface-plasmon-
polariton (SPP) propagation have been known to
enable efficient confinement of EM energy
19-22
.
Specifically, waveguides with a 3D taper that rel
y on adiabatic compression of the SPP mode inside
the MIM gap have been shown to provide extreme
volumetric nanoscale confinement of EM fields
23-27
.
While extremely promising for energy confinement
and transfer, the potential of 3D-tapered designs
for coupling with fluorescent emitters and bioassays had
remained unrealized primarily due to closed
monolithic structures which prevente
d molecular integration with th
ese devices. Furthermore, as
electromagnetic field intensity on a plasmonic surfa
ce is maximum at the metal-dielectric interface,
close packing of multiple metal-dielectric interfaces
such as in very thin meta
l-insulator-metal (MIM)
gaps, can result in integration of these multiple
field profiles within the gap creating a more
homogeneous field distribution. Therefore, a 3D
-tapered structure provides unutilized potential
towards these goals, for enabling confinement of a
large amount of incident electromagnetic energy
into a tiny MIM gap. In order to take advantage
of the previously known as well as unexplored
abilities of 3D-tapered MIM devices, we designed an
d fabricated a fluidic channel-like 3D-tapered
gap plasmon nanocavity, allowing ready access of th
e nanostructures to molecules in solution
(Figure 1a).”
Page 9: “
We demonstrate 3D-tapered gap
plasmon nanocavities which pr
ovide one of the highest
enhancement of fluorescence obtained by plasmonic n
anostructures (EF: ~ 2200 with figure of merit
~ 260), independent of the size of the molecular a
ssemblies used in the assa
y. Overcoming molecule
size and placement-dependent extreme variation in
plasmonic fluorescence enhancement has been a
major challenge restricting widespread application
of this method in bioassays. The nanostructure
geometry presented in this manuscript demons
trates a way to overcome this limitation thereby
improving the consistency and range
of plasmon-enhanced emission for diversely sized assembly of
molecules. Simultaneously, we also demonstrate capt
ure and visualization of single antibodies at the
tip as well as sensing of proteins at low concen
trations (10 pM). These
advantages can be readily
transferred towards applications such as highly
sensitive biosensing using molecular labels of
varying sizes and analysis of single molecules or tigh
tly controlled arrays of molecules for protein
orientation, protein function, and
biological polymer formation studies
32-34
.”
There are parameter studies using the FDTD ca
lculation in the supporting information.
However, overall experimental st
udies are insufficient (Fig. 2 and Fig. 3). They only show
simple experimental results for the 3D tapered
nanocavity, the tip only structure, and the
conventional bowtie nanoantenna. It would be
better to show and discuss experimental results
for the 3D tapered nanocavities with different ph
ysical dimensions as shown in the numerical
results.
Response
– We have added the experimental results to
the manuscript now with the change in taper
angle and body width (Figure S2-2). We have al
so added a plot showing the improved performance
with respect to the tip length (Figure 4e).
Figure S2-2:
Device optimization.
(a)
Experimental results sh
owing that the highest
fluorescence intensity from the devi
ces was obtained for taper angle (
α
) close to 20°, with the
body width maintained at 150 nm, tip length as 500 nm and width as 20 nm. (b)
Experimental results showing that devi
ces with body width around 150 nm had higher
fluorescence emission as compared to devices
with wider body widths. Device taper angle
was maintained at 20° for these tests. The e
xperimental results support the parameters
indicated by simulations
Figure 4(e):
Increase in mean enhancement factors
with decrease in tip length to 20 nm.
2.
The device coated with biotin can capture the stre
ptavidin high concentration. What is the limit
of low concentration?
Response
– In terms of number of molecules, we can detect presence and emission from a single
molecule at the end of taper (Figure 3d).
In terms of concentration, the detection limit is
simply governed by the time
taken for a single target
molecule to reach the hotspot such that it is captu
red there. In the current model, where there is no
convective flow or continuous stirring, diffusion g
overns the limit of detection. We were able to
detect signal from 10 pM solution, which was added as a 10 μL droplet onto the chip.
This plot has been added as Figure S7-1.
Page 6: “
Limit of detection for biomolecules on the device was examined using two types of sensing
experiments. Firstly, detection of low concentrati
on protein molecules in solution (10 pM – 1 nM)
was performed, indicating device suitability fo
r on-chip diagnostics of rare disease-specific
biomarkers (Figure S7-1). The detection limit in th
is case is governed by diffusive transport of
molecules to the plasmonic hotspot and can be a
ccelerated by improving molecule transport using
convective flow, magnetic or dielectrophoretic tr
apping. Secondly, we examined the capture and
detection of individual or small array of molecules at the hotspot, while high concentration of
molecules were present in solution.
User-controlled analysis of single or small array of molecules
remains an important target for high-resoluti
on analysis of protein function and behavior
32-34
. This is
specially important in cases where ligands and biom
olecules are physiologically present at higher
(micro – to millimolar) concentrations
35
. We control the number of mo
lecules captured within the
tips by altering the tip length.
After formation of the biotin
monolayer within the 3D-tapered
nanocavities, we utilized an anti-biotin IgG prima
ry antibody (tagged with DyLight 755 ~ spectral
response similar to AF-750) fo
r performing fluorescence assays
36
. In order to compare effect of tip
length on capture of molecules, we performed
these antibody binding experiments using devices with
l
tip
= 500 nm as well as l
tip
= 20 nm (Figure 3c, d). The dimensi
ons of a 3D-tapered nanocavity with a
short tip (l
tip
= 20 nm, w
tip
= 20 nm) compared to those of an IgG antibody (length ~ 15 – 20 nm)
36,37
indicate that a single antibody s
hould be specifically trapped within the tip region. Our AFM studies
demonstrated uniform monolayer of PEG-biotin a
nd antibodies on flat silica surfaces and indicated
that only one antibody should
specifically occupy the 20 nm × 20 nm tip (Figure S7-2).”
Figure S7-1:
Detection of low concentration molecu
les on 3D-tapered waveguides (tip
length 500 nm). (a) Log-log plot showing increase
in signal with increase in concentration of
added Streptavidin (10 pM – 1000 pM). (b) Diffe
rence in signal obtained from two devices
after addition of 10 pM streptav
idin-AF 750. Negative control de
vice had no biotin layer.
3.
They demonstrated specific and single mol
ecular capture using the nanocavity which has
similar dimensions with the target molecule. In
this paper, they capture the IgG antibody
(length 15~20 nm) using the nanocavity with 20 nm
x 20 nm size. If this single molecule capture
depends on the size of nanocavity and the target
molecule, additional experimental results for
different sizes (at least two or three) can support
the principle of single molecule capture
.
Response
– For the tip size shown in the manuscript (i.e. width = 20 nm and length = 20 nm), we
demonstrate single protein capture for IgG anti
bodies with provided molecular weight around 255
kDa and size ~ 15 nm. These IgG antibodies are comm
only used for bioassays and are the largest
among the molecules we tested. The signal increases
as expected for longer tips due to presence of
more molecules and falls dramatically for tip
length ~ 10 nm indicating the lack of antibody
molecules in the tip (Figure S9).
The difference in fluorescence inte
nsity between tip length 20 nm
and 500 nm also fits well with calculations based
on energy density within the tips as shown in Figure
S3b.
Figure S9:
Fluorescence intensity (background subtra
cted) obtained using tips of various
lengths after performing binding assay with AF-750.
For the current fabrication method we were limite
d by the FIB fabrication resolution and it was
trickier to reliably perform single molecule tests w
ith smaller proteins such as streptavidin (size ~ 5
nm) or even smaller molecules. In order to pe
rform single molecule volumetric traps with even
smaller molecules we plan on changing the fabricati
on protocol to e-beam or nanoimprint lithography
for higher resolution small cavities.
Since the current demonstration specifically demonstrates antibody-based single molecule binding,
we have made this clearer in the manuscript now. We
thank the reviewer for helping us clarify this
point further.
Abstract: “
The 3D-tapered device provides fluorescence e
nhancement factors clo
se to 2200 uniformly
for various molecular assemblies ranging from few
angstroms to 20 nanometers in size. Furthermore,
our nanostructure allows detection of low con
centration (100 pM) biomarkers as well as specific
capture of single antibody molecules at the nanoc
avity tip for high resolution molecular binding
analysis.
”
Page 3: “
We specifically trap and observe single antib
ody molecules or arrays of molecular
assemblies within the device, as well as detect lo
w concentration (10 pM) pr
otein molecules diffusing
in solution.
”
Page 9: “
Simultaneously, we also demonstrate capture and vi
sualization of single antibodies at the tip
as well as sensing of proteins at low concentrations (10 pM).
”
In addition, the authors should ca
refully proofread the manuscript.
Response
– We have proofread the manu
script to eliminate these errors.
For example,
-Please insert scale bars for all figures of 2D E-field intensity profiles.
Response
– All field intensity profile figures have scale bars included now.
-In Figure 3, the alphabets (numbers) for sub-fi
gures and descriptions don’t match. In addition,
it would be better to effectively displa
y their results to place the results under the
corresponding schematic illustrations fo
r effective delivery of results.
Response
– The descriptions and figures
have been changed accordingly.
On page 4, Finite-Domain Time-Difference is incorrect terminology for FDTD. Finite
Difference Time Domain (FDTD) is correct terminology.
Response
– The typo has been corrected.
In Figure S4b, the direction of arrow is strang
e. It should be in the opposite direction
.
Response
– We have changed the direction of the arrow
to indicate that the molecule is coming out.
Once again we would like to thank the reviewer fo
r careful assessment of our work and helping us
improve the depth and clarity of our manuscript. We
hope our revised version satisfies their concerns.
Reviewer #3(Remarks to the Author):
The authors present an interesting concept to im
prove the fluorescent enhancement intensity using
a plasmonic nanotapered cavity. The concept is n
ovel, and will be of interest to the biosensing
research community, and also to other branches of
photonics reserach. The work is very thorough,
and the results are convincing.
However, the manuscript is not
written very well, and a more
balanced view of the importance of the work should be presented.
Response:
We thank the reviewer for their supportive
and constructive comments. We found their
suggestions extremely helpful to revise our ma
nuscript and have made changes accordingly.
Comments regarding writing and presentation of results
Some of the examples about the writing and organization are described below
a) In page 3 of the manuscript the authors mentioned
“This engineered hotspot within the device tip was
used for highly efficient and uniform fluorescent
enhancement – the enhancement factor up to
1100 times and 40x improvement in uniformity
compared to a bowtie antenna-....”
Reading this, my impression was the 1100 enhancem
ent was compared to the bowtie antenna. But
from page 6 of the Supplementary Materials, I und
erstand that the enhancement is compared to a
flat silica surface. Such ambiguity about one of the central claims of the manuscript should be
avoided.
Response –
We have clarified the enhancement statemen
ts through the manuscript now. We used the
standard reporting methodology of reporting the enha
ncement with respect to
a non-enhancing substrate
(with similar chemistry, in this case silica). We
have now made it clear throughout the manuscript.
Page 3 “
The enhancement factor (EF) was calculated with
respect to non-enhancing silica substrate such
as glass chips commonl
y used in bioassays...”
Page 7: “
We analyzed the enhancement of fluorescence experi
enced by an emitter within the tip, as
compared to a non-enhancing subs
trate (silica or glass surface)
.”
We have also updated the analysis and
enhancement values in the manuscript.
b)
In Figure 1 the authors show the schematic of a three dimensional structure and in Figure 2 they
the discussed the field distribution over its cross
section. But since the coordinate system was not
clearly defined in these figures, one has to go throug
h the text and caption trying to confirm if x and
y are in the lateral and vertical directions.
Response
– The coordinate system has now been defined in the images.