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Breaking the diffusion limit with super-hydrophobic delivery of molecules to

plasmonic nanofocusing SERS structures

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 ARTICLES
PUBLISHED ONLINE: 18 SEPTEMBER 2011 | DOI: 10.1038/NPHOTON.2011.222

Breaking the diffusion limit with

super-hydrophobic delivery of molecules to
plasmonic nanofocusing SERS structures
F. De Angelis1,2, F. Gentile1,2, F. Mecarini1, G. Das1, M. Moretti1, P. Candeloro2, M. L. Coluccio1,2,
G. Cojoc2, A. Accardo1,2, C. Liberale1, R. P. Zaccaria1, G. Perozziello1, L. Tirinato2, A. Toma1, G. Cuda2,
R. Cingolani1 and E. Di Fabrizio1,2 *

The detection of a few molecules in a highly diluted solution is of paramount interest in fields including biomedicine,
safety and eco-pollution in relation to rare and dangerous chemicals. Nanosensors based on plasmonics are promising
devices in this regard, in that they combine the features of high sensitivity, label-free detection and miniaturization.
However, plasmonic-based nanosensors, in common with general sensors with sensitive areas on the scale of nanometres,
cannot be used directly to detect molecules dissolved in femto- or attomolar solutions. In other words, they are diffusion-
limited and their detection times become impractical at such concentrations. In this Article, we demonstrate, by combining
super-hydrophobic artificial surfaces and nanoplasmonic structures, that few molecules can be localized and detected even
at attomolar (10218 mol l21) concentration. Moreover, the detection can be combined with fluorescence and Raman
spectroscopy, such that the chemical signature of the molecules can be clearly determined.

through them7,8. Despite the great success of these techniques, the

E
arly diagnosis in medicine is a field in which nanotechnology
can sensibly offer important and unprecedented benefits. For ‘diffusion limit’ still prevents their efficient exploitation in the
example, although it is well understood that blood contains a femto/attomolar range.
number of molecules and biomarkers that may reveal the presence Regardless of the detection technique applied, the challenge to be
of a disease, their identification or recognition is still challenging met is to find a way to drive molecules towards sensitive areas, or in
because of their very low concentrations. There is therefore an other words, overcome the diffusion limit. In this Article, for the
urgent need for novel devices with (i) superior sensing capabilities first time, surface nanostructuring is used to redesign and fabricate
and/or (ii) increased spatial localization. new and highly sensitive sensors in which super-hydrophobic sur-
In the recent past, the combination of plasmonics and Raman faces and plasmonics nanostructures are combined in a synergistic
spectroscopy has led to extraordinary success in few-molecule inves- way to allow single-molecule detection in highly diluted solutions,
tigations by concentrating optical radiation energy in hot spots with even at femto- or attomolar (10215/10218 mol l21) levels. Super-
areas of just a few nm2 (refs 1–4). Such electric-field hot spots are hydrophobic surfaces allow us to drive and concentrate molecules
active only in the near-field region, that is, within a few nanometres over the sensing nano-area, where plasmonic electric-field hot
of the surface. Unfortunately, in most practical applications, mol- spots are used to carry out molecule detection. The diffusion limit
ecules are dispersed in solutions and are free to diffuse into the can then be overcome and time accumulation reduced to a few
liquid volume, far from the plasmonic sensitive surfaces. Large- minutes, even for concentrations at attomolar levels.
area sensors provide a high geometrical cross-section, but very We first show how a super-hydrophobic surface can be used to
poor signal-to-noise ratio when only few molecules are investigated localize molecules in highly diluted solutions at a specific position.
(only a small portion of the sensitive area is actively involved). On A set of spectroscopic experiments, based on fluorescence and
the other hand, the reduction of the sensitive area down to few Raman scattering, will be presented to demonstrate the extreme
nm2 renders an encounter of the molecules and nanosensor very sensing capability and wide applicability of our method. Finally,
unlikely. It has been demonstrated5,6 that when the concentration an advanced plasmonic nanosensor expressly conceived to fully
of the solution is close to femtomolar and the linear size of the exploit the advantage of our approach will be presented.
sensor is in the range of a few tens of nanometres, the accumulation
time for the detection of a few molecules is on the scale of days. In Device design and working principle
other words, nanosensors cannot be used for low-concentration Super-hydrophobicity9–11 is a phenomenon in which a drop placed
detection (below picomolar concentrations) because the accumu- on a surface adopts a quasi-spherical shape with a contact angle
lation time is far beyond practical timescales. Different approaches greater than 1508, rather than spreading or wetting indefinitely
have been investigated to overcome these limitations, for example, the plane of contact (Supplementary Movie M1). The present
by using plasmonic nanoparticles that can diffuse through the sol- work takes advantage of the possibility of designing super-
ution and meet the molecules of interest, microfluidic channels hydrophobic surfaces with high contact angles and low friction
that drive the molecules towards the sensitive surfaces, and mem- forces (low friction coefficient), independent of the radius of the
brane nanopores acting as sieves that force molecules to pass drop. Figure 1 shows that, when a drop of an extremely diluted

1
Department of Nanostructrures, Istituto Italiano di Tecnologia (IIT), via Morego 30, I16163 Genova, Italy, 2 BIONEM Lab, University of Magna Graecia,
Campus S. Venuta, Germaneto, viale Europa, I88100 Catanzaro, Italy. * e-mail: [email protected]

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ARTICLES NATURE PHOTONICS DOI: 10.1038/NPHOTON.2011.222

a b

1.2 mm (s)

ϕ = 1.2 mm

25 μm

t = 25 min t = 35 min
c t = 10 min t = 15 min

150 μm 150 μm 150 μm 25 μm

Figure 1 | High contact angle and evaporation process. a, Sketch representing the high contact angle and evaporation process with no pinning of the drop
and no solute left on the substrate during drop concentration. b, SEM images of the footprint diameter of the drop and the suspended deposition of the
solute on pillars. Notice that the whole content of the drop, with an initial contact area of 1.2 mm (original diameter, 2 mm), is localized on a triangle with
lateral sides of 25 mm. c, Contact angle measurements during evaporation at four different times (optical images taken with a microscope contact angle of
908 geometry), showing the sliding and evaporation mechanism of the drop on the super-hydrophobic surface. Note that the drop slips on the surface,
keeping the contact angle and the shape of the drop constant. The last image shows the collapsing condition (optical image taken at lower magnification to
better show the volume reduction; see also Supplementary Movies M1 and M2).

solution is deposited on a textured, super-hydrophobic substrate accumulation of plasmon polaritons in the region of deposition to
and is allowed to evaporate, the drop will reduce in volume while allow a few-molecule investigation by means of Raman scatter-
maintaining its quasi-spherical shape (self-similar geometrical ing16–24. The interest in this spectroscopy is related to its ability to
transformation). This behaviour is a result of the low adhesion provide a clear physical and chemical insight into the molecules
forces between the drop and the surface; during evaporation, these under study. In the following, we report the use of such micro–
allow the drop to slide on the surface, thereby avoiding it being nano hybrid devices to detect various molecules and biomolecules
pinned at its initial contact point12–15. During evaporation, the sol- (rhodamine, lambda DNA, lysozyme) starting from solutions with
ution therefore becomes more and more concentrated. At the end of concentrations ranging from femto- to attomolar. The geometries
the process, when the shape and concentration reach a condition of used were chosen for having stable hydrophobic conditions and
instability, the drop collapses and the solute deposits in a suspended appropriate plasmonic structure. In Supplementary Fig. S1.6, the
confined region with an area of a few square micrometres contact angle u ce is reported as a function of pillar diameter d and
(Supplementary Movie M2). When designed properly (for details distance d for the hexagonal lattice. In particular, it is shown that
on surface design and its calculations, see Supplementary Section a region of stability is achieved when a contact angle between
1), the precipitation region and the sensitive area of the nanosensor 1608 and 1708 is obtained for a wide range of d and d between
are coincident and the detection of a few molecules (or a single mol- tens of nanometres and a few micrometres. We note here that
ecule) is possible, even for solutions with initial concentrations on super-hydrophobic surfaces are reproducible and can be scaled up
an attomolar scale. for high-throughput production using micro- and nanofabrication
Figure 2 presents scanning electron microscopy (SEM) images of techniques. Moreover, independent of the analytical technique
super-hydrophobic surfaces made of silicon micropillar arrays with used (fluorescence, Raman, and other spectroscopies or techniques),
a typical periodicity of 30 mm, duty cycle (fraction of air to solid) of they provide an easy and fast concentration and localization method
3:1 and aspect ratio of 10. Through an appropriate combination fully compatible with existing protocols in biology and medicine.
of micro- and nanofabrication (Supplementary Section 2), super-
hydrophobic surfaces and plasmonic nanosensors can be realized
Detection of rhodamine at femto- to attomolar concentrations
on the same chip. In this work, we fabricated different kinds of
As an introductory experiment to show the potential of this pro-
plasmonic nanostructures on the top of a silicon micropillar, includ-
cedure, we report the detection of rhodamine from water solutions
ing simple rough silver surfaces (Fig. 2b), regular arrays of silver
below a concentration of 1 fM (see Supplementary Section 3 for
nanoparticles (Fig. 2c), silver nanocones with circular grating
sample preparation). Evaporation of the drop (on bare super-
coupler (Fig. 2d), and an advanced plasmonic nanosensor fully
hydrophobic surfaces, Fig. 2a) was followed over time until precipi-
embedded in the micropillar array, which will be described in
tation of the solute. The solute molecules were confined into a small
detail later (Fig. 2e).
area of a few tens of micrometres square. Solutions of progressively
decreasing concentration were investigated. Figure 3a presents an
Device functions SEM image of the residual solute of rhodamine 6G at the end of a
As mentioned above, the functions of these devices include (i) the process of evaporation starting from a drop with a diameter of
concentration and localization of an initially greatly diluted solute 2 mm. Notice the strong concentration and localization of rhoda-
into a very small region of the plane and (ii) the generation and mine, which precipitates onto a small area bridging the pillars.

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NATURE PHOTONICS DOI: 10.1038/NPHOTON.2011.222 ARTICLES
a b c

2 μm 2 μm

d e

30 μm 2 μm 5 μm

Figure 2 | Architecture of the four different devices fabricated in this work. a, Periodical array designed to have a high contact angle (.1508) and high drop
stability. b, The first type of pillar, with its top decorated with silver nanoparticles applied by electroless deposition. Random nanoparticle deposition is the
simplest way of combining super-hydrophobicity and plasmonic enhancement. c, The second type of pillar, with the plasmonic nanostructures arranged in a
regular array. d, The plasmonic structure represented by a nanocone coupled with a circular grating for efficient generation of plasmon polaritons on the
cone. e, The plasmonic structure embedded in the super-hydrophobic array, with a single pillar used for plasmonic enhancement and detection.

a b c
100

intensity (a.u.)
Fluorescence
75

50

25

30 μm 30 μm 400 450 500 550

Wavelength (nm)

d e f
1,000
Raman intensity (a.u.)
Raman intensity (a.u.)

1650
1

500
0
200
0
yp

100 0
)
os

μm
iti

n( 1,200 1,400 1,600 1,800

o

30 μm io
n

0 sit
(μ

130 o Raman shift (cm−1)

m

xp
)

Figure 3 | Localization and spectroscopic measurements. a, SEM image of solute precipitation from a 1 fM solution of rhodamine. b,c, Fluorescence
measurements of rhodamine (b) and its spectral signature (c). d, SEM image of solute precipitation from a 10 aM solution of rhodamine. e,f, Raman mapping
measurement of rhodamine (e) and its spectral signature (f).

In each experiment the residual solute was localized in a region with Considering that the quantity of solution initially deposited was
a linear extension smaller than a few tens of micrometres. In the case 20 ml (initial drop diameter, 2 mm, containing R6G molecules),
presented in Fig. 3a, the initial concentration of rhodamine was these devices reveal or detect 100 molecules, starting from attomo-
1 × 10215 M. The fluorescent image in Fig. 3b confirms the identi- lar concentrations, with relative ease of use. We note that when the
fication of the residue as rhodamine, and it is clear that no appreci- same drop was deposited on a conventional flat plasmonic substrate,
able quantity is left around the precipitate on the neighbouring the drop expanded over the metal surface and, at the end of evapor-
pillars. To further substantiate the method for concentrations as ation, R6G molecules were deposited over an area of 5 × 5 mm2.
low as 10 aM (10217 M), micro-Raman mapping measurements This indicates a concentration factor of at least 1 × 104
were performed using the device of Fig. 2b. The results are reported (5 × 5 mm2/50 × 50 mm2) between the two arrangements.
in Fig. 3d–f. The intensity and contrast of the Raman signal are very
strong due to the presence of the silver nanostructures. A mapping Single lambda DNA molecule from attomolar concentration
analysis (Fig. 3e) was performed, with detection of the band centred Lambda DNA molecules were chosen as a test system for the recog-
at 1,650 cm21, and overlaps clearly with the SEM image of Fig. 3d, nition and localization of single molecules because of their clear and
in which the entire precipitate is seen to lie on only three pillars. unambiguous imaging results. Lambda DNA solutions were

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ARTICLES NATURE PHOTONICS DOI: 10.1038/NPHOTON.2011.222

a b c e

5 μm 10 μm

d f

20 μm 20 μm 10 μm 10 μm

g h i

15 μm 10 μm 10 μm

Figure 4 | Single lambda-DNA localization and detection from a 10 aM solution. a, SEM image showing a single molecule of DNA on an area of 150 ×
150 mm2. b,c,e, SEM images showing a single suspended DNA molecule from different viewpoints. d,f, Optical fluorescent measurements of the single DNA
molecule. g, Two lambda-DNA bridges are localized. h, A network is obtained by increasing the lambda-DNA concentration in the starting solution. i, Detail
of a suspended node as a result of a few lambda-DNA molecule interaction.

prepared following a staining and serial dilution protocol cell-free protein expression30, or even to study DNA motors or
(Supplementary Section 3). Preliminary experiments demonstrated walkers31 to explore the design of drug transport and release.
that a double-stranded lambda DNA molecules (48,502 bp) can be
deposited, suspended and stretched between two or more super- Lysozyme molecules by advanced plasmonic devices
hydrophobic pillars (Supplementary Section 4). As a result of As introduced above, super-hydrophobic substrates and plasmonic
surface super-hydrophobicity, dewetting of the DNA-containing structures can be designed independently. In other words, given
solution occurred on the top surface, allowing the DNA to link the pillar architecture, which is designed in the stable super-
exactly on the top of the pillars. An attomolar concentration of hydrophobic region (Supplementary Section 1), it is possible to
DNA stained with YOYO-1 dye (Supplementary Section 3) in a choose separately the textures covering the pillars that locally
3 ml drop was evaporated on the substrates for 20 min (a 3 ml enhance the electric field. In recent years, plasmonic tips and anten-
drop with a concentration of 1 × 10218 mol l21 contains, statisti- nas made of noble metals have led to few-molecule investigations
cally, two DNA molecules). Samples were analysed both in epifluor- using Raman spectroscopy32–36. To fully exploit the advantage of
escence optical and SEM microscopy. The results are shown in our approach, we designed super-hydrophobic surfaces with a plas-
Fig. 4. Fluorescent DNA was clearly visible (Fig. 4d,f ). SEM monic cone directly embedded in a micropillar array. As shown in
images of the same sample showed a correspondence at the same Fig. 5, the cone was fabricated at the centre of the array as a replace-
position with the fluorescent DNA stretches (Fig. 4b,c,e). The fila- ment for a micropillar. For symmetry reasons, the cone can be
ments were 10–30 mm long. From Fig. 4b, we found that filaments viewed as a spatial defect that breaks the spatial symmetry defined
longer than 15 mm were probably due to DNA overlapping. In other by the pillars. As a result, the hydrophobic radial forces cause the
words, unpaired overlapping of lambda filaments makes a longer collapse of the drop on the cone tip (Supplementary Section 1).
filament. Moreover, in a more accurate observation in SEM In other words, during evaporation, such a local defect drives the
microscopy the filament appears to have two distinct diameters. drop towards the tip itself, as a result of the reduced hydrophobic
In Fig. 4a, a wider SEM snapshot over an area of 150 × 150 mm2 effect. As a consequence, molecules initially dispersed in solution
does not reveal the presence of other DNA molecules, which is will be progressively guided towards the plasmonic tip, where they
consistent with the nominal concentration of 1 × 10218 mol l21. will be deposited at the end of the evaporation process.
This concentration and localization process can be accurately The plasmonic tip, consisting of a silver nanocone with a grating
controlled and, by varying the dilution, it is possible to deposit an on one side, was fabricated by focused ion beam milling and
increasing number of molecules, as in Fig. 4g, and create a sus- electron-beam-induced deposition (see Supplementary Section 2
pended regular lambda DNA network, as shown in Fig. 4h,i for details). Device simulation was performed with commercial soft-
(Supplementary Section 4). ware (www.lumerical.com) using a finite-difference time-domain
We envisage that the devices we have presented, prepared (FDTD) approach. The results are shown in Fig. 5e (see
entirely without any chemical modification of the DNA and isolated Supplementary Section 5 for details). The lateral grating was
from interaction with the substrate, could be used in wide range of designed to provide an efficient coupling (even if the symmetry of
applications. These might include the design of DNA wires for the plasmonic mode in the cone was not fully fulfilled) between
DNA-template electronic devices25, the positioning and study of the incoming laser (l ¼ 532 nm) and the nanocone. Surface
protein interactions and enzyme cascading26, the rational assembly plasmon polaritons are launched from the grating (480 nm period)
of nanoarrays27,28, for structuring live cell friendly immobilization towards the tip, where electric-field enhancement occurs. For a tip
beads29, as a building base for the design of DNA hydrogels for with a 10 nm radius of curvature, the calculated electric-field

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NATURE PHOTONICS DOI: 10.1038/NPHOTON.2011.222 ARTICLES

enhancement (log scale) enhancement (log scale)

a 1.5
b d e 0
Incoming laser

Electric field
Height (nm)
linear polarization −40 0.5

SPP λ = 532 nm −80

concentration NA = 0.9 −0.5
−120
Grating
5 μm −10 0 10
period
Width (nm)
480 nm −2
c 0.6

nm

Electric field
Height (nm)
g 40
−4

coatin
−1.2
−6
SPP

r
Silve
20 μm 1 μm −8 −3.0
−750 0 750
Width (nm)

Figure 5 | Super-hydrophobic nanostructure. a, Super-hydrophobic device with embedded plasmonic nanostructure. b,c, Details of the device.
d, Measurement principle. After evaporation, the solute precipitates on the tip of the nanocone. Through a scanning procedure an optimal
illumination/detection of the grating/sample is obtained. e, FDTD simulation results, showing an asymmetrical intensity field, in good agreement
with the experimental imaging measurements reported in the Fig. 6b.

a b 1,400 z = 3 μm 1,400 z = 1.6 μm 1,400 z = 0.3 μm 1,400 z = 0 μm

1,000 1,000 1,000 1,000

600 600 600 600

Raman intensity (a.u.)

Raman intensity (a.u.)

Raman intensity (a.u.)

Raman intensity (a.u.)

200 200 200 200

0 1,000 2,000 3,000 0 1,000 2,000 3,000 0 1,000 2,000 3,000 0 1,000 2,000 3,000
Raman shift (cm−1) Raman shift (cm−1) Raman shift (cm−1) Raman shift (cm−1)

1 μm

Figure 6 | Super-hydrophobic device with embedded plasmonic nanostructure. a, SEM image after lysozyme precipitation. b, Optical images and
corresponding Raman measurements while scanning along the optical axis. Optimal conditions are reached at z ¼ 0 mm, where a lysozyme high-contrast
Raman signal is obtained.

enhancement is 35 and the Raman enhancement is 1.5 × 106, direction (z-axis), the Raman signal is observed to acquire a very
strong enough for the detection of a few molecules located in the high contrast when the coupling with the grating and the detection
proximity of the tip end. In Fig. 6b (bottom), activation of a conditions are optimized (Supplementary Movie M3). In these con-
surface plasmon polariton is clearly shown when the illumination ditions, the Raman sensitivity and spectra details increase strongly.
and imaging of the grating/nanocone are both properly satisfied Figure 6b (bottom) clearly shows (as a function of z scan) activation
(0 ≤ z ≤ 1.6 mm). In these conditions, the Raman spectra of lyso- of the plasmon polaritons when optimal coupling/detection
zyme were simultaneously collected as described in the following. is reached.
A 160 nl droplet was deposited on the surface of the device of The spectra taken for lysozyme when focused 3 mm out of the
Fig. 5 using a micro-injector system. The droplet contained lyso- optimal position shows very limited information about the sub-
zyme at a concentration of 1 fM (100 molecules in 160 nl stance, as only three weak bands are observed centred at 2,940,
drops). At the end of the evaporation process (which lasted a few 2,184, and 1,600 cm21, attributed to C–Hx, –S–CHx and aromatic
seconds in this case because the volume of the solution was 30 bands (Phe, phenylalanine; Trp, tryptophan). As the focal point
times smaller than in previous experiments), lysozyme accumulated moves towards the optimal illumination (z ≤ 1.6 mm), various
on the silver cone, as can be seen in Fig. 6a. Notice that the material characteristic bands of lysozyme start appearing, providing rich
along the cone surface includes additional environmental debris chemical information about the protein (Fig. 6b, z , 3 mm).
deposited in a dry condition after drop evaporation. As demon- Vibrational bands are observed centred at 1,610 (Phe, Trp),
strated in previous works32, only molecules deposited in close proxi- 1,555–1,568 (amide II), 1,450 (C–Hx), 1,350 (Trp), 1,230–1,295
mity to the tip are efficiently detected because the enhancement (Amide III), 1,069 and 1,126 (C–N stretching), 990 (a-helix), 895
decreases very rapidly away from the apex. We estimate that, statisti- (Trp), 650 (C–S stretching) and 620 cm21 (Phe breathing), in
cally and for geometrical reasons (the cone radius of curvature is addition to the broad band centred around 3,000 cm21 (C–Hx
10 nm), fewer than five molecules are deposited on the tip stretching), which confirm the presence of proteins at the tip.
vertex (lysozyme can be described as a slightly deformed sphere Furthermore, the most significant band centred at 2,140 cm21,
with a diameter of 4 nm; refs 37–40). The spectra of the lysozyme attributed to the –S–CHx, can be clearly observed. This band is rela-
are shown in Fig. 6b as a function of z scan position (z is the scan tively less visible in the protein spectrum from the liquid sample.
direction along the optical axis of the illuminating objective). For The broad band around 3,350 cm21, which is related to the N–Hx
different positions of illumination/acquisition along the vertical stretching (amine groups), as shown in Fig. 6b (z ¼ 0), is also

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 ARTICLES NATURE PHOTONICS DOI: 10.1038/NPHOTON.2011.222

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