Spectral tagging by integrated photonic crystal resonators for highly sensitive and
parallel detection in biochips
Luigi Martiradonna1, Ferruccio Pisanello2,3, Tiziana Stomeo2, Antonio Qualtieri1,2, Giuseppe Vecchio1,
Stefania Sabella1, Roberto Cingolani1, Massimo De Vittorio1,2, Pier Paolo Pompa1
1
Istituto Italiano di Tecnologia (IIT), Center for Bio-Molecular Nanotechnology, Via Barsanti 1, Arnesano
(Lecce) 73010 (Italy).
2
National Nanotechnology Laboratory, CNR/INFM, Scuola superiore ISUFI, Università del Salento, 16 Via
Arnesano, Lecce 73100, Italy.
3
Université Pierre et Marie Curie, Laboratoire Kastler Brossel, CNRS UMR8552, Ecole Normale
Supérieure, 4 place Jussieu, 75252 Paris Cedex 05, France.
SUPPLEMENTARY MATERIAL
The investigated two-dimensional photonic crystal (PhC) pattern consists of a
triangular lattice of air-holes obtained in a Si3N4 slab in air-bridge configuration. The
photonic band gap (PBG) has been designed by using a Plane Wave Expansion (PWE)
algorithm and optimized as a function of the filling factor a/r, where a is the period of the
lattice and r the radius of the holes. PWE simulations have been restricted to transverse
electric like modes (TE-like) with significant components of the electric field in the xy
plane of the structure and only one component of the magnetic field along z. The
resulting band diagram for a slab thickness of t = λ/(2n), shown if Figure S1(a), presents
a PBG below the light line between the 1st and the 2nd order mode. t has been then
increased in order to obtain a closed bandgap [Ref. 26 in main text]. Figure S1(b),
obtained for t=1.55a, clearly shows that both the 1st and the 2nd order modes fold down
leading to the closing band gap condition. In agreement with the results of ref.26 in main
text, this strategy allows to increase the quality factor (Q-factor) of the two degenerated
dipole modes allowed in a H1 cavity, obtained in such a PhC slab by removing a single
hole from the periodic pattern in the PhC slab. In the following text, we will refer to these
two modes as x- and y-pole modes, since they present the main component of the inplane wavevector along x and y axis in the PhC plane, respectively.
Figure S1. Photonic band diagrams of a triangular lattice of air holes in a Si3N4 slab with
a thickness t= λ/(2n) (a) and t=1.55a (b). The blue regions represent the light cone
whereas the photonic band gap is represented in (a) by the light blue area. (c): Q-factor
(red curve) and Purcell Factor (black curve) of the H1 cavity as a function of the
membrane thickness. (d) Dependence of the y-pole Q-factor as function of the hole shift.
The inset reports the definition of S.
The electromagnetic behaviour of the x- and y-pole modes, and the following
optimization of the cavity geometry have been investigated by exploiting 3D Finite
Differences Time Domain (FDTD, in the commercial implementation FullWAVE, Rsoft
Design Group) algorithm over a calculation domain of 50a x 50a in xy plane. The Qfactor has been estimated by evaluating the time decay of the energy stored in the cavity.
As reported in Figure S1(c) for y-pole mode, Q presents a local maximum for t=1.55a; a
similar result has been found for x-pole mode, with a maximum value of Q at the same t.
In the proposed approach, the electromagnetic cavity has the purpose to enhance the
fluorescence of a luminescent emitter deposited on top of the PhC slab with high spectral
selectivity which is accomplished by increasing the spontaneous emission rate of the
cavity, namely its Purcell factor. According to the definition of Purcell factor, the
spontaneous emission rate of an emitter weakly coupled to a cavity is increased of a
factor F=4/3 π-2 Q/V (λ/n)3 , where V represents the modal volume of the coupled
photonic state. The cavity tagging effect on the fluorophores emission spectrum is
enhanced with the increase of F. As reported in Figure S1(c), both Q and F present a
maximum for t=1.55a equal to Q~700 and F~140, respectively.
Furthermore, two holes have been moved according with the inset of Figure S1(d),
leading to a further increase of the Q-factor of the y-pole mode due to smoothening of the
electric field modal function (as reported also in Refs 10,26 of the main text) , with
negligible effects on V. The dependence of y-pole Q-factor on the modal volume is
reported in Figure S1(d). This modification allowed us to increase Q and F to 810 and
154, respectively.
We investigated the angular and spectral dependence of leaky eigenstates localized in
our PhC pattern by evaluating the diffraction efficiency of the structure. This was
accomplished by using a Rigorous Coupled Wave Analysis (RCWA) technique.
The reflectivity maps computed for the PhC presented in this work are reported in
Figure S2; these maps have been calculated for two different polarizations of the electric
field wavevector (hereafter referred to a S and P, as defined by the vectors Es and Ep in
Figure S2(a)) and for the crystalline directions -M and -K. The typical behavior of a
PhC slab reflection (with radius equal to 0.308a and thickness equal to 1.55a) is clearly
visible in Figure S2(b-e): narrow sharp peaks are superimposed to a smooth background
[Ref.s 16, 29,30 of the main text].
Figure S2. Simulation of angular and spectral dependences of leaky modes in the
modeled PhC pattern. (a): sketch of the excitation and collection conditions of our
experiment; (b)-(c): reflectivity maps for s-and p-polarized incident light along 
direction; (d)-(e): reflectivity maps for s- and p-polarized incident light along 
direction.
As described in the main text, Cy3 and TRITC fluorophores have been deposited on
the patterned surfaces. We have found that a limited influence of leaky modes on the
excitation light (equal to 514 nm, in our case) is expected for incident angles higher than
40 degrees, while no interaction at all is observed in the emission spectral ranges of both
Cy3 and TRITC. Moreover, being the laser focused by means of an objective lens with
numerical aperture equal to 0.85, which corresponds to a continuous of incident beams in
a broad angular range, the diffraction efficiency in the case of our setup is more
realistically described by averaging the scattered light in the whole range. As a
consequence of these numerical and theoretical results we conclude that excitation
efficiency enhancement due to leaky modes has been observed but it results to be almost
two order of magnitude lower than the one reported in Ref 16 of the main text.
The two-dimensional photonic crystal (2D-PhC) nanocavities were fabricated into a
400-nm-thick Si3N4 layer deposited on a silicon substrate by means of Plasma Enhanced
Chemical Vapor Deposition (PECVD) technique. Si3N4 refractive index was measured
through
spectrophotometric
methods
(performed
with
a
Varian
Cary
5000
spectrophotometer), giving a value of nSiN = 1.93 @  = 600 nm.
The PhC geometry was defined using a Raith150 e-beam lithography tool. A
thickness of 400 nm of ZEP520-A resist was chosen to ensure sufficient durability as a
mask for pattern transfer into the underlying Si3N4 and, at the same time, to ensure good
resolution of e-beam writing. The patterns defined in the ZEP were then transferred into
the Si3N4 layer using inductive coupled plasma reactive ion etching (ICP-RIE) in fluorine
chemistry until the silicon substrate surface was reached. The membrane structure was
released by wet etching of the underlying Si substrate in a TetraMethylAmmonium
Hydroxide (TMAH) solution.
Each sample consisted of an array of H1 nanocavities, whose dimensions were scaled
according to the lattice period a (in the range a = 257nm – 277nm) thus allowing spectral
shifting of the resonant wavelength.
Figure S3 shows Scanning Electron Microscopy
micrographs of the fabricated
structures, with details of the modified H1 cavity (left inset) and of the cross-sectional
profile of the suspended Si3N4 membranes.
Figure S3. Realization of a PhC–nanocavities biochip prototype. SEM micrograph of an
array of PhC cavities fabricated on Si3N4. Insets: (left) detail of the defect zone; (right)
cross section after cleavage of the structure. Si3N4 layer and Si substrate have been
colored in blue and brown, respectively, for a better readability.
Si3N4 surfaces containing 2D-PhC nanocavities were chemically modified by means
of a solid state bioconjugation chemistry yielding primary amino-groups on the surface to
link biomolecules. Before reacting with appropriate solutions, samples were thoroughly
washed with acetone, isopropanol, MilliQ water for 10 min each and then cleaned for 30
min by UV/Ozone (UV-tip Cleaner, BioForce Nanosciences, Inc.). Afterwards, by
reacting the surface with a freshly prepared aqueous solution of APTES (0.5% v/v for 5
min) (Sigma–Aldrich), a layer of primary amino groups was introduced. Substrates were
then abundantly rinsed with MilliQ water and stored overnight in a vacuum desiccator to
evaporate physisorbed APTES aggregates. Subsequently, the amino modified layer was
linked with glutaraldehyde (GTA), an homo-functional linker suitable to anchor onto the
surface aminated probes, such as ssDNA or antibodies. Hence, solutions of GTA at a
concentration of 2.5 % (v/v) in 100 mM phosphate buffer were incubated onto the layer
and reactions were carried out at 4 °C in the dark for 2 h. Afterward, substrates were
abundantly washed with MilliQ water and dried in air. GTA functionalized 2D-PhC
nanocavities were then covalently linked with two class of probe biomolecules, namely
aminated-ssDNA and antibodies. Specifically, the DNA probes (Thermo Fischer) (1 μM
in 1X TE buffer) were amino modified at 5’ end (5’ – NH2 – CGC AGG ATG GCA TGG
GGG AG – 3’) and spotted for 2 h onto the surface at 25 °C. After washing procedures
(TE solutions at a concentration of 1X for 3 min), the hybridization experiments were
carried out using complementary target DNA sequences (match: 5’ – Cy3 – CTC CCC
CAT GCC ATC CTG CG – 3’). DNA targets (10 M in 1X PCR buffer) were left to
hybridize with probes for 10 min at 37 °C. A single mismatch sequence (5’ – Cy3 – CTC
CCC CAT ACC ATC CTG CG – 3’) was also tested giving a significantly lower
fluorescence signal (15-20% with respect to the reference complementary sequence).
Similarly, antibody
solutions (mouse monoclonal Anti-Vinculin, Clone hVIN-1,
purchased from Sigma Aldrich) at the concentration of 1 g/mL in 100 mM phosphate
buffer were spotted for 1h onto the glutaraldehyde-activated substrates. The immunomodified surfaces were then thoroughly rinsed with 0.02 % of Tween20 and 1X PBS
buffer, and incubated for 1h with a solution of Rhodamine (TRITC)-conjugated
AffiniPure F(ab')2 Fragment Goat Anti-Mouse IgG (H+L) at a concentration of 1:200 in
1X PBS (purchased from Jackson ImmunoResearch Laboratories, INC.). Finally,
substrates were abundantly washed in 1X PBS buffer and dried in air.
The surface density of both probe biomolecules (i.e., ssDNA and antibodies) was
assessed and optimized by fluorescence measurements. Standard solutions of Cy3modified ssDNA probes (ranging from 0.001 to 50 µM) and antibodies (from 1:1 to
1:2500) were spotted onto GTA functionalized 2D-PhC nanocavities. By confocal
microscopy analyses (Leica, TCS-SP5 AOBS), we found out that the highest
fluorescence signal was obtained with 1 µM for DNA and a dilution of 1:200 for the
antibody.
The samples were analyzed by a confocal microscope system (Olympus Fluoview
1000). The excitation laser (CW @ λ=514nm) was focused on the sample by means of an
objective lens (Numerical Aperture NA=0.85). The obtained photoluminescence signal
was sent into a spectral resolved section composed by a 300 mm spectrometer equipped
with a nitrogen cooled Charge Coupled Device (CCD). A polarization cube and an half
wavelength plate have been introduced in the optical path in order to exploit polarization
resolved measurements and to distinguish between the two resonant modes in the PhC
cavity. This allowed to increase the signal-to-noise ratio among the polarized signal
coming from markers coupled to a specific dipole mode and the unpolarized noise
coming from uncoupled markers.