**4. Integration of biophotonics and superhydrophobic devices for the detection of few bio-molecules**

In biomedical disciplines including medicine, biology or biotechnology, there is a huge interest in understanding the pathogenesis of diseases, and to develop new biomarkers for their diagnosis. In particular, early detection can lead to significant benefits in terms of efficient and timely treatment. In these regards, the blood contains a multitude of unstudied and unknown biomarkers that could reflect the ongoing physiologic state of tissues and organs. The lowmolecular weight region of the blood proteome is an important source of diagnostic markers (Posadas et al., 2001). Nevertheless, this 'mine' of data for diagnosis is often practically inaccessible by conventional approaches, in that the solutions at study are extremely diluted, and contain a multitude of substances that would blur the signal of interest. To exploit this 'mine' of data for diagnosis, new devices are required with very low detection limits. *Nanotechnology*, as a whole, offers unprecedented opportunities to realize such devices, thus increasing the efficacy of diagnosis (Liotta et al., 2003). In the paragraphs above, the theory of plasmons and the use of plasmonic devices have been largely described. Here, the integration of this technology with superhydrophobic surfaces (SHSs) is introduced.

The aim of this paragraph is to expound on the contributions that this combination can possibly offer to clinical medicine, specifically in the discipline of early diagnostics. In particular, it revolves around the study and the micro/nano fabrication of SHSs which

hexagonal geometry of the holes. Important features emerging from the simulations are both the mean (Emean) and the effective (Eeff) value of the electric field outside the holes (shaded regions in Fig. 3.3c). The former is simply defined as the average field whereas the latter is given by the difference between the maximum and minimum values in the hole.

In conclusion, home-built AuAPA substrates with pore size of 60 nm and wall thickness of 40 nm was utilized for large area SERS substrates. CV was deposited using chemisorption technique, using which a monolayer of molecules can be achieved. CST simulations of the electric field distribution on these ordered and reproducible SERS substrates were also performed, keeping the structural parameters as close as possible to the experimental honey-comb nanostructures. Theoretical results follow the same trend of experimental findings. The major advantage of using nanoporous alumina substrates, as compared to the traditional colloidal coating or lithographic processing, is a good trade-off between the high enhancement factor '*G*' obtained and the large surface area produced. The respective SERS

Further research should be made in order to optimize '*G*' on the basis of the substrate parameters of pore size, wall thickness, and thickness of the gold coating. The next step towards reproducible micro-fabricated SERS devices would be the transfer of APA to technological substrates such as optical glass or silicon wafer, and combining APA patterning with optical lithography. In this way, large-area SERS bio- and chemical sensors assays for e.g. parallel biomedical screening of different body liquids or even tissues could be carried out, conveniently. The easy and inexpensive processing required for APA SERS fabrication would also make these substrates disposable, opening the way to their large scale applications. Such a high SERS enhancement as demonstrated here can provide single

molecule sensitivity in a sensor based on labeling using fluorescence dyes.

of this technology with superhydrophobic surfaces (SHSs) is introduced.

**4. Integration of biophotonics and superhydrophobic devices for the** 

In biomedical disciplines including medicine, biology or biotechnology, there is a huge interest in understanding the pathogenesis of diseases, and to develop new biomarkers for their diagnosis. In particular, early detection can lead to significant benefits in terms of efficient and timely treatment. In these regards, the blood contains a multitude of unstudied and unknown biomarkers that could reflect the ongoing physiologic state of tissues and organs. The lowmolecular weight region of the blood proteome is an important source of diagnostic markers (Posadas et al., 2001). Nevertheless, this 'mine' of data for diagnosis is often practically inaccessible by conventional approaches, in that the solutions at study are extremely diluted, and contain a multitude of substances that would blur the signal of interest. To exploit this 'mine' of data for diagnosis, new devices are required with very low detection limits. *Nanotechnology*, as a whole, offers unprecedented opportunities to realize such devices, thus increasing the efficacy of diagnosis (Liotta et al., 2003). In the paragraphs above, the theory of plasmons and the use of plasmonic devices have been largely described. Here, the integration

The aim of this paragraph is to expound on the contributions that this combination can possibly offer to clinical medicine, specifically in the discipline of early diagnostics. In particular, it revolves around the study and the micro/nano fabrication of SHSs which

enhancement factor '*G*' is estimated to be ~4106.

**detection of few bio-molecules**

incorporate biophotonic devices. Well assessed mechanisms such as (i) superhydrophobicity and (ii) SERS (Surface Enhanced Raman Scattering) are recapitulated and integrated into a single micro- nano- system. The combination of these two would represent a boost towards the detection and the analysis of few (or single) molecules. In the following, SHSs and nano optics based photonic devices are treated separately. After an introduction of the most important features of these, their combination and the effects thereof are discussed. Notice that, differently from plasmonic devices, SHSs are exposed to a good detail in that they are introduced here for the first time in the text.

In short, the major novelty, here, is the simultaneous use of wetting mechanisms (that arise due to the superhydrophobic surfaces, SHSs) and sensitive materials (that include, randomly distributed silver nanograin aggregates, regular arrays of metallic nano dots, adiabatic nanofocusing cones) to increase the response of nano optics based spectroscopy devices (see the cartoon representation of Fig. 4.1). By doing so, otherwise inaccessible information about the biological moieties at study is disclosed.

Fig. 4.1. Raman spectroscopy through super-hydrophobicity.

#### **4.1 Superhydrophobic surfaces**

It is well known that a drop post upon a solid surface develops a contact with the solid described by the sole parameter e (Fig. 4.2A) that is the equilibrium contact angle at the interface between the liquid and the solid. e obeys the celebrated Young equation (Young, 1805):

$$\cos \theta\_e = \frac{\chi\_{SV} - \chi\_{SL}}{\mathcal{Y}\_{LV}} \tag{4.1}$$

where ij is the surface tension between the phase i and j, and the letters S, L, V stand for the solid, liquid and vapour, and thus equation (1) may be regarded as a simple balance of

Photonic Crystals for Plasmonics: From Fundamentals to Superhydrophobic Devices 273

In the Cassie state the drop would roll upon the surface and it would progressively reduce its contact area during a process of evaporation. The drop would then maintain the Cassie state over time, thus avoiding collapse and any irreversible transition to Wenzel until a critical radius of impalement is achieved. The mechanisms inducing collapse in a slowly evaporating droplet are two and, namely, (i) the drop could either touch the surface below the posts, or (ii) the surface free energy gained as the drop collapses wins over the surface free energy lost by increased contact with the hydrophobic posts. While the first mechanism regards surfaces decorated with short posts or pillars, the second relies upon an energetic argument and is independent on the pillars' height. In many practical situations one should consider the latter criterion solely (De Angelis et al., 2011). In particular, both analytical calculations and numerical simulations show that the critical radius of impalement depends

min

*r*

cos *<sup>e</sup>*

and thus the closer the pillars the smaller the final area of contact. From the analysis above it stems out that surfaces with large (that is, with sufficiently dense pillars), would guarantee a stable Cassie configuration. Notice though that large would induce small contact angles, and thus the choice for the best design parameters is not trivial, and it is indeed a matter of

SHSs retain unique properties in terms of wettability that can be reviewed as follows: (i) SHSs have superior adhesive properties, in the sense that they exhibit vanishing friction coefficients; (ii) a droplet, post upon these surfaces, would accordingly preserve a quasispherical shape while evaporates, and the contact area at the interface would thus progressively reduce; (iii) SHSs can be artificially reproduced using micro and nano fabrication techniques. Using the properties above, micro textured surfaces may be successfully exploited to concentrate tiny amounts of moities over micrometric areas, and consequently measure these moieties with unprecedented accuracy (De Angelis et al., 2011;

Accardo et al., 2011; Gentile et al., 2011a; Gentile et al., 2011b; Di Fabrizio et al., 2008).

SHSs typically comprise a *periodic* hexagonal lattice of cylindrical Si micro pillars with a certain diameter and pitch (Fig. 4.3A). Due to the surface patterning the drop experiences an increased contact angle that can be theoretically predicted (Fig. 4.3B). The diameter *d* of the pillars and the distance between the pillars (gap) may be arbitrarily imposed, and can be chosen in accordance to a criterion of optimal design. This criterion would guarantee the best trade-off between sufficiently dense forests of pillars, that prevent the early collapse of the drop, and diluted structures, that instead assure large contact angles (De Angelis et al., 2011). The height of the pillars h is chosen to be as large as, at least, two times d, and this would avoid the spontaneous impalement of the drop. For the present configuration, d=10m,=20 m, and h=20 m. The pillars are arranged to recover a honeycomb lattice; notice that when these are combined with adiabatic nanofocusing cones the symmetry of the pattern is broken and, on account of this, the biomolecules are enforced to deposit upon the cones. The micro pillars are typically realized combining optical lithography, electroless

(4.4)

upon the distance between the pillars and e as

optimization.

**4.1.1 Fabrication of SHSs** 

forces. For sufficiently small droplets the dominant force becomes the liquid-vapor surface tension and gravitation may be neglected. The dimensionless Bond number can be consequently introduced as Bo=×g×R2/LV, where is the density of the liquid, R is the radius of the spherical drop prior the deposition upon the surface, and g is the acceleration due to gravity. When Bo<<1 gravitational effects vanish and the shape of the droplet may be assumed spherical everywhere. For a drop of water with LV=72.9 mJ/m2, =1000 kg/m3, and diameter d=2×R=1 mm, it follows that Bo~0.035, and thus the physics of micrometric or submillimetric drops is correctly governed by surface tension solely.

*Superhydrophobicity* (Fig. 4.2B) is a phenomenon whereby a drop post upon a surface would preserve its original spherical shape rather than spreading or wetting indefinitely the plane of contact (Lafuma & Quéré, 2003; Blossey, 2003). The theoretical conundrum explaining this mechanism dates back to the mid forties of the last century and is very well assessed. In the celebrated model of Cassie, a surface would be superhydrophobic on account of the pockets of air that remain trapped between the liquid and the substrate (Fig. 4.2C), and the smaller the fraction of solid in contact with the drop () the larger the apparent contact angle. In particular, the increased contact angle <sup>e</sup> c>e the drop experiences may be theoretically written as (Cassie & Baxter, 1944)

$$\cos \theta\_{\varepsilon}^{\varepsilon} = -1 + \phi\_{s} \left( \cos \theta\_{\varepsilon} + 1 \right) \tag{4.2}$$

where s is the fraction of solid in contact with the drop. The less s the larger the apparent contact angle. At the limit of s0, the drop would paradoxically float in air. The Cassie model is intuitive in that predicts that a drop upon a patterned surface 'sees' a contact angle that is proportional to the fraction of air in contact with the drop. A flat surface would have s=1 and accordingly <sup>e</sup> c=e. Another mechanism (Wenzel model) explains the increased contact angle on account of the roughness, and thus relies upon geometric effects solely (Fig. 4.2D). The modified angle <sup>e</sup> w is related to the unmodified contact angle e as (Lafuma & Quéré, 2003):

$$r\cos\theta\_{\varepsilon}^{\text{av}} = r\cos\theta\_{\varepsilon} \tag{4.3}$$

Where *r* is the solid roughness defined as the ratio between the real the projected surface, and thus the effect of surface roughness is to amplify the wetting. Noticeably, when e</2, *r* increases the hydrophilicity of the surface, whereas for e>/2 roughness promotes hydrophobicity. In spite of the fact that they do both induce hydrophobicity, these situations are very different when considering their adhesive properties. In Wenzel state drops are found to be highly pinned. On the contrary, in the Cassie state the drop sits mainly upon air, and this increases the contact angle.

Fig. 4.2. Superhydrophobicity and contact angle.

In the Cassie state the drop would roll upon the surface and it would progressively reduce its contact area during a process of evaporation. The drop would then maintain the Cassie state over time, thus avoiding collapse and any irreversible transition to Wenzel until a critical radius of impalement is achieved. The mechanisms inducing collapse in a slowly evaporating droplet are two and, namely, (i) the drop could either touch the surface below the posts, or (ii) the surface free energy gained as the drop collapses wins over the surface free energy lost by increased contact with the hydrophobic posts. While the first mechanism regards surfaces decorated with short posts or pillars, the second relies upon an energetic argument and is independent on the pillars' height. In many practical situations one should consider the latter criterion solely (De Angelis et al., 2011). In particular, both analytical calculations and numerical simulations show that the critical radius of impalement depends upon the distance between the pillars and e as

$$r\_{\min} = \frac{\mathcal{S}}{\cos \theta\_{\varepsilon}} \tag{4.4}$$

and thus the closer the pillars the smaller the final area of contact. From the analysis above it stems out that surfaces with large (that is, with sufficiently dense pillars), would guarantee a stable Cassie configuration. Notice though that large would induce small contact angles, and thus the choice for the best design parameters is not trivial, and it is indeed a matter of optimization.

SHSs retain unique properties in terms of wettability that can be reviewed as follows: (i) SHSs have superior adhesive properties, in the sense that they exhibit vanishing friction coefficients; (ii) a droplet, post upon these surfaces, would accordingly preserve a quasispherical shape while evaporates, and the contact area at the interface would thus progressively reduce; (iii) SHSs can be artificially reproduced using micro and nano fabrication techniques. Using the properties above, micro textured surfaces may be successfully exploited to concentrate tiny amounts of moities over micrometric areas, and consequently measure these moieties with unprecedented accuracy (De Angelis et al., 2011; Accardo et al., 2011; Gentile et al., 2011a; Gentile et al., 2011b; Di Fabrizio et al., 2008).

#### **4.1.1 Fabrication of SHSs**

272 Photonic Crystals – Innovative Systems, Lasers and Waveguides

forces. For sufficiently small droplets the dominant force becomes the liquid-vapor surface tension and gravitation may be neglected. The dimensionless Bond number can be consequently introduced as Bo=×g×R2/LV, where is the density of the liquid, R is the radius of the spherical drop prior the deposition upon the surface, and g is the acceleration due to gravity. When Bo<<1 gravitational effects vanish and the shape of the droplet may be assumed spherical everywhere. For a drop of water with LV=72.9 mJ/m2, =1000 kg/m3, and diameter d=2×R=1 mm, it follows that Bo~0.035, and thus the physics of micrometric or

*Superhydrophobicity* (Fig. 4.2B) is a phenomenon whereby a drop post upon a surface would preserve its original spherical shape rather than spreading or wetting indefinitely the plane of contact (Lafuma & Quéré, 2003; Blossey, 2003). The theoretical conundrum explaining this mechanism dates back to the mid forties of the last century and is very well assessed. In the celebrated model of Cassie, a surface would be superhydrophobic on account of the pockets of air that remain trapped between the liquid and the substrate (Fig. 4.2C), and the smaller the fraction of solid in contact with the drop () the larger the apparent contact angle. In

cos 1 cos 1 *<sup>c</sup>*

 

where s is the fraction of solid in contact with the drop. The less s the larger the apparent contact angle. At the limit of s0, the drop would paradoxically float in air. The Cassie model is intuitive in that predicts that a drop upon a patterned surface 'sees' a contact angle that is proportional to the fraction of air in contact with the drop. A flat surface would have s=1 and

on account of the roughness, and thus relies upon geometric effects solely (Fig. 4.2D). The

cos cos *<sup>w</sup> e e*

Where *r* is the solid roughness defined as the ratio between the real the projected surface, and thus the effect of surface roughness is to amplify the wetting. Noticeably, when e</2, *r* increases the hydrophilicity of the surface, whereas for e>/2 roughness promotes hydrophobicity. In spite of the fact that they do both induce hydrophobicity, these situations are very different when considering their adhesive properties. In Wenzel state drops are found to be highly pinned. On the contrary, in the Cassie state the drop sits mainly upon air,

*r*

c=e. Another mechanism (Wenzel model) explains the increased contact angle

w is related to the unmodified contact angle e as (Lafuma & Quéré, 2003):

c>e the drop experiences may be theoretically

(4.3)

*e se* (4.2)

submillimetric drops is correctly governed by surface tension solely.

particular, the increased contact angle <sup>e</sup>

written as (Cassie & Baxter, 1944)

and this increases the contact angle.

Fig. 4.2. Superhydrophobicity and contact angle.

accordingly <sup>e</sup>

modified angle <sup>e</sup>

SHSs typically comprise a *periodic* hexagonal lattice of cylindrical Si micro pillars with a certain diameter and pitch (Fig. 4.3A). Due to the surface patterning the drop experiences an increased contact angle that can be theoretically predicted (Fig. 4.3B). The diameter *d* of the pillars and the distance between the pillars (gap) may be arbitrarily imposed, and can be chosen in accordance to a criterion of optimal design. This criterion would guarantee the best trade-off between sufficiently dense forests of pillars, that prevent the early collapse of the drop, and diluted structures, that instead assure large contact angles (De Angelis et al., 2011). The height of the pillars h is chosen to be as large as, at least, two times d, and this would avoid the spontaneous impalement of the drop. For the present configuration, d=10m,=20 m, and h=20 m. The pillars are arranged to recover a honeycomb lattice; notice that when these are combined with adiabatic nanofocusing cones the symmetry of the pattern is broken and, on account of this, the biomolecules are enforced to deposit upon the cones. The micro pillars are typically realized combining optical lithography, electroless

Photonic Crystals for Plasmonics: From Fundamentals to Superhydrophobic Devices 275

properties are also different, thus creating well defined and distinguished tracks or patterns, and the drop could be exteriorly positioned into a precise area or point of the substrate. This argument is the ground for novel droplet-based microfluidic systems as reported in a number or publications and patents, and these surface would be in fact lab-on-chip systems which deliver the ability of performing bio-chemical detections and reactions with incomparable accuracy and efficiency. Recently, the use of superhydrophobic surfaces to

Biophotonic nanostructures can be integrated to SHSs to obtain devices with advanced sensing capabilities. Here we shall recapitulate in short specific types of nano-geometry based plasmonic device, and namely (i) electroless grown random assemblies of silver nanograins; (ii) regular arrays of metallic nano dots; (iii) adiabatic nanofocusing cones. Nevertheless, the method can be extended to a number of different plasmonic nanostructures, as those broadly described in the paragraphs above (De Angelis et al., 2011; Accardo et al., 2011; Gentile et al.,

The electroless growth is a process whereby ionic silver is reduced and deposited as metallic silver upon silicon, via a redox reaction, and according to a mechanism that may be adequately described by a diffusion limited aggregation model. A satisfactory comprehension of the method may be found in (Coluccio et al., 2009). Here it is briefly recalled that, conveniently employing this process, and depending upon the growth parameters, silver nanograins may be obtained with an average size as small as few tens of

concentrate and localize a solute has been proposed.

**4.2.1 Electroless grown silver nanograins** 

**4.2.2 Regular arrays of metallic nano dots** 

2011a; Gentile et al., 2011b; Di Fabrizio et al., 2008; Gentile et al., 2010).

nanometers, which compose efficient SERS substrates (Fig. 4.4A).

Fig. 4.4. SEM images of a metallic photonic crystal and plasmonic device.

These are gold or silver hemisphere positioned upon the substrate as to reproduce a regular square lattice. Optical lithography, electron beam lithography, Reactive Ion Etching, evaporation techniques are routinely used for the fabrication of these devices. Differently from the case above, the shape, dimension, position of the dots is deterministic in the limit of the fabrication process. Thereby SERS effects arise from the rational design of the nano

**4.2 BioPhotonic devices** 

growth and Bosch Reactive Ion Etching (RIE) techniques. Non conventional biophotonic nanostructures as those described below, conveniently positioned upon the pillars, would complete a hierarchical structure thus permitting the identification of proteins in the single molecule regime (Fig. 4.3C). These 'two-stages' micro nano structures function as SHSs with an increased contact angle ranging from 155° to about 175°.

Fig. 4.3. SEM image of a photonic crystal for superhydrophobicity applications.

#### **4.1.2 Current applications of SHSs**

Many applications exist revolving around the use of superhydrophobic surfaces and mainly founding upon two key features of these, that are the limited contact area and the ability of a drop to slide or slip upon such surfaces (Li et al., 2007). Water repellent coatings for radar domes, satellite dishes and glass are widely reported and currently in use. These coatings provide self-cleaning under the action of rain in that they mimic the self-cleaning action of the Lotus leaf. Other utilizations include antibiofouling paints for boats, antisticking of snow for antennas and windows, self-cleaning windshields for automobiles, the separation of water and oil, and the manufacture of water-proof fireretardant clothes. The latter would represent examples of a target market that is the huge household-commodity sector, nevertheless new analytical potentials also do exist which carry the promise of unparalleled opportunities in life sciences and in the high tech field of biotechnology. In Biotechnology many efforts are devoted to manipulate, control and analyse relevant molecules as DNA and proteins, and artificially (nanotech) fabricated superhydrophobic surfaces could be an effective advance towards this objective. Let's consider, for instance, the case of microarrays. These are libraries of biological or chemical entities (probes) immobolised in a grid on a solid surface, the probes would be then interacting with targets (i.e., geneses, proteins, cDNA...) to provide deep insight into DNA sequences. It is desirable that the drop containing the targets would evaporate avoiding pinning and the formation of noxious ring like structures (it is in fact well known that pinning during evaporations causes final solute formations disposed in doughnut shape, and this is commonly known as the coffee-ring effect, and here suitable superhydrophobic substrates come into play.

The transport, separation and mixing of moieties dispersed in droplets has an enormous interest likewise. Superhydrophobic surfaces are slippy, and thus a droplet would move upon the application of an infinitesimal external force field: tilting the surface (and thus gravitation) or electrostatic or surface waves induced force fields would move the droplet in a controllable way (see also below in materials and methods). On the other hand, variations in the patterning or texture of the surface would generate regions where the adhesive properties are also different, thus creating well defined and distinguished tracks or patterns, and the drop could be exteriorly positioned into a precise area or point of the substrate. This argument is the ground for novel droplet-based microfluidic systems as reported in a number or publications and patents, and these surface would be in fact lab-on-chip systems which deliver the ability of performing bio-chemical detections and reactions with incomparable accuracy and efficiency. Recently, the use of superhydrophobic surfaces to concentrate and localize a solute has been proposed.
