**3. Construction basics**

microtoroid shaped resonators on a chip, the stand-alone dielectric microspheres and the so called capillary-based opto-fluidic ring resonators are the most common examples. The chip-based ring resonators present some advantages which include the capability of opto‐ electronic integration, but, apart from the microtoroid configuration, they usually present problems of low quality factors (Q-factor), which are designated as all intrinsic and extrin‐ sic losses occurred in the optical resonant cavity system [52] and in this case, these prob‐ lems are related to their surface roughness. These types of ring resonators are very well

This class of biosensors is, in fact, an evolution of the optical fiber based ones. They are formed by photonic crystal microcavities, obtained by introducing a defect in periodically organized microstructured holes, usually of silica, by altering their dimensions. Some can be embedded with molecules, which are responsible for the occurrence of a change in the re‐ fractive index of the biosensor, leading to a detectable signal in the form of a spectral shift of the resonant wavelength of the photonic crystal cavity. Also, polymers can be used as a coat‐ ing layer for the photonic crystal cavities, as showed by Chakravarty et al., [53] which doped the photonic crystal microcavities with a quantum dot and coated it with anion-selective polymer. With this procedure, they were able to construct a sensor with good properties, such as a very specific and accurate detection for changes of perchlorate anions and calcium cations at submicro concentrations in solution, while Lee et al. [54] presented a photonic crystal suitable for protein and single particle detection. In their experimental and theoreti‐

sented a limit of detection as small as 1 fg. They also determined its performance for

In a recent work, Aroua et al.[55] have studied, also by experimental and theoretical means, a label-free biosensor in order to determine it characteristics, the field intensity and the reso‐ nant wavelength shift when the nanocavities of the photonic crystal are filled with blood plasma, water or dried air. With this protocol, they showed that the enhancement on sensi‐

Some new materials had also found a great deal of applications, especially in biosensing, such as carbon nanotubes and lately, graphene. As for single-walled carbon nanotubes, (SWNTs), they are known to exhibit unique intrinsic properties, which include a semiconductive behav‐ ior and photophysical propertiesdependent of their structure. For example, nanotubes with some chirality, band gap fluorescence is observed, as well as strong resonance Raman scatter‐ ing.In this way, hybrid materials of SWCNTs and biomolecules is a way to obtain good materi‐ als for biosensing applications, since the fluorescence band-gap of SWNTs is highly sensitive to its environment and show shifts when the nanotube is in contact with other molecules.

In their work, Jin et al. [56] proposed the construction of a platform for selectively deter‐ mine the hydrogen peroxide efflux from living cells, in order to biosensing human carci‐

, and that it pre‐

cal work, they claimed their device achieved a sensing volume of 0.15 µm3

tivity is related to the photonic crystal design parameters.

**2.8. Carbon nanotubes and graphene-based biosensors**

particles in the size range of a variety of viruses, using latex spheres as models.

presented and discussed in Fan´s review. [46]

**•** Photonic crystal

126 State of the Art in Biosensors - General Aspects

The devices architectures that have been found in the literature and in the market are as nu‐ merous as the applications that they find. As well resumed by Reardon et al., [61] optical sensing opens a wide range of methodologies that can be based in either one of the electro‐ magnetic wave parameters analysis, as their phase, polarization and amplitude. Analysis and detection methods applied to the amplitude parameters are the most common, due to the direct information that they can give about the system and due to the possibility of cou‐ pling this information to those obtained by polarized systems or phase optics.

There are no limits to explore regarding to optical properties or to instrumentation to be ap‐ plied, alone or combined. Nevertheless, the most common optical approach is to promote the interaction of the analyte with light to produce light as response. Conventional methods permit the detection of absorption, reflection or scattering of light, through which it is possi‐ ble to infer about the interaction between light and the analyte through signal changes.

Most elegant methods employ the detection (and promotion) of emitted light from the ana‐ lyte, which in general relies on larger wavelengths that those used to illuminate the sample. The wavelength shift can be caused by either nonlinear interaction processes between light and analyte, as harmonic or inharmonic oscillations, such as vibrational modes that give rise to Raman shifts or by the electronic excitation of a system that result in the loss of a portion of the excitation energy as photons through luminescent phenomena that occur at shifted wavelength range. These luminescent processes include fluorescence and phosphorescence, which differ from each other with respect to the quantum levels involved in each electronic transition and that are affected by the interaction with the environment and, consequently, to analyte concentration.

There are diverse new methods of optical detection involving combined techniques to deliv‐ er real-time imaging of the sample. In this sense, fluorescence or Raman confocal microscop‐ ies are coupled to time-resolved fluorescence or to Raman spectrophotometers and images obtained can be evaluated in terms of concentration, energetic processes, charge transfers and localized events.Once detection methods and finality of the biosensor are defined, the size scale must be determined and, then, convenient materials for the construction of the bi‐ osensor must be selected and tested.

With respect to materials, it is important to consider environmental, economical and sustain‐ able issues when choosing the ideal material combination for a biosensor. It is important to elect safe and ease of processing materials that can lead to a cheap method of fabrication and, in addition, can be recycled. Also, it needs to be chemically stable and permits a good selectivity and sensibility to the biosensor. Thus, the functional nanostructures can be com‐ posed of metals, semiconductors, magnetic materials, quantum dots, molecular or polymeric dyes and some hybrid materials, proposed to achieve any specific property that is an issue in other materials. [62]

Electrodes must be also adequately elected, with respect to work function as well as to proc‐ essing. Usually, gold surface is preferred due to its possibility of modification with sulfur containing biological elements, such as SH-protein and antibodies, but also due to its optical and low potential properties, which enables refractive index measurements by several tech‐ niques, as mentioned in previous sections. Nevertheless, there are some alternative electro‐ des, such as metal-modified carbon electrodes, as those described by Wang et al. in which carbon electrodes were modified with rhodium [63], ruthenium [64] and platinum [65] to achieve lower potentials. More recently, some works presented Indium Tin Oxide thin lay‐ ers as alternative electrodes for optoelectrical biosensors, with transmittance similar to the glass substrate. In their work, Choi et al. [66] showed that a 100 nm thick ITO layer as elec‐ trode permits simultaneous optoelectric measurements to record optical images and micro‐ impedance to examine time-dependent cellular growth.

sensing opens a wide range of methodologies that can be based in either one of the electro‐ magnetic wave parameters analysis, as their phase, polarization and amplitude. Analysis and detection methods applied to the amplitude parameters are the most common, due to the direct information that they can give about the system and due to the possibility of cou‐

There are no limits to explore regarding to optical properties or to instrumentation to be ap‐ plied, alone or combined. Nevertheless, the most common optical approach is to promote the interaction of the analyte with light to produce light as response. Conventional methods permit the detection of absorption, reflection or scattering of light, through which it is possi‐ ble to infer about the interaction between light and the analyte through signal changes.

Most elegant methods employ the detection (and promotion) of emitted light from the ana‐ lyte, which in general relies on larger wavelengths that those used to illuminate the sample. The wavelength shift can be caused by either nonlinear interaction processes between light and analyte, as harmonic or inharmonic oscillations, such as vibrational modes that give rise to Raman shifts or by the electronic excitation of a system that result in the loss of a portion of the excitation energy as photons through luminescent phenomena that occur at shifted wavelength range. These luminescent processes include fluorescence and phosphorescence, which differ from each other with respect to the quantum levels involved in each electronic transition and that are affected by the interaction with the environment and, consequently,

There are diverse new methods of optical detection involving combined techniques to deliv‐ er real-time imaging of the sample. In this sense, fluorescence or Raman confocal microscop‐ ies are coupled to time-resolved fluorescence or to Raman spectrophotometers and images obtained can be evaluated in terms of concentration, energetic processes, charge transfers and localized events.Once detection methods and finality of the biosensor are defined, the size scale must be determined and, then, convenient materials for the construction of the bi‐

With respect to materials, it is important to consider environmental, economical and sustain‐ able issues when choosing the ideal material combination for a biosensor. It is important to elect safe and ease of processing materials that can lead to a cheap method of fabrication and, in addition, can be recycled. Also, it needs to be chemically stable and permits a good selectivity and sensibility to the biosensor. Thus, the functional nanostructures can be com‐ posed of metals, semiconductors, magnetic materials, quantum dots, molecular or polymeric dyes and some hybrid materials, proposed to achieve any specific property that is an issue

Electrodes must be also adequately elected, with respect to work function as well as to proc‐ essing. Usually, gold surface is preferred due to its possibility of modification with sulfur containing biological elements, such as SH-protein and antibodies, but also due to its optical and low potential properties, which enables refractive index measurements by several tech‐ niques, as mentioned in previous sections. Nevertheless, there are some alternative electro‐ des, such as metal-modified carbon electrodes, as those described by Wang et al. in which

pling this information to those obtained by polarized systems or phase optics.

to analyte concentration.

128 State of the Art in Biosensors - General Aspects

in other materials. [62]

osensor must be selected and tested.

Transduction methods are responsible for the identity of the biosensor and, thus for address their applicability. Since the first step on the optical transduction in a biosensor is the chemi‐ cal interaction between the analyte and the indicator phase (the recognition element) that will produce the optically detectable signal, it is critical because it will determine the biosen‐ sor stability, selectivity and sensitivity as well as the optical region in which the effect will be observed and the best detection means. Also, it is important to take into account whatev‐ er the biosensor will be used in continuous measurements or in simple detection. It will in‐ form if strong interactions will be needed and in what conditions the biosensor can be employed. If chemical reactions are the signal origin, such it is in catalysis-based biosensors, an immobilized enzyme is preferred, once it can permits steady-state measurements to ex‐ plore the sample. In this case, high selectivity can be achieved using antibodies as reagents and basing sensing on competitive binding. Also, methods to immobilize the recognition el‐ ement include adsorption on solid substrates, covalent bonding to a substrate and confine‐ ment by membranes with selective permeability. [67]

In optical fiber-based biosensors, immobilized recognition elements are also needed, as well as the optical fiber to enable remote measurements. The immobilization technique and the reagent must be well selected to avoid undesired effects such attenuation on fiber´s trans‐ mission efficiency, coupling of light into the fiber and attenuation characteristics of the fiber itself. Nevertheless, this is a good choice for transduction since it enables a range of photo‐ physical processes to be detected, including light absorption, absorption followed by lumi‐ nescence, light reflection and scattering, among others. Optical instrumentation is then selected, based on the most important photophysical effect. [68]

Finally, when proposing a biosensor, it is always good to make two considerations: first, nanoscaled materials are not really small to living bodies, they can be sensed as intruder‐ sand be attacked and second, biosensors, in some way, will be employed in a living body. Issues related to toxicity, lifetime, stability, durability, mechanical properties, body adjust‐ ment, coherence and adaptability must be evaluated by a scientific and systematic method. It is not unusual to find information that was mistakenly collected, and conflicting results can be found on the same subject, leading to confusion. For example, carbon nanotubes are said to be health safe, nevertheless it is known that their structure is very similar to that of asbestos, great villain to miner´s lungs. Actually, aspired nanosize fibers of asbestos are rec‐ ognized by the lung cells as an aggressive agent, which can cause lung cancer, pleural disor‐ ders, pleural plaques, pleural thickening, and pleural effusions. Another aspect is that asbestos present some properties such as electricity insulate and is a chemically resistant material and it is quickly incorporated in many materials.
