5.2 Sample control and fluidics
5.2.3 Optofluidics and RWG
In addition to permeable pores, nanofluidics in the context of optofluidics also uses small volume pits to which molecules are attached or pulled. This is the case for one of the most important applications so far: a novel single molecule-based sequencing
holes, pits and clefts are the focus now rather than long channels that conduct fluids, as seen with microfluidics. On the other hand, much is known about nanopores in nature, e.g. the aforementioned ion channels. Importantly, when structural features are made this small, as small as the molecules themselves, theoretical methods can change from the physical modelling of fluids to computational chemistry and molecular dynamics. However, the grey zone (10-100 nm) between the micron scale and truly nanometre scale pores, is still between different approaches. In this section, pores below 10 nm are mainly discussed.
Biological membranes are largely impermeable for water, ions, and other electrostatically charged or polar molecules;
therefore, membranes are perforated with the variety of biological nanopores, with higher or lower selectivity and working passively or consuming or producing energy. They cover size scale from several nanometres of the nuclear pore complex (spanning two lipid bilayers) down to the size of ion channels with pore sizes as small as ion radii without solvation.
Remarkably, natural water conducting pores, aquaporins, can be impermeable to single H+ ions but permeable to water and much larger polar molecules (Wu & Beitz, 2007). The underlying mechanism for such selective water-conducting must be based on evolutionarily selected amino acid (aa) residues (AAs) that allow water molecules to form favourable transient hydrogen bonding. Similar function could be achieved by immobilising aa or other molecules into the walls of a very narrow channel. That could drastically change the permeability for different molecules trying to pass through the manmade structure.
Indeed, with this approach, nanofluidic biological pores through membranes are constructed with self-assembled DNA structures (Burns et al., 2013) and artificial ion pumps have been made (Zhang et al., 2013). Furthermore, manmade pores have been covered with lipid layers, resulting in lower clogging in the device and the translocation of proteins through the channel/pore (Yusko et al., 2011). Lipids and other long chain
alkyls containing molecules can be practical due to their tendency to self-organise to layers (Love et al., 2005). If such molecules contain a thiol group at the end that faces the substrate, they readily bind covalently to the substrate. In turn, via the selection of the part facing the solvent, interactions with passing molecules within the channel or pore would be affected.
Carbon-based materials are good examples of macroscopically hydrophobic materials that show interesting properties at the nanoscale. For example, while a micron-sized capillary made of carbon would hardly conduct water flow within ambient conditions, theoretical simulations can reveal different behaviours for their nanosized counterparts (Hummer et al., 2001); carbon nanotubes can toggle between filled and empty states and hence conduct water in amounts that are comparable to natural water channels (Zeidel et al., 1992), the aquaporins.
A lot of research has been conducted on the unique electrical and optical properties of graphene sheets (Avouris, 2010) and carbon tubes (Avouris & Chen, 2006). In addition to their specific thermal and mechanical properties, their impermeability for small molecules and gases (Bunch et al., 2008) is especially interesting in the nanofluidic context, particularly since the impermeability can be cancelled by perforating a graphene sheet with nanopores. That can make them capable of separating small molecules by size (Jiang et al., 2009) or working like ion channels (Sint et al., 2008). Graphene sheets can be combined with structural surfaces (Bunch & Dunn, 2012) and optical properties; all of these reasons makes it an attractive material for nano optical and fluidic devices.
5.2.3OPTOFLUIDICS AND RWG
In addition to permeable pores, nanofluidics in the context of optofluidics also uses small volume pits to which molecules are attached or pulled. This is the case for one of the most important applications so far: a novel single molecule-based sequencing
method (Eid et al., 2009). The method is optically based on the so-called zero-mode waveguides (Levene et al., 2003), which means that no modes of propagating waves are supported by the metallic nanostructure for certain wavelengths. The pits are extremely small (zeptolitre) in volumes where the reactions by single molecules are performed and monitored with the aid of a high intensity field. The method has already been commercialised and is part of the latest wave of the revolution of the sequencing methods. This indeed demonstrates the potential of nano and optofluidics.
Although the definition of optofluidics can vary, one describes it as a combination of microfluidics and optics or photonics (Schmidt & Hawkins 2011). However, together with plasmonic membranes, nanofluidics, light-inducible chemistry (Klajn et al., 2007), and liquid waveguides (Wolfe et al., 2005) among others, it holds no unambiguous definition. Nevertheless, since the current manufacturing methods and materials are not limiting the development—quite the contrary, optofluidics will probably have a long history ahead.
An example of an imaginary optofluidic device, combining RWG and plasmonic membranes, is presented in Figure 10. Such a two compartment fluidic device could, for instance, support the manipulation of optical functions by the distinct or variable RI of the liquids, as noted earlier (last paragraph in Chapter 5.2.1). Herein, nanofluidics means the diffusion of soluble analytes through pores perforating the middle layer. In this case, it is made of metal, but it could be carbon as well, as both support plasmonics.
Figure 10. Imaginary optofluidic device. The orange arrow shows the direction of the micron scale flow, while the green ones show one possible direction of nanoscale diffusion. The middle component is functionalised with a self-assembled monolayer and the close-up is shown left, where the letter S indicates the employment of thiol chemistry while the letter X represents a chemical group facing the liquid. Super- and substrates would be good targets for imprinted electrodes (yellow bars) or printed micro and nano optics. Spacers could be fabricated as integral parts of the layers (as in the superstrate). Alternatively, they could be added as polymer fibres (light pink bars between two bottom layers).
The middle layer in this imaginary scheme (Figure 10) is accompanied with tangential flow through the space above it.
This, in comparison to placing nanopores at a dead-end position, would result in a lower level of concentration polarisation i.e.
the accumulation of particles or molecules that cannot pass the membrane through these pores.
Summing up, micro- and nanofluidics have been materialised in nature during multicellular evolution, which has created apparatuses like the mammalian kidney, where microfluidic channels work in concert with nanoscale pores.
From such, we have learned the principles of how to separate and sort molecules from larger particles. The foundations of artificial sorting molecules and particles, in turn, rest on over a century of history of the molecular biology. Today’s micro- and nano-methods allow us to fabricate devices combining both micro- and nano-fluidics, and to integrate electronics and optics into them. Thus, molecules, cells and droplets are recognised and even sorted with the optics of a device. Such man-made devices can be further functionalised with “smart” chemicals or
method (Eid et al., 2009). The method is optically based on the so-called zero-mode waveguides (Levene et al., 2003), which means that no modes of propagating waves are supported by the metallic nanostructure for certain wavelengths. The pits are extremely small (zeptolitre) in volumes where the reactions by single molecules are performed and monitored with the aid of a high intensity field. The method has already been commercialised and is part of the latest wave of the revolution of the sequencing methods. This indeed demonstrates the potential of nano and optofluidics.
Although the definition of optofluidics can vary, one describes it as a combination of microfluidics and optics or photonics (Schmidt & Hawkins 2011). However, together with plasmonic membranes, nanofluidics, light-inducible chemistry (Klajn et al., 2007), and liquid waveguides (Wolfe et al., 2005) among others, it holds no unambiguous definition. Nevertheless, since the current manufacturing methods and materials are not limiting the development—quite the contrary, optofluidics will probably have a long history ahead.
An example of an imaginary optofluidic device, combining RWG and plasmonic membranes, is presented in Figure 10. Such a two compartment fluidic device could, for instance, support the manipulation of optical functions by the distinct or variable RI of the liquids, as noted earlier (last paragraph in Chapter 5.2.1). Herein, nanofluidics means the diffusion of soluble analytes through pores perforating the middle layer. In this case, it is made of metal, but it could be carbon as well, as both support plasmonics.
Figure 10. Imaginary optofluidic device. The orange arrow shows the direction of the micron scale flow, while the green ones show one possible direction of nanoscale diffusion. The middle component is functionalised with a self-assembled monolayer and the close-up is shown left, where the letter S indicates the employment of thiol chemistry while the letter X represents a chemical group facing the liquid. Super- and substrates would be good targets for imprinted electrodes (yellow bars) or printed micro and nano optics. Spacers could be fabricated as integral parts of the layers (as in the superstrate). Alternatively, they could be added as polymer fibres (light pink bars between two bottom layers).
The middle layer in this imaginary scheme (Figure 10) is accompanied with tangential flow through the space above it.
This, in comparison to placing nanopores at a dead-end position, would result in a lower level of concentration polarisation i.e.
the accumulation of particles or molecules that cannot pass the membrane through these pores.
Summing up, micro- and nanofluidics have been materialised in nature during multicellular evolution, which has created apparatuses like the mammalian kidney, where microfluidic channels work in concert with nanoscale pores.
From such, we have learned the principles of how to separate and sort molecules from larger particles. The foundations of artificial sorting molecules and particles, in turn, rest on over a century of history of the molecular biology. Today’s micro- and nano-methods allow us to fabricate devices combining both micro- and nano-fluidics, and to integrate electronics and optics into them. Thus, molecules, cells and droplets are recognised and even sorted with the optics of a device. Such man-made devices can be further functionalised with “smart” chemicals or
biomolecules attached to its surfaces. By combining them with carbon-based materials, further options for the photonic design are opened up. Importantly, they enable artificial pore design for small molecule sorting. The theoretical management of such complex devices, however, might be beyond the reach of the existing tools, and hence, in addition to simple trial-and-error experimental work, one may need to find novel approaches to the future development.
6 Conclusions
Enhancement of the fluorescence signal with RWG is shown here to be two orders of magnitude higher than without the RWG when using the laser beam for the illumination. Over one order of magnitude higher enhancement was still reached with conical, broad-band illumination in a microscope. Similarly, Raman signal was enhanced in a microscope when RWG was combined with an SERS substrate. Together, the latter two studies imply that neither collimated beams nor complicated measurement set-ups are required for efficient detection of the fluorescence or Raman signals of biomolecules. Several future studies are suggested. These include life-time and polarisation resolved measurements, the use of multi-photon excitation, emission manipulation schemes and combining RWG with new kinds of plasmonic structures. Lastly, the use of RWG as a part of novel micro- and nano-fluidic sensor devices was envisaged.
Such a device would enable the simultaneous manipulation of complex samples during optical detection. This means that the analytes of interest could be purified with the aid of the functional component surfaces, by light or other forces, during the measurement. Current manufacturing methods are not directly limiting the development of integrated lab-on-chip devices for in vitro analytics, but it could further benefit from new tools and approaches in the zones between disciplines and size scales.
biomolecules attached to its surfaces. By combining them with carbon-based materials, further options for the photonic design are opened up. Importantly, they enable artificial pore design for small molecule sorting. The theoretical management of such complex devices, however, might be beyond the reach of the existing tools, and hence, in addition to simple trial-and-error experimental work, one may need to find novel approaches to the future development.
6 Conclusions
Enhancement of the fluorescence signal with RWG is shown here to be two orders of magnitude higher than without the RWG when using the laser beam for the illumination. Over one order of magnitude higher enhancement was still reached with conical, broad-band illumination in a microscope. Similarly, Raman signal was enhanced in a microscope when RWG was combined with an SERS substrate. Together, the latter two studies imply that neither collimated beams nor complicated measurement set-ups are required for efficient detection of the fluorescence or Raman signals of biomolecules. Several future studies are suggested. These include life-time and polarisation resolved measurements, the use of multi-photon excitation, emission manipulation schemes and combining RWG with new kinds of plasmonic structures. Lastly, the use of RWG as a part of novel micro- and nano-fluidic sensor devices was envisaged.
Such a device would enable the simultaneous manipulation of complex samples during optical detection. This means that the analytes of interest could be purified with the aid of the functional component surfaces, by light or other forces, during the measurement. Current manufacturing methods are not directly limiting the development of integrated lab-on-chip devices for in vitro analytics, but it could further benefit from new tools and approaches in the zones between disciplines and size scales.
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