Control válvula de expansión EKC 315A

In Section 4.3, the integration of photonic crystal fibre into PDMS microfluidic chips will be discussed for applications in optical chromatography, a distinct category of passive optical sorting. As first reported by T. Imasakaet al. in 1995 [126], optical

chromatography was demonstrated to be capable of fractionating dielectric particles by size, and later by refractive index [127] and by hydrodynamic profile (predomi- nantly shape) [128]. In this first experiment, a dielectric particle was introduced into a capillary tube by a laminar liquid flow. A lightly focused counter-propagating free- space laser beam was aligned parallel to this fluid flow. The optical gradient forces

1.4. Fractionation of Microscopic Particles using Optical Fields 26

Figure 1.11: The basic sorting scheme of optical chromatography, where the balance of the

Stokes fluid drag force and the optical scattering force forms increased retention distances (or equilibrium distances) for larger particles or those with higher refractive indices.[125]

trapped the particle along the centre of the laser beam and accelerated it against the fluid flow, away from the beam waist due to the radiation pressure. Counter to this, the Stokes fluid drag force decelerated the particle until finding an equili- brium position where the fluid drag force and radiation force balanced. The distance between the equilibrium position and the beam waist was termed theretention dis- tance and is dependent upon the intrinsic optical and hydrodynamic properties of

the particle. The retention distance is also a function of the laser power and fluid flow rate [9,129].

A later paper [129] demonstrated the first biological applications of optical chromatography, where human erythrocytes were shown to be split into two sub- populations using the technique. The potential of optical chromatography as a bio- logical tool was thoroughly embedded through another paper by the group, where the technique was applied to immunoassay of protein [106]. Polymer beads coated in antibody (anti-mouse IgG) were flowed into the optical chromatography setup and the retention distance was observed. A concentration of antigen (mouse IgG) was then flowed in, generating bead-bead binding through the presence of the antigen. Bound coagulated beads exhibited an increased radiation pressure thus receiving an increase in retention distance by up to 500 µmcompared to the unbound free beads.

The ratio of free to bound beads was calculated providing a reaction probability, and protein concentration of 10’sng mL−1 could readily be measured. The reversibility

of the reaction in real time was also investigated by monitoring the exchange to and from the bound and unbound as visualised on camera. As such, optical chromato- graphy provided the capability to study for the first time a single bond reversible reaction, as well as the capacity to watch the reaction in real time. A follow-up pa- per [130] improved the sensitivity of the experiment down to 1 ng mL−1 by altering

1.4. Fractionation of Microscopic Particles using Optical Fields 27

in dissociation.

As well as supplying a means of separating particles according to retention dis- tances dependent upon particle properties, optical chromatography has branched into three not so dissimilar approaches, namely theoptical funnel, theoptical chan- nel and the optical chromatography filter. The various names are more of an indi-

cation of a different application rather than a change in experimental setup. The optical funnel is a technique for measuring the force a biological particle can exert to escape the gradient forces of the optical field within an optical chromatography setup. This was first applied to fresh water bacteria Trachelomonas volvocina [131] and later to sperm cells for varying medium pH [132] and allowed for large numbers (100’s) of cells to be characterised in a few hours.

The optical channel is another variation on the optical chromatography for mea- suring the elasticity of a cell. Unlike the work on the dual-beam fibre trap optical stretcher by J. Guck and coworkers [133], here the fluid flow gives rise to the stretch of a trapped cell, rather than the optical field. Briefly, a flow of cells is flowed against a lightly diverging optical beam, which traps cells on axis. The cell is allowed to flow through the beam focus, at which point it elongates in one direction due to the fluid shear stresses, and the stretch is measured from a CCD image giving a one dimensional shear strength measurement of the cell. Here, erythrocyte shear strength was measured as a function of the age of the cell [134].

The most recent variation on optical chromatography is the optical filter, which is the result of developments from S.J. Hart, A.V. Terray and coworkers in the field of optical chromatography. The first paper on optical chromatography published by this group demonstrated sorting by refractive index [127]. Separations of several hundred of microns were exhibited for a polydisperse sample of silica, PMMA and polystyrene spheres. This was followed by the exciting report of the separation of the etiological agent of the mammalian disease anthrax, Bacillus anthracis bacteria, from both its spore and from the common environmental interferent, pollen. The separation of mulberry and ragweed pollen was also demonstrated. The publication was of utmost interest to the biodefense area as B. Anthracis is a common biological warfare threat. In another publication a distinct optical chromatographic separa-

1.4. Fractionation of Microscopic Particles using Optical Fields 28

tion between B. Anthracis and its close relative B. Thuringiensis was reported [128], caused by a combination of subtle differences in their morphologies. On close inspec- tion, B. Thuringiensis was observed to be more of an oblate spheroid in shape and also exhibited a larger exosporium, giving rise to changes in both the hydrodynamic and optical forces involved.

Using fluid channels of varying dimensions allows for specific separation of spe- cies to be enhanced [135]. S.J. Hart, A.V. Terray and coworkers used customised PDMS chips with varying channel dimensions to enhance the optical chromatogra- phy separations, demonstrated on colloidal particles and spores of B. Anthracis and Mulberry pollen. The focusing lens was adjusted to move the beam waist and posi- tion the separation such that the two particles laid either side of a point where the channel width increased rapidly. The linear fluid flow is slower in a wider channel, hence sees a reduced Stokes fluid force and an increased retention distance compared to that in the narrower flow channel region. As a result, the retention distance of one species can be artificially increased to accentuate an already present difference in retention between two species.

In 2007 S.J. Hart, A.V. Terray and coworkers reported a new chip design and application that they termed an optical chromatographic filter [136]. Here, instead of holding two or more species at different retention distances, the laser and flow parameters are altered such that one species is held up and the others are allowed to flow through. The channel is made narrower and shorter, and the beam fills the entire channel ensuring that all particles feel the optical force. These adjustments evade the common issues with previous designs, where only a small fraction of particles are trapped by the beam, and allows for a large number to be held up without producing instabilities in the equilibrium positions due to re-scattering of the beam due to the particles. The new setup allows for separations of particles, or as demonstrated with B. Anthracis, a sample can be enriched to produce a more concentrated pure sample. The technique represents a more robust experimental setup, capable of dealing with much higher number of particles, as demonstrated in their later publication where separation efficiencies of 99% were achieved for thousands of polymer and silica particles compared to tens in previous setups. The

1.4. Fractionation of Microscopic Particles using Optical Fields 29

particle trajectories in an optical chromatography filter setup has also been modelled [137], using a finite element method for solving the Navier-Stokes equations and a ray optics model for the optical field.