Nicole Pamme

Continuous flow separations in microfluidic devices

Biochemical sample mixtures are commonly separated in batch processes, such as filtration, centrifugation, chromatography or electrophoresis. In recent years, however, many research groups have demonstrated continuous flow separation methods in microfluidic devices. Such separation methods are characterised by continuous injection, real-time monitoring, as well as continuous collection, which makes them ideal for combination with upstream and downstream applications. Importantly, in continuous flow separation the sample components are deflected from the main direction of flow, either by means of a force field (electric, magnetic, acoustic, optical etc.), or by intelligent positioning of obstacles in combination with laminar flow profiles. Sample components susceptible to deflection can be spatially separated. A large variety of methods has been reported, some of these are miniaturised versions of larger scale methods, others are only possible in microfluidic regimes. Researchers now have a diverse toolbox to choose from and it is likely that continuous flow methods will play an important role in future point-of-care or in-the-field analysis devices.

On-chip bioanalysis with magnetic particles

Magnetic particles can combine two very selective processes in bioanalysis: the specific binding of analytes to the particle surface based on molecular recognition and the specific isolation of magnetic objects from complex sample mixtures. They have found numerous applications including cell isolation, immunoassays or DNA extraction. In this review recent trends in the use of magnetic particles are presented. Integrated sample-in-answer-out lab-on-a-chip systems often employ magnetic particles for at least one of the reaction steps. Several groups have shown on-particle processing in continuous flow for assays and DNA extractions. Other researchers have demonstrated the manoeuvring and splitting of magnetically functionalised droplets for various bioapplications. Improvements in magnet configuration now allow for sorting of magnetically labelled cells within mL volumes in minutes.

Diamagnetic repulsion—A versatile tool for label-free particle handling in microfluidic devices

We report the exploration of diamagnetic repulsion forces for the selective manipulation of microparticles inside microfluidic devices. Diamagnetic materials such as polymers are repelled from magnetic fields, an effect greatly enhanced by suspending a diamagnetic object in a paramagnetic Mn(2+) solution. The versatility of diamagnetic repulsion is demonstrated for the trapping, focussing and deflection of polystyrene particles for three example applications. Firstly, magnet pairs with unlike poles facing each other were arranged along a microcapillary to trap plugs of differently functionalised particles for a simultaneous surface-based assay in which biotin was selectively bound to a plug of streptavidin coated particles utilising only 22nL of reagent. Secondly, by slightly modifying the magnetic field design, the rapid focussing of particles into a narrow central stream at a flow rate of 650microms(-1) was accomplished for particle pre-concentration. In a third application, 5 and 10microm polystyrene particles were separated from each other in continuous flow by passing the particle mixture through a microfluidic chamber with a perpendicular magnetic field, a method termed diamagnetophoresis. The separation was investigated between flow rates of 20-100microL h(-1), with full resolution of the particle populations being achieved at 20microL h(-1). These experiments show the potential of diamagnetic repulsion for simple, label-free manipulation of particles and other diamagnetic objects such as cells for a range of bioanalytical techniques.

Simultaneous bioassays in a microfluidic channel on plugs of different magnetic particles.

Magnetic particles coated with specific biomolecules are often used as solid supports for bioassays but conventional test tube based techniques are time consuming and labour intensive. An alternative is to work on magnetic particle plugs immobilised inside microfluidic channels. Most research so far has focussed on immobilising one type of particle to perform one type of assay. Here we demonstrate how several assays can be performed simultaneously by flushing a sample solution over several plugs of magnetic particles with different surface coatings. Within a microchannel, three plugs of magnetic particles were immobilised with external magnets. The particles featured surface coatings of glycine, streptavidin and protein A, respectively. Reagents were then flushed through the three plugs. Molecular binding occurred between matching antigens and antibodies in continuous flow and was detected by fluorescence. This first demonstration opens the door to a quicker and easier technique for simultaneous bioassays using magnetic particles.

Rapid on-chip multi-step (bio)chemical procedures in continuous flow : manoeuvring particles through co-laminar reagent streams

We introduce a novel and extremely versatile microfluidic platform in which tedious multi-step biochemical processes can be performed in continuous flow within a fraction of the time required for conventional methods.

Rapid, multistep on-chip DNA hybridisation in continuous flow on magnetic particles.

DNA hybridisation is an important tool for bioanalytical research and clinical diagnostics; conventional methods, however, require long incubation times and numerous washing steps, rendering the procedure time consuming and labour intensive. In this paper, we report on a rapid method for DNA hybridisation and isolation within a microfluidic device, where all reaction and washing steps are performed in continuous flow in an automated fashion within less than two minutes. Magnetic particles were used as a solid support and manipulated through laminar flow streams containing reagents and buffers by means of an external magnet. Thus, hybridisation, washing, intercalation, fluorescence detection and isolation were performed in continuous flow on the surface of the particles. Initially, the sensitivity of the system was investigated for a one-step DNA hybridisation of Alexa Fluor 555 labelled target DNA to a capture probe immobilised on the particle surface. Hybridisation and washing steps were performed in half a minute and target DNA was readily detected down to 20 nmol L(-1). Then a two-step assay, label-free DNA hybridisation followed by intercalation with PicoGreen was performed. All reaction and washing steps were carried out in continuous flow with a total assay time of about 1 min. This is a significant reduction in procedural time compared to conventional methods and opens the door for developing fully automated continuous flow integrated DNA analysis platforms.

Radiochemistry on chip

We have developed an integrated microfluidic platform for producing 2-[(18)F]-fluoro-2-deoxy-D-glucose ((18)F-FDG) in continuous flow from a single bolus of radioactive isotope solution, with constant product yields achieved throughout the operation that were comparable to those reported for commercially available vessel-based synthesisers (40-80%). The system would allow researchers to obtain radiopharmaceuticals in a dose-on-demand setting within a few minutes. The flexible architecture of the platform, based on a modular design, can potentially be applied to the synthesis of other radiotracers that require a two-step synthetic approach, and may be adaptable to more complex synthetic routes by implementing additional modules. It can therefore be employed for standard synthesis protocols as well as for research and development of new radiopharmaceuticals.

On-chip processing of particles and cells via multilaminar flow streams

The processing of particles, cells, and droplets for reactions, analyses, labeling, and coating is an important aspect of many microfluidic workflows. However, performing multi-step processes is typically a laborious and time-consuming endeavor. By exploiting the laminar nature of flow within microchannels, such procedures can benefit in terms of both speed and simplicity. This can be achieved either by manipulating the flow streams around the objects of interest, particularly for the localized perfusion of cells, or by manipulating the objects themselves within the streams via a range of forces. Here, we review the variety of methods that have been employed for performing such “multilaminar flow” procedures on particles, cells, and droplets.

On-chip diamagnetic repulsion in continuous flow

Abstract We explore the potential of a microfluidic continuous flow particle separation system based on the repulsion of diamagnetic materials from a high magnetic field. Diamagnetic polystyrene particles in paramagnetic manganese (II) chloride solution were pumped into a microfluidic chamber and their deflection behaviour in a high magnetic field applied by a superconducting magnet was investigated. Two particle sizes (5 and 10 μm) were examined in two concentrations of MnCl2 (6 and 10%). The larger particles were repelled to a greater extent than the smaller ones, and the effect was greatly enhanced when the particles were suspended in a higher concentration of MnCl2. These findings indicate that the system could be viable for the separation of materials of differing size and/or diamagnetic susceptibility, and as such could be suitable for the separation and sorting of small biological species for subsequent studies.

On-chip pre-concentration and complexation of [18F]fluoride ions via regenerable anion exchange particles for radiochemical synthesis of Positron Emission Tomography tracers

Microfluidic approaches have demonstrated a relevant impact on radiochemical reactions involving Positron Emission Tomography (PET) nuclides, due to shorter reaction times and smaller precursor quantities. However, little attention has been given to the integration of the initial pre-concentration and drying of radioactive [(18)F]fluoride ions, required for the labeling of radiotracer compounds. In this work we report the design, fabrication and implementation of a glass microfluidic device filled with recyclable anion exchange particles for the repeated recovery of [(18)F] and [(19)F]fluoride ions. The device was first tested with non radioactive [(19)F]fluoride ions and it was shown to repeatedly trap and elute >95% fluoride over 40 successive experimental runs with no decrease in efficiency. The same device was then tested for the trapping and release of [(18)F]fluoride ions over 20 experiments with no measurable decrease in performance. Finally, the [(18)F]fluoride ions were eluted as a K(18)F/K2.2.2 complex, dried by repeated dissolution in acetonitrile and evaporation of residual water, and reacted with ethyl ditosylate (EtDT) leading to the desired product ([(18)F]fluoroethyltosylate) with 96 ± 3% yield (RCY). The overall time needed for conditioning, trapping, elution and regeneration was less than 6 min. This approach will be of great benefit towards an integrated platform able to perform faster and safer radiochemical synthesis on the micro-scale.