Magnetic particle plug-based assays for biomarker analysis

Conventional immunoassays offer selective and quantitative detection of a number of biomarkers, but are laborious and time-consuming. Magnetic particle-based assays allow easy and rapid selection of analytes, but still suffer from the requirement of tedious multiple reaction and washing steps. Here, we demonstrate the trapping of functionalised magnetic particles within a microchannel for performing rapid immunoassays by flushing consecutive reagent and washing solutions over the trapped particle plug. Three main studies were performed to investigate the potential of the platform for quantitative analysis of biomarkers: (i) a streptavidin-biotin binding assay; (ii) a sandwich assay of the inflammation biomarker, C-reactive protein (CRP); and (iii) detection of the steroid hormone, progesterone (P4), towards a competitive assay. Quantitative analysis with low limits of detection was demonstrated with streptavidin-biotin, while the CRP and P4 assays exhibited the ability to detect clinically relevant analytes, and all assays were completed in only 15 min. These preliminary results show the great potential of the platform for performing rapid, low volume magnetic particle plug-based assays of a range of clinical biomarkers via an exceedingly simple technique

Continuous flow processes on single magnetic and diamagnetic particles in microfluidic devices

Magnetic microparticles have seen increasing interest in (bio)chemical processes in recent years due to their various surface functionalities, high surface-to-volume ratio, small sizes, and ease of manipulation via magnetic fields. However, conventional reactions and assays that use magnetic particles as solid supports are typically performed in multi-step procedures that require consecutive reaction and washing steps. While offering high capture efficiencies, these are batch processes that, due to the consecutive steps required, are typically time-consuming and laborious. Their incorporation into microfluidic devices has brought about benefits including finer control over the movement of particles and reagent/sample solutions, as well as the ability to place a magnet closer to the area of interest. However, most instances of on-chip magnetic particle based procedures rely on trap-and-release methodology, essentially requiring the same stepwise routine as with conventional systems. A method of reducing these inefficiencies is to perform the reaction or separation in continuous flow, thereby allowing continuous sample introduction and analysis of the process in rapid times, and with minimal reagent consumption and waste production.Two methods of performing continuous flow procedures on single particles in microfluidic devices via the application of magnetic forces were investigated: 1) the use of magnetic microparticles as mobile solid supports for performing rapid separations, reactions, and immunoassays via magnetic attraction, and 2) the use of diamagnetic repulsion forces for performing similar procedures on non-magnetic particles, with a view to the label-free processing of diamagnetic species such as polymer particles and biological cells, based on their intrinsic properties.For the magnetic attraction experiments, a study into the effect of temperature on magnetic particle deflection behaviour and separations was performed, whereupon it was found that an increased temperature of the system yielded increased deflection distances and separation resolution due to the reduced viscous drag. This was followed by several investigations into the deflection of particles through laminar flow streams containing alternating reagents and washing buffers for performing multistep reactions and assays. The setup was used to demonstrate amide bond formation and polyelectrolyte deposition in continuous flow, before being used to detect clinically relevant levels (5 and 10µg mL-1) of the inflammatory biomarker, C-reactive protein. Thus, these findings show great potential for rapid, continuous processing of particles for a number of chemical and biological applications, as well as in clinical diagnostics.For the diamagnetic repulsion studies, diamagnetic polystyrene particles were suspended in paramagnetic media and deflected away from a magnetic field in continuous flow. The effect of particle size and the magnetic susceptibility of the paramagnetic media on particle deflection were investigated using high magnetic fields, where it was found that larger particles in a medium with higher susceptibility yielded the greatest deflection. This work was extended via a proof-of-principle setup in which polystyrene particles were repelled out of a reagent stream and into a buffer stream using permanent magnets, with a view to performing continuous flow reactions through laminar flow reagent and washing buffer streams, akin to those achieved via magnetic attraction. Finally, flow focussing of polystyrene particles and label-free cells was achieved via diamagnetic repulsion forces applied by permanent magnets, demonstrating the ability to manipulate cells in continuous flow by magnetic forces based on their intrinsic properties. This work could be applied to the label-free processing of particles and cells for separations, reactions, and assays

Tailoring pH-responsive acrylic acid microgels with hydrophobic crosslinks for drug release

Amphiphilic microgels based on the hydrophilic acrylic acid (AA) and hydrophobic crosslinks of different compositions were synthesised using a lab-on-a-chip device. The microgels were formed by polymerising hydrophobic droplets. The droplets were generated via a microfluidic platform and contained a protected form of AA, a hydrophobic crosslinker (ethylene glycol dimethacrylate, EGDMA) and a free radical initiator in an organic solvent. Following photopolymerisation and subsequent hydrolysis, AA based microgels of amphiphilic nature were produced and it was demonstrated that they can successfully deliver both hydrophilic as well as hydrophobic moieties. The model drug delivery and the swelling ability of the microgels were influenced by the pH of the aqueous solution as well as the crosslinking density and hydrophobic content of the microgels

Microfluidically fabricated pH-responsive anionic amphiphilic microgels for drug release

© 2016 The Royal Society of Chemistry. Amphiphilic microgels of different composition based on the hydrophilic, pH-responsive acrylic acid (AA) and the hydrophobic, non-ionic n-butyl acrylate (BuA) were synthesised using a lab-on-a-chip device. Hydrophobic droplets were generated via a microfluidic platform that contained a protected form of AA, BuA, the hydrophobic crosslinker, ethylene glycol dimethacrylate (EGDMA), and a free radical initiator in an organic solvent. These hydrophobic droplets were photopolymerised within the microfluidic channels and subsequently hydrolysed, enabling an integrated platform for the rapid, automated, and in situ production of anionic amphiphilic microgels. The amphiphilic microgels did not feature the conventional core-shell structure but were instead based on random amphiphilic copolymers of AA and BuA and hydrophobic crosslinks. Due to their amphiphilic nature they were able to encapsulate and deliver both hydrophobic and hydrophilic moieties. The model drug delivery and the swelling ability of the microgels were influenced by the pH of the surrounding aqueous solution and the hydrophobic content of the microgels

Diamagnetic repulsion of particles for multilaminar flow assays

© The Royal Society of Chemistry. We demonstrate diamagnetic repulsion forces for performing continuous multilaminar flow assays on particles based on their intrinsic properties and with a simple setup. The platform could be applied to sandwich assays on polystyrene particles, and to cell-based assays via their suspension in biologically benign magnetic media

On-chip determination of C-reactive protein using magnetic particles in continuous flow

We demonstrate the application of a multilaminar flow platform, in which functionalized magnetic particles are deflected through alternating laminar flow streams of reagents and washing solutions via an external magnet, for the rapid detection of the inflammatory biomarker, C-reactive protein (CRP). The two-step sandwich immunoassay was accomplished in less than 60 s, a vast improvement on the 80−300 min time frame required for enzyme-linked immunosorbent assays (ELISA) and the 50 min necessary for off-chip magnetic particle-based assays. The combination of continuous flow and a stationary magnet enables a degree of autonomy in the system, while a detection limit of 0.87 μg mL−1 makes it suitable for the determination of CRP concentrations in clinical diagnostics. Its applicability was further proven by assaying real human serum samples and comparing those results to values obtained using standard ELISA tests

Phaseguide assisted liquid lamination for magnetic particle-based assays

We have developed a magnetic particle-based assay platform in which functionalised magnetic particles are transferred sequentially through laminated volumes of reagents and washing buffers. Lamination of aqueous liquids is achieved via the use of phaseguide technology; microstructures that control the advancing air–liquid interface of solutions as they enter a microfluidic chamber. This allows manual filling of the device, eliminating the need for external pumping systems, and preparation of the system requires only a few minutes. Here, we apply the platform to two on-chip strategies: (i) a one-step streptavidin–biotin binding assay, and (ii) a two-step C-reactive protein immunoassay. With these, we demonstrate how condensing multiple reaction and washing processes into a single step significantly reduces procedural times, with both assay procedures requiring less than 8 seconds

Control design of uncertain quantum systems with fuzzy estimators

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Temperature-based tuning of magnetic particle separation by on-chip free-flow magnetophoresis

This paper was presented at the 2nd Micro and Nano Flows Conference (MNF2009), which was held at Brunel University, West London, UK. The conference was organised by Brunel University and supported by the Institution of Mechanical Engineers, IPEM, the Italian Union of Thermofluid dynamics, the Process Intensification Network, HEXAG - the Heat Exchange Action Group and the Institute of Mathematics and its Applications.Free-flow magnetophoresis provides a fast and efficient means of continuous flow magnetic separation for the detection of biological analytes, due to the wide variety of magnetic particle surface properties available for binding specific targets. Here, we investigate the effect of temperature changes on the deflection behaviour of magnetic particles in a microfluidic magnetophoresis separation chamber. It was found that the extent of deflection was greatly increased at higher temperatures due to decreased solution viscosity and thus reduced resistance against particle motion. This concept was used to improve the resolution of the separation of 2.8 μm and 1 μm diameter magnetic particles. Hence, controlling the temperature of the separation system provides a simple but highly effective means of enhancing magnetic separation efficiency. This concept could also be applied to the temperature-based tuning of microparticle trajectories in many others types of continuous flow processes, such as those using optical, electrical or acoustic forces.This study is funded by the Engineering and Physical Sciences Research Council (EPSRC)

Positron detection in silica monoliths for miniaturised quality control of PET radiotracers

We demonstrate the use of the miniaturised Medipix positron sensor for detection of the clinical PET radiotracer, [⁶⁸Ga]gallium-citrate, on a silica-based monolith, towards microfluidic quality control. The system achieved a far superior signal-to-noise ratio compared to conventional sodium iodide-based radio-HPLC detection and allowed real-time visualisation of positrons in the monolith