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

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

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

Microcapsules as assay compartments formed through layer-by-layer deposition

We investigate the potential of using microcapsules, generated through layer-by-layer assembly, as reaction compartments for bioassays. A streptavidin-biotin and an esterase-fluorescein diacetate (FDA) assay were employed for proof-of-concept. Streptavidin was incorporated into the microcapsules via two methods: (i) physical adsorption onto MnCO3 microcrystal templates or (ii) co-precipitation with CaCO3 to form mixed microcrystals. The streptavidin-loaded template microparticles were then coated with five bi-layers of polyelectrolytes (polystyrene sulfonate/polyalylamine hydrochloride) (PAH/PSS)5 and the templated cores were dissolved using EDTA to form polyelectrolyte microcapsules loaded with streptavidin. Streptavidin loading of around 77 mg streptavidin per mol of CaCO3 was achieved using the co-precipitation method, compared to 5.1 mg streptavidin per mol of MnCO3 using physical adsorption onto the manganese carbonate crystal surface. The microencapsulated streptavidin was allowed to bind to an analyte which was able to freely diffuse through the polyelectrolyte shell from the aqueous media. Biotin-4-fluorescein was added to the streptavidin-loaded microcapsules, with streptavidin-free capsules serving as the control sample. It was found that both types of microcapsules exhibited a similar fluorescence signal intensity, likely due to non-specific binding of biotin-4-fluorescein to charged groups on the polyelectrolyte shell. Therefore, an alternative esterase-FDA assay was investigated as fluorescence is only produced when FDA penetrates the capsule shell and reacts with the esterase enzyme inside which leads to its hydrolysis. Esterase was loaded into the LbL capsules templated on CaCO3 via co-precipitation. FDA was added to both the esterase-loaded capsules and esterase-free capsules. It was found that the capsules containing esterase fluoresced, while the esterase-free capsules did not. This indicates that the hydrolysis reaction of FDA with esterase occurred inside the LbL microcapsules. These experiments demonstrate the potential of compartmentalised assays to be carried out inside microcapsules which could be applied in complex matrices or in multiplexing experiments, wherein different barcoded microcapsules contain different reagents to provide independent readouts without interference from larger biomolecules present in the surrounding media

High-speed imaging of ice nucleation in water proves the existence of active sites

Understanding how surfaces direct nucleation is a complex problem that limits our ability to predict and control crystal formation. We here address this challenge using high-speed imaging to identify and quantify the sites at which ice nucleates in water droplets on the two natural cleavage faces of macroscopic feldspar substrates. Our data show that ice nucleation only occurs at a few locations, all of which are associated with micron-size surface pits. Similar behavior is observed on α-quartz substrates that lack cleavage planes. These results demonstrate that substrate heterogeneities are the salient factor in promoting nucleation and therefore prove the existence of active sites. We also provide strong evidence that the activity of these sites derives from a combination of surface chemistry and nanoscale topography. Our results have implications for the nucleation of many materials and suggest new strategies for promoting or inhibiting nucleation across a wide range of applications

The ice-nucleating ability of quartz immersed in water and its atmospheric importance compared to K-feldspar

Mineral dust particles are thought to be an important type of ice-nucleating particle (INP) in the mixedphase cloud regime around the globe. While K-rich feldspar (K-feldspar) has been identified as being a particularly important component of mineral dust for ice nucleation, it has been shown that quartz is also relatively ice-nucleation active. Given quartz typically makes up a substantial proportion of atmospheric desert dust, it could potentially be important for cloud glaciation. Here, we survey the ice-nucleating ability of 10 α-quartz samples (the most common quartz polymorph) when immersed in microlitre supercooled water droplets. Despite all samples being α-quartz, the temperature at which they induce freezing varies by around 12 ◦C for a constant active site density. We find that some quartz samples are very sensitive to ageing in both aqueous suspension and air, resulting in a loss of ice-nucleating activity, while other samples are insensitive to exposure to air and water over many months. For example, the ice-nucleation temperatures for one quartz sample shift down by ∼ 2 ◦C in 1 h and 12 ◦C after 16 months in water. The sensitivity to water and air is perhaps surprising, as quartz is thought of as a chemically resistant mineral, but this observation suggests that the active sites responsible for nucleation are less stable than the bulk of the mineral. We find that the quartz group of minerals is generally less active than K-feldspars by roughly 7 ◦C, although the most active quartz samples are of a similar activity to some K-feldspars with an active site density, ns(T ), of 1 cm−2 at −9◦C. We also find that the freshly milled quartz samples are generally more active by roughly 5 ◦C than the plagioclase feldspar group of minerals and the albite end member has an intermediate activity. Using both the new and literature data, active site density parameterizations have been proposed for freshly milled quartz, K-feldspar, plagioclase and albite. Combining these parameterizations with the typical atmospheric abundance of each mineral supports previous work that suggests that K-feldspar is the most important ice-nucleating mineral in airborne mineral dust

On-chip polyelectrolyte coating onto magnetic droplets-towards continuous flow assembly of drug delivery capsules

Polyelectrolyte (PE) microcapsules for drug delivery are typically fabricated via layer-by-layer (LbL) deposition of PE layers of alternating charge on sacrificial template microparticles, which usually requires multiple incubation and washing steps that render the process repetitive and time-consuming. Here, ferrofluid droplets were explored for this purpose as an elegant alternative of templates that can be easily manipulated via an external magnetic field, and require only a simple microfluidic chip design and setup. Glass microfluidic devices featuring T-junctions or flow focusing junctions for the generation of oil-based ferrofluid droplets in an aqueous continuous phase were investigated. Droplet size was controlled by the microfluidic channel dimensions as well as the flow rates of the ferrofluid and aqueous phases. The generated droplets were stabilised by a surface active polymer, polyvinylpyrrolidone (PVP), and then guided into a chamber featuring alternating, co-laminar PE solutions and wash streams, and deflected across them by means of an external permanent magnet. The extent of droplet deflection was tailored by the flow rates, the concentration of magnetic nanoparticles in the droplets, and the magnetic field strength. PVP-coated ferrofluid droplets were deflected through solutions of polyelectrolyte and washing streams using several iterations of multilaminar flow designs. This culminated in an innovative "Snakes-and-Ladders" inspired microfluidic chip design that overcame various issues of the previous iterations for the deposition of layers of anionic poly(sodium-4-styrene sulfonate) (PSS) and cationic poly(fluorescein isothiocyanate allylamine hydrochloride) (PAH-FITC) onto the droplets. The presented method demonstrates a simple and rapid process for PE layer deposition in <30 seconds, and opens the way towards rapid layer-by-layer assembly of PE microcapsules for drug delivery applications.The authors thank the Royal Embassy of Saudi Arabia Cultural Bureau in London and Albaha University in Saudi Arabia for funding. J.G.-P., E.B. and I.O. acknowledge financial support from the Spanish Ministry of Economy and Competitiveness (project CTQ2015-66078-R (MINECO/FEDER) and FPI postgraduate research grant (BES-2013-064415). The authors thank Dr Stephen Clark for fabrication of the microfluidic devices