Magnetic forces for surface-based bioanalysis in microfluidic devices

Biochemical analysis is a critical part of clinical diagnostics, forensic science and environmental monitoring. Typically, this form of analysis involves the use of bioanalytical procedures which are extremely sensitive and highly specific. However, such assays often involve many different reaction and washing steps, which means that a single analysis could take hours or even days to perform. There is an urgent demand for high through-put analysis systems that are rapid, simple to use and can be utilised in the field or at the point-of-care. Microfluidic technology has gone some way to producing such systems, however many of the current devices still incorporate batch methods of analysis, which are still time consuming or do not integrate all steps of the analysis on one device.Two aspects of particle handling utilising magnetic forces were investigated. 1) The use of single magnetic particles as mobile solid-supports in a continuous flow system for bioanalysis. 2) The use of diamagnetic repulsion forces for label-free on-chip particle handling.For the continuous flow system, a magnetic particle type was selected from eight commercially available brands of particles and characterised using on and off-chip measurements. Dynabead M-270 particles were ultimately used as mobile solidsupports for performing entire bioanalytical processes in continuous flow, for the first time. This was achieved by generating multiple laminar flow streams along the length of a rectangular chamber and applying a magnetic field gradient perpendicular to flow. Each reagent stream contained reagents for a molecular binding assay and functionalised magnetic particles were deflected across the width of the chamber. As the particles were deflected through reagent streams, washing and reaction steps were performed on the surface of the particle in one operation in continuous flow. The system was used to perform a one-step streptavidin - biotin binding assay with an LOD of 20 µg mL-1 , a two-step mouse IgG immunoassay with an LOD of 0.1 µg mL-1 and a qualitative sandwich immunoassay for C-reactive protein (CRP). All three procedures were performed within one minute with no manual intervention.For the diamagnetic repulsion forces for label-free particle handling, 10 µm polystyrene particles were trapped into plugs on a glass capillary by suspending them in a paramagnetic solution and creating an area of high magnetic field gradient between two magnets. Particles were repelled by the field and formed a plug upstream from the magnets. The system was used to simultaneously perform positive and negative controls on a sample of fluorescently labelled biotin using two plugs; one containing streptavidin functionalised polystyrene particles and one containing plain polystyrene particles. In addition, diamagnetic repulsion was used to deflect polystyrene particles from flow inside a square deflection chamber. A particle mixture of 5 µm and 10 µm particles were separated into different exits at a flow rate of 20 µLh-1 based on the difference in their volume, termed free-flow diamagnetophoresis. Potential applications include label-free cell manipulation

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

Lipid coated liquid crystal droplets for the on-chip detection of antimicrobial peptides

We describe a novel biosensor based on phospholipid-coated nematic liquid crystal (LC) droplets and demonstrate the detection of Smp43, a model antimicrobial peptide (AMP) from the venom of North African scorpion Scorpio maurus palmatus. Mono-disperse lipid-coated LC droplets of diameter 16.7 ± 0.2 μm were generated using PDMS microfluidic devices with a flow-focusing configuration and were the target for AMPs. The droplets were trapped in a bespoke microfluidic trap structure and were simultaneously treated with Smp43 at gradient concentrations in six different chambers. The disruption of the lipid monolayer by the Smp43 was detected (<6 μM) at concentrations well within its biologically active range, indicated by a dramatic change in the appearance of the droplets associated with the transition from a typical radial configuration to a bipolar configuration, which is readily observed by polarizing microscopy. This suggests the system has feasibility as a drug-discovery screening tool. Further, compared to previously reported LC droplet biosensors, this LC droplet biosensor with a lipid coating is more biologically relevant and its ease of use in detecting membrane-related biological processes and interactions has the potential for development as a reliable, low-cost and disposable point of care diagnostic tool

Physical biomarkers of disease progression:on-chip monitoring of changes in mechanobiology of colorectal cancer cells

Disease can induce changes to subcellular components, altering cell phenotype and leading to measurable bulk-material mechanical properties. The mechanical phenotyping of single cells therefore offers many potential diagnostic applications. Cells are viscoelastic and their response to an applied stress is highly dependent on the magnitude and timescale of the actuation. Microfluidics can be used to measure cell deformability over a wide range of flow conditions, operating two distinct flow regimes (shear and inertial) which can expose subtle mechanical properties arising from subcellular components. Here, we investigate the deformability of three colorectal cancer (CRC) cell lines using a range of flow conditions. These cell lines offer a model for CRC metastatic progression; SW480 derived from primary adenocarcinoma, HT29 from a more advanced primary tumor and SW620 from lymph-node metastasis. HL60 (leukemia cells) were also studied as a model circulatory cell, offering a non-epithelial comparison. We demonstrate that microfluidic induced flow deformation can be used to robustly detect mechanical changes associated with CRC progression. We also show that single-cell multivariate analysis, utilising deformation and relaxation dynamics, offers potential to distinguish these different cell types. These results point to the benefit of multiparameter determination for improving detection and accuracy of disease stage diagnosis

Impedimetric biosensor based on extracellular matrix protein-adhesin binding

Data about ECM protein-adhesin based biosensor for whole pathogen detection. The information found in the data corresponds to the most relevant findings of the research aimed to be published

Cells Under Stress : An Inertial-Shear Microfluidic Determination of Cell Behavior

The deformability of a cell is the direct result of a complex interplay between the different constituent elements at the subcellular level, coupling a wide range of mechanical responses at different length scales. Changes to the structure of these components can also alter cell phenotype, which points to the critical importance of cell mechanoresponse for diagnostic applications. The response to mechanical stress depends strongly on the forces experienced by the cell. Here, we use cell deformability in both shear-dominant and inertia-dominant microfluidic flow regimes to probe different aspects of the cell structure. In the inertial regime, we follow cellular response from (visco-)elastic through plastic deformation to cell structural failure and show a significant drop in cell viability for shear stresses >11.8 kN/m2. Comparatively, a shear-dominant regime requires lower applied stresses to achieve higher cell strains. From this regime, deformation traces as a function of time contain a rich source of information including maximal strain, elastic modulus, and cell relaxation times and thus provide a number of markers for distinguishing cell types and potential disease progression. These results emphasize the benefit of multiple parameter determination for improving detection and will ultimately lead to improved accuracy for diagnosis. We present results for leukemia cells (HL60) as a model circulatory cell as well as for a colorectal cancer cell line, SW480, derived from primary adenocarcinoma (Dukes stage B). SW480 were also treated with the actin-disrupting drug latrunculin A to test the sensitivity of flow regimes to the cytoskeleton. We show that the shear regime is more sensitive to cytoskeletal changes and that large strains in the inertial regime cannot resolve changes to the actin cytoskeleton

Characterisation of Liposome-Loaded Microbubble Populations for Subharmonic Imaging

Therapeutic microbubbles could make an important contribution to the diagnosis and treatment of cancer. Acoustic characterisation was performed on microfluidic generated microbubble populations that either were bare or had liposomes attached. Through the use of broadband attenuation techniques (3–8 MHz), the shell stiffness was measured to be 0.72 ± 0.01 and 0.78 ± 0.05 N/m and shell friction was 0.37 ± 0.05 and 0.74 ± 0.05 × 10−6 kg/s for bare and liposome-loaded microbubbles, respectively. Acoustic scatter revealed that liposome-loaded microbubbles had a lower subharmonic threshold, occurring from a peak negative pressure of 50 kPa, compared with 200 kPa for equivalent bare microbubbles. It was found that liposome loading had a negligible effect on the destruction threshold for this microbubble type, because at a mechanical index >0.4 (570 kPa), 80% of both populations were destroyed

Magnetic forces for surface-based bioanalysis in microfluidic devices

Biochemical analysis is a critical part of clinical diagnostics, forensic science and environmental monitoring. Typically, this form of analysis involves the use of bioanalytical procedures which are extremely sensitive and highly specific. However, such assays often involve many different reaction and washing steps, which means that a single analysis could take hours or even days to perform. There is an urgent demand for high through-put analysis systems that are rapid, simple to use and can be utilised in the field or at the point-of-care. Microfluidic technology has gone some way to producing such systems, however many of the current devices still incorporate batch methods of analysis, which are still time consuming or do not integrate all steps of the analysis on one device. Two aspects of particle handling utilising magnetic forces were investigated. 1) The use of single magnetic particles as mobile solid-supports in a continuous flow system for bioanalysis. 2) The use of diamagnetic repulsion forces for label-free on-chip particle handling. For the continuous flow system, a magnetic particle type was selected from eight commercially available brands of particles and characterised using on and off-chip measurements. Dynabead M-270 particles were ultimately used as mobile solidsupports for performing entire bioanalytical processes in continuous flow, for the first time. This was achieved by generating multiple laminar flow streams along the length of a rectangular chamber and applying a magnetic field gradient perpendicular to flow. Each reagent stream contained reagents for a molecular binding assay and functionalised magnetic particles were deflected across the width of the chamber. As the particles were deflected through reagent streams, washing and reaction steps were performed on the surface of the particle in one operation in continuous flow. The system was used to perform a one-step streptavidin - biotin binding assay with an LOD of 20 µg mL-1 , a two-step mouse IgG immunoassay with an LOD of 0.1 µg mL-1 and a qualitative sandwich immunoassay for C-reactive protein (CRP). All three procedures were performed within one minute with no manual intervention. For the diamagnetic repulsion forces for label-free particle handling, 10 µm polystyrene particles were trapped into plugs on a glass capillary by suspending them in a paramagnetic solution and creating an area of high magnetic field gradient between two magnets. Particles were repelled by the field and formed a plug upstream from the magnets. The system was used to simultaneously perform positive and negative controls on a sample of fluorescently labelled biotin using two plugs; one containing streptavidin functionalised polystyrene particles and one containing plain polystyrene particles. In addition, diamagnetic repulsion was used to deflect polystyrene particles from flow inside a square deflection chamber. A particle mixture of 5 µm and 10 µm particles were separated into different exits at a flow rate of 20 µLh-1 based on the difference in their volume, termed free-flow diamagnetophoresis. Potential applications include label-free cell manipulation