Luke Rajah

Proliferation of amyloid-β42 aggregates occurs through a secondary nucleation mechanism

The generation of toxic oligomers during the aggregation of the amyloid-β (Aβ) peptide Aβ42 into amyloid fibrils and plaques has emerged as a central feature of the onset and progression of Alzheimer’s disease, but the molecular pathways that control pathological aggregation have proved challenging to identify. Here, we use a combination of kinetic studies, selective radiolabeling experiments, and cell viability assays to detect directly the rates of formation of both fibrils and oligomers and the resulting cytotoxic effects. Our results show that once a small but critical concentration of amyloid fibrils has accumulated, the toxic oligomeric species are predominantly formed from monomeric peptide molecules through a fibril-catalyzed secondary nucleation reaction, rather than through a classical mechanism of homogeneous primary nucleation. This catalytic mechanism couples together the growth of insoluble amyloid fibrils and the generation of diffusible oligomeric aggregates that are implicated as neurotoxic agents in Alzheimer’s disease. These results reveal that the aggregation of Aβ42 is promoted by a positive feedback loop that originates from the interactions between the monomeric and fibrillar forms of this peptide. Our findings bring together the main molecular species implicated in the Aβ aggregation cascade and suggest that perturbation of the secondary nucleation pathway identified in this study could be an effective strategy to control the proliferation of neurotoxic Aβ42 oligomers.

Microfluidic diffusion analysis of the sizes and interactions of proteins under native solution conditions

Characterizing the sizes and interactions of macromolecules under native conditions is a challenging problem in many areas of molecular sciences, which fundamentally arises from the polydisperse nature of biomolecular mixtures. Here, we describe a microfluidic platform for diffusional sizing based on monitoring micron-scale mass transport simultaneously in space and time. We show that the global analysis of such combined space-time data enables the hydrodynamic radii of individual species within mixtures to be determined directly by deconvoluting average signals into the contributions from the individual species. We demonstrate that the ability to perform rapid noninvasive sizing allows this method to be used to characterize interactions between biomolecules under native conditions. We illustrate the potential of the technique by implementing a single-step quantitative immunoassay that operates on a time scale of seconds and detects specific interactions between biomolecules within complex mixtures.

Integration and characterization of solid wall electrodes in microfluidic devices fabricated in a single photolithography step

We describe the fabrication and characterization of solid 3-dimensional electrodes in direct contact with microfluidic channels, implemented using a single photolithography step, allowing operation in high-dielectric constant media. Incorporation and self-alignment of electrodes is achieved by combining microsolidic approaches with exploitation of the surface tension of low melting point alloys. Thus the metal forms the walls flanking the channel. We show that this approach yields electrodes with a well-defined, reproducible morphology and stable electronic properties when in contact with biochemical buffers. By combining calibration of the electric field with free-flow electrophoresis we quantify the net solvated charges of small molecules.We describe the fabrication and characterization of solid 3-dimensional electrodes in direct contact with microfluidic channels, implemented using a single photolithography step, allowing operation in high-dielectric constant media. Incorporation and self-alignment of electrodes is achieved by combining microsolidic approaches with exploitation of the surface tension of low melting point alloys. Thus the metal forms the walls flanking the channel. We show that this approach yields electrodes with a well-defined, reproducible morphology and stable electronic properties when in contact with biochemical buffers. By combining calibration of the electric field with free-flow electrophoresis we quantify the net solvated charges of small molecules.

Single-molecule measurements of transient biomolecular complexes through microfluidic dilution

Single-molecule confocal microscopy experiments require concentrations which are low enough to guarantee that, on average, less than one single molecule resides in the probe volume at any given time. Such concentrations are, however, significantly lower than the dissociation constants of many biological complexes which can therefore dissociate under single-molecule conditions. To address the challenge of observing weakly bound complexes in single-molecule experiments in solution, we have designed a microfluidic device that rapidly dilutes samples by up to one hundred thousand times, allowing the observation of unstable complexes before they dissociate. The device can interface with standard biochemistry laboratory experiments and generates a spatially uniform dilution that is stable over time allowing the quantification of the relative concentrations of different molecular species.

Scaling behaviour and rate-determining steps in filamentous self-assembly

A general reaction network for filamentous self-assembly unifies mechanistic descriptions and links the overall scaling behaviour to the underlying rate-determining steps.

Particle-Based Monte-Carlo Simulations of Steady-State Mass Transport at Intermediate Péclet Numbers

Abstract Conventional approaches for simulating steady-state distributions of dilute particles under diffusive and advective transport involve solving the diffusion and advection equations in at least two dimensions. Here, we present an alternative computational strategy by combining a particle-based rather than a field-based approach with the initialisation of particles in proportion to their flux. This method allows accurate prediction of the steady state and is applicable even at intermediate and high Péclet numbers (Pe>1)

Highly Non-linear Microfluidic Resistor Elements for Flow Rate-dependent Addressing of Microchannels

(Pe \gt 1)

Twisting Transition between Crystalline and Fibrillar Phases of Aggregated Peptides

where traditional particle-based Monte-Carlo methods starting from randomly initialised particle distributions fail. We demonstrate that generating a flux of particles according to a predetermined density and velocity distribution at a single fixed time and initial location allows for accurate simulation of mass transport under flow. Specifically, upon initialisation in proportion to their flux, these particles are propagated individually and detected by summing up their Monte-Carlo trajectories in predefined detection regions. We demonstrate quantitative agreement of the predicted concentration profiles with the results of experiments performed with fluorescent particles in microfluidic channels under continuous flow. This approach is computationally advantageous and readily allows non-trivial initial distributions to be considered. In particular, this method is highly suitable for simulating advective and diffusive transport in microfluidic devices, for instance in the context of diffusive sizing.

Latent analysis of unmodified biomolecules and their complexes in solution with attomole detection sensitivity

Abstract We have developed a microfluidic resistor element exhibiting a hydrodynamic resistance that is strongly dependent on the pressure at the inlet of the device. Such an element can be used to construct microfluidic circuits that possess a highly non-linear dependence between the pressure and the flow rate, in contrast to conventional microfluidic resistors. We show that this effect can be exploited to create valves which do not require an external control line, but are actuated directly through the pressure difference between the single inlet and outlet of the device. Furthermore, we show that the flux to each branch of the circuit controlled by the valve can be fixed for high or low flow rates by modifying the fixed resistances internal to the device.