Microfluidic Tools for Advanced Biomolecular Characterisation

Proteins – the key building blocks of life – are responsible for the majority of the processes behind biological function. To understand what role proteins play in health and disease, how they operate and interact, it is vital to have tools for biomolecular detection, quantification and fundamental physicochemical characterisation. In this thesis, I have focused on the development of new microfluidic approaches enabling quantitative analysis of biomolecules. First, I describe a microfluidic spray device, developed for a controlled deposition of analyte on surfaces. Due to the small micron-scale droplet size, the evaporation happens in a few milliseconds, thus, leaving only the solvent-free solutes. This method has been vital for depositing biomolecules on a scanning-probe microscopy-imaging substrate, enabling quantitative measurements of heterogeneous protein mixtures. Afterwards, I present the spray combination with gravimetric sensors, such as micro-cantilevers, for a label-free protein detection. I show that this technique can be used for a protein-solution concentration measurement in a quantitative manner. Currently, one of the main issues of diagnostic platforms is the analysis of heterogeneous mixtures. A number of protein-separation techniques have been developed; however, most of the characterisation requires an offline analysis which can introduce artefacts and reequilibration. In the second part of this dissertation, I bridge the gap between liquid chromatography, microfluidic characterisation and mechanical-sensor detection. Specifically, I demonstrate the serial combination between liquid chromatography and analyte deposition by a microfluidic spray nozzle. By depositing analytes onto a quartz-crystal microbalance, I perform a specific label-free analysis of protein mixtures. Furthermore, I present a fluidic interface, facilitating a combination of separation at fast liquid flow with microfluidic size and electrophoretic-mobility measurements. This method allows for a simultaneous measurement of molecule size and charge and acts as an additional chromatographic detector. I demonstrate that this method works for both label-free and labelled biomolecule characterisation and suggests ways to perform scalable mass-spectrometry analysis on a chip

Label-Free Protein Analysis Using Liquid Chromatography with Gravimetric Detection.

The detection and analysis of proteins in a label-free manner under native solution conditions is an increasingly important objective in analytical bioscience platform development. Common approaches to detect native proteins in solution often require specific labels to enhance sensitivity. Dry mass sensing approaches, by contrast, using mechanical resonators, can operate in a label-free manner and offer attractive sensitivity. However, such approaches typically suffer from a lack of analyte selectivity as the interface between standard protein separation techniques and micro-resonator platforms is often constrained by qualitative mechanical sensor performance in the liquid phase. Here, we describe a strategy that overcomes this limitation by coupling liquid chromatography with a quartz crystal microbalance (QCM) platform by using a microfluidic spray dryer. We explore a strategy which allows first to separate a protein mixture in a physiological buffer solution using size exclusion chromatography, permitting specific protein fractions to be selected, desalted, and subsequently spray-dried onto the QCM for absolute mass analysis. By establishing a continuous flow interface between the chromatography column and the spray device via a flow splitter, simultaneous protein mass detection and sample fractionation is achieved, with sensitivity down to a 100 μg/mL limit of detection. This approach for quantitative label-free protein mixture analysis offers the potential for detection of protein species under physiological conditions.ERC EPSRC Frances and Augustus Newman Foundation Oppenheimer Early Career Fellowship Nanotechnologies Doctoral Training Centre Fluidic Analytics Lt

Rapid highly sensitive general protein quantification through on-chip chemiluminescence.

Protein detection and quantification is a routinely performed procedure in research laboratories, predominantly executed either by spectroscopy-based measurements, such as NanoDrop, or by colorimetric assays. The detection limits of such assays, however, are limited to μ M concentrations. To establish an approach that achieves general protein detection at an enhanced sensitivity and without necessitating the requirement for signal amplification steps or a multicomponent detection system, here, we established a chemiluminescence-based protein detection assay. Our assay specifically targeted primary amines in proteins, which permitted characterization of any protein sample and, moreover, its latent nature eliminated the requirement for washing steps providing a simple route to implementation. Additionally, the use of a chemiluminescence-based readout ensured that the assay could be operated in an excitation source-free manner, which did not only permit an enhanced sensitivity due to a reduced background signal but also allowed for the use of a very simple optical setup comprising only an objective and a detection element. Using this assay, we demonstrated quantitative protein detection over a concentration range of five orders of magnitude and down to a high sensitivity of 10 pg mL - 1 , corresponding to pM concentrations. The capability of the platform presented here to achieve a high detection sensitivity without the requirement for a multistep operation or a multicomponent optical system sets the basis for a simple yet universal and sensitive protein detection strategy.Engineering and Physical Sciences Research Council Schmidt Science Fellows program in partnership with the Rhodes Trust European Research Council Newman Foundatio

Label-Free Protein Analysis Using Liquid Chromatography with Gravimetric Detection.

The detection and analysis of proteins in a label-free manner under native solution conditions is an increasingly important objective in analytical bioscience platform development. Common approaches to detect native proteins in solution often require specific labels to enhance sensitivity. Dry mass sensing approaches, by contrast, using mechanical resonators, can operate in a label-free manner and offer attractive sensitivity. However, such approaches typically suffer from a lack of analyte selectivity as the interface between standard protein separation techniques and micro-resonator platforms is often constrained by qualitative mechanical sensor performance in the liquid phase. Here, we describe a strategy that overcomes this limitation by coupling liquid chromatography with a quartz crystal microbalance (QCM) platform by using a microfluidic spray dryer. We explore a strategy which allows first to separate a protein mixture in a physiological buffer solution using size exclusion chromatography, permitting specific protein fractions to be selected, desalted, and subsequently spray-dried onto the QCM for absolute mass analysis. By establishing a continuous flow interface between the chromatography column and the spray device via a flow splitter, simultaneous protein mass detection and sample fractionation is achieved, with sensitivity down to a 100 μg/mL limit of detection. This approach for quantitative label-free protein mixture analysis offers the potential for detection of protein species under physiological conditions.ERC EPSRC Frances and Augustus Newman Foundation Oppenheimer Early Career Fellowship Nanotechnologies Doctoral Training Centre Fluidic Analytics Lt

Enhanced Quality Factor Label-free Biosensing with Micro-Cantilevers Integrated into Microfluidic Systems.

Microelectromechanical systems (MEMS) have enabled the development of a new generation of sensor platforms. Acoustic sensor operation in liquid, the native environment of biomolecules, causes, however, significant degradation of sensing performance due to viscous drag and relies on the availability of capture molecules to bind analytes of interest to the sensor surface. Here, we describe a strategy to interface MEMS sensors with microfluidic platforms through an aerosol spray. Our sensing platform comprises a microfluidic spray nozzle and a microcantilever array operated in dynamic mode within a closed loop oscillator. A solution containing the analyte is sprayed uniformly through picoliter droplets onto the microcantilever surface; the micrometer-scale drops evaporate rapidly and leave the solutes behind, adding to the mass of the cantilever. This sensing scheme results in a 50-fold increase in the quality factor compared to operation in liquid, yet allows the analytes to be introduced into the sensing system from a solution phase. It achieves a 370 femtogram limit of detection, and we demonstrate quantitative label-free analysis of inorganic salts and model proteins. These results demonstrate that the standard resolution limits of cantilever sensing in dynamic mode can be overcome with the integration of spray microfluidics with MEMS

Resolving protein mixtures using microfluidic diffusional sizing combined with synchrotron radiation circular dichroism

Circular dichroism spectroscopy has become a powerful tool to characterise proteins and other biomolecules. For heterogeneous samples such as those present for interacting proteins, typically only average spectroscopic features can be resolved. Here we overcome this limitation by using free-flow microfluidic size separation in-line with synchrotron radiation circular dichroism to resolve the secondary structure of each component of a model protein mixture containing monomers and fibrils. To enable this objective, we have integrated far-UV compatible measurement chambers into PDMS-based microfluidic devices. Two architectures are proposed so as to accommodate for a wide range of concentrations. The approach, which can be used in combination with other bulk measurement techniques, paves the way to the study of complex mixtures such as the ones associated with protein misfolding and aggregation diseases including Alzheimer’s and Parkinson’s diseases

Squalamine and Its Derivatives Modulate the Aggregation of Amyloid-β and α-Synuclein and Suppress the Toxicity of Their Oligomers.

The aberrant aggregation of proteins is a key molecular event in the development and progression of a wide range of neurodegenerative disorders. We have shown previously that squalamine and trodusquemine, two natural products in the aminosterol class, can modulate the aggregation of the amyloid-β peptide (Aβ) and of α-synuclein (αS), which are associated with Alzheimer's and Parkinson's diseases. In this work, we expand our previous analyses to two squalamine derivatives, des-squalamine and α-squalamine, obtaining further insights into the mechanism by which aminosterols modulate Aβ and αS aggregation. We then characterize the ability of these small molecules to alter the physicochemical properties of stabilized oligomeric species in vitro and to suppress the toxicity of these aggregates to varying degrees toward human neuroblastoma cells. We found that, despite the fact that these aminosterols exert opposing effects on Aβ and αS aggregation under the conditions that we tested, the modifications that they induced to the toxicity of oligomers were similar. Our results indicate that the suppression of toxicity is mediated by the displacement of toxic oligomeric species from cellular membranes by the aminosterols. This study, thus, provides evidence that aminosterols could be rationally optimized in drug discovery programs to target oligomer toxicity in Alzheimer's and Parkinson's diseases

Trodusquemine displaces protein misfolded oligomers from cell membranes and abrogates their cytotoxicity through a generic mechanism

Funder: Listed in manuscript for all authors.Abstract: The onset and progression of numerous protein misfolding diseases are associated with the presence of oligomers formed during the aberrant aggregation of several different proteins, including amyloid-β (Aβ) in Alzheimer’s disease and α-synuclein (αS) in Parkinson’s disease. These small, soluble aggregates are currently major targets for drug discovery. In this study, we show that trodusquemine, a naturally-occurring aminosterol, markedly reduces the cytotoxicity of αS, Aβ and HypF-N oligomers to human neuroblastoma cells by displacing the oligomers from cell membranes in the absence of any substantial morphological and structural changes to the oligomers. These results indicate that the reduced toxicity results from a mechanism that is common to oligomers from different proteins, shed light on the origin of the toxicity of the most deleterious species associated with protein aggregation and suggest that aminosterols have the therapeutically-relevant potential to protect cells from the oligomer-induced cytotoxicity associated with numerous protein misfolding diseases

Trodusquemine displaces protein misfolded oligomers from cell membranes and abrogates their cytotoxicity through a generic mechanism

Funder: Listed in manuscript for all authors.Abstract: The onset and progression of numerous protein misfolding diseases are associated with the presence of oligomers formed during the aberrant aggregation of several different proteins, including amyloid-β (Aβ) in Alzheimer’s disease and α-synuclein (αS) in Parkinson’s disease. These small, soluble aggregates are currently major targets for drug discovery. In this study, we show that trodusquemine, a naturally-occurring aminosterol, markedly reduces the cytotoxicity of αS, Aβ and HypF-N oligomers to human neuroblastoma cells by displacing the oligomers from cell membranes in the absence of any substantial morphological and structural changes to the oligomers. These results indicate that the reduced toxicity results from a mechanism that is common to oligomers from different proteins, shed light on the origin of the toxicity of the most deleterious species associated with protein aggregation and suggest that aminosterols have the therapeutically-relevant potential to protect cells from the oligomer-induced cytotoxicity associated with numerous protein misfolding diseases