Microfluidics for protein biophysics

Microfluidics has the potential to transform experimental approaches across the life sciences. In this review, we discuss recent advances enabled by the development and application of microfluidic approaches to protein biophysics. We focus on areas where key fundamental features of microfluidics open up new possibilities and present advantages beyond low volumes and short time-scale analysis, conventionally provided by microfluidics. We discuss the two most commonly used forms of microfluidic technology, single-phase laminar flow and multiphase microfluidics. We explore how the understanding and control of the characteristic physical features of the microfluidic regime, the integration of microfluidics with orthogonal systems and the generation of well-defined microenvironments can be used to develop novel devices and methods in protein biophysics for sample manipulation, functional and structural studies, detection and material processing

High-dimensional metric combining for non-coherent molecular signal detection

In emerging Internet-of-Nano-Thing (IoNT), information will be embedded and conveyed in the form of molecules through complex and diffusive medias. One main challenge lies in the long-tail nature of the channel response causing inter-symbolinterference (ISI), which deteriorates the detection performance. If the channel is unknown, existing coherent schemes (e.g., the state-of-the-art maximum a posteriori, MAP) have to pursue complex channel estimation and ISI mitigation techniques, which will result in either high computational complexity, or poor estimation accuracy that will hinder the detection performance. In this paper, we develop a novel high-dimensional non-coherent detection scheme for molecular signals. We achieve this in a higher-dimensional metric space by combining different noncoherent metrics that exploit the transient features of the signals. By deducing the theoretical bit error rate (BER) for any constructed high-dimensional non-coherent metric, we prove that, higher dimensionality always achieves a lower BER in the same sample space, at the expense of higher complexity on computing the multivariate posterior densities. The realization of this high-dimensional non-coherent scheme is resorting to the Parzen window technique based probabilistic neural network (Parzen-PNN), given its ability to approximate the multivariate posterior densities by taking the previous detection results into a channel-independent Gaussian Parzen window, thereby avoiding the complex channel estimations. The complexity of the posterior computation is shared by the parallel implementation of the Parzen-PNN. Numerical simulations demonstrate that our proposed scheme can gain 10dB in SNR given a fixed BER as 10-4, in comparison with other state-of-the-art methods

Flow Rate Independent Multiscale Liquid Biopsy for Precision Oncology

Immunoaffinity-based liquid biopsies of circulating tumour cells (CTCs) hold great promise for cancer management, but typically suffer from low throughput, relative complexity and post-processing limitations. Here we address these issues simultaneously by decoupling and independently optimising the nano-, micro- and macro-scales of a CTC enrichment device that is both simple to fabricate and operate. At its core is a scalable macroscale mesh with optimised micropores, nano-functionalised with antibodies against cell surface proteins. Unlike other affinity-based liquid biopsies, optimum capture can be achieved independently of the flow rate, as demonstrated with constant capture efficiencies, above 75%, between 50-200 uL/min. The device achieved 96% sensitivity and 100% specificity when used to detect CTCs in the blood of 79 cancer patients and 20 healthy controls. To demonstrate its post-processing capabilities, we used immunofluorescence labelling to identify PD-L1+ CTCs in 36% of patients (n=33) as potential responders to immune checkpoint inhibition therapy. Finally, our device achieved an 80% positive match in the identification of HER2+ breast cancer (n=26) compared to clinical standard FISH on solid biopsy. The results suggest that our approach, which overcomes major limitations previously associated with affinity-based liquid biopsies, could provide a versatile tool to improve cancer management.Comment: 21 pages, 5 figures (+ supplementary materials: 11 pages, 10 figures

Mechanism of droplet-formation in a supersonic microfluidic spray device

Spray drying is an approach employed in automotive, food, and pharmaceutical industries as a robust and cost efficient liquid atomization technique offering direct control over droplet dimensions. The majority of commercially available spray nozzles are designed for large throughput spray drying applications or uniform surface coating, but microfluidic nebulizers have recently been developed as small scale alternatives. Here, we explore the physical parameters that define the droplet size and formation under supersonic flow conditions commonly found in microfluidic spray drying systems. We examined the spray nozzle operation using high speed imaging and laser scattering measurements, which allowed us to describe the spray regimes and droplet size distributions. It was determined that by using this spray nozzle device, droplets with diameters of 4–8 μm could be generated. Moreover, we show that the supersonic de Laval nozzle model can be used to predict the average droplet size. Our approach can be used as a platform for interfacing fluid microprocessing with gas phase detection and characterization

Enhancing the resolution of micro free flow electrophoresis through spatially controlled sample injection

Free flow electrophoresis is a versatile technique for the continuous separation of mixtures with both preparative and analytical applications. Microscale versions of free flow electrophoresis are particularly attractive strategies because of their fast separation times, ability to work with small sample volumes and large surface area to volume ratios facilitating rapid heat transfer, thus minimising the detrimental effects of Joule heating even at high voltages. The resolution of microscale free flow electrophoresis, however, is limited by the broadening of the analyte beam in the microfluidic channel - an effect that becomes especially pronounced when the analyte is deflected significantly away from its original position. Here we describe and demonstrate how by spatially restricting the sample injection and collection to the regions where the gradients in the velocity distribution of the carrier medium are the smallest, this broadening effect can be substantially suppressed and hence the resolution of microscale free flow electrophoresis devices increased. To demonstrate this concept we fabricated microfluidic free flow electrophoresis devices with spatially restricted injection nozzles implemented via the use of multilayer soft-photolithography and further integrated quartz based observation areas for fluorescent detection and imaging. With these devices we demonstrated a five fold reduction in the beam broadening extent compared to conventional free flow electrophoresis approaches with non-restricted sample introduction. The manifold enhancement in the achievable resolution of microscale free flow electrophoresis devices opens up the possibility of rapid separation and analysis of more complex mixtures

Heating characterization of low energy consumption Lab-on-a-Chip

The ultimate goal of this study is to develop an effective and user-friendly form of thermal cycler to perform Polymerase Chain Reaction (PCR). This paper research the ability to manufacture a thermal cycler using conventional methods and readily available components to achieve a significant performance result with low production cost compared with commercially available counterparts. Commonly, thermal cyclers use an aluminum heating block as a heating vessel for samples, to achieve fast temperature changes and uniformity, but Polydimethylsiloxanes (PDMS) lab-on-a-chip (LoC) is utilized in contrast to silver as a heating vessel in this study to achieve biocompatibility with various samples. The raspberry-Pi pocket computer was used to achieve the necessary control with room for future capabilities addition and improvement. Characterization of the heating and PCR processes was done to understand the heat transfer phenomenon and thermal cycling speed in PDMS environment. This paper also includes analysis of the cycle speed corresponding to the control parameters of the manufactured thermal cycler. Ultimately, the characterization and analysis results will show the achieved performance capabilities of the thermal cycler

Low-Cost Microfabrication Tool Box.

Microsystems are key enabling technologies, with applications found in almost every industrial field, including in vitro diagnostic, energy harvesting, automotive, telecommunication, drug screening, etc. Microsystems, such as microsensors and actuators, are typically made up of components below 1000 microns in size that can be manufactured at low unit cost through mass-production. Yet, their development for commercial or educational purposes has typically been limited to specialized laboratories in upper-income countries due to the initial investment costs associated with the microfabrication equipment and processes. However, recent technological advances have enabled the development of low-cost microfabrication tools. In this paper, we describe a range of low-cost approaches and equipment (below £1000), developed or adapted and implemented in our laboratories. We describe processes including photolithography, micromilling, 3D printing, xurography and screen-printing used for the microfabrication of structural and functional materials. The processes that can be used to shape a range of materials with sub-millimetre feature sizes are demonstrated here in the context of lab-on-chips, but they can be adapted for other applications. We anticipate that this paper, which will enable researchers to build a low-cost microfabrication toolbox in a wide range of settings, will spark a new interest in microsystems

Graphene FET sensors for Alzheimer’s disease protein biomarker clusterin detection

We report on the fabrication and characterisation of Graphene field-effect transistor (GFET) Biosensors for detecting clusterin, a prominent protein biomarker of Alzheimer’s disease (AD). There are approximately 54 million people currently living with dementia worldwide and this is expected to rise to 130 million by 2050. Although there are over 400 different types of dementia, AD is the most common type, affecting between 50–75% of those diagnosed with dementia. Diagnosis of AD can take up to 2 years currently using MRI, PET, CT scans and memory tests. There is, therefore, an urgent need to develop low-cost, accurate, non-invasive and point-of-care (PoC) sensors for early diagnosis of AD. The GFET sensors we are developing to address this challenge were fabricated on Si/SiO2 substrate through processes of photolithographic patterning and metal lift-off techniques with evaporated chromium and sputtered gold contacts. Raman Spectroscopy was performed on the devices to determine the quality of the graphene. The GFETs were annealed to improve their performance before the channels were functionalized by immobilising the graphene surface with a linker molecule and anti-clusterin antibody. The detection was achieved through the binding reaction between the antibody and varying concentrations of clusterin antigen from 1 pg/mL to 1 ng/mL. The GFETs were characterized using 4-probe direct current (DC) electrical measurements which demonstrated a limit of detection of the biosensors to be below 1 pg/mL

Flow rate-independent multiscale liquid biopsy for precision oncology

Immunoaffinity-based liquid biopsies of circulating tumor cells (CTCs) hold great promise for cancer management but typically suffer from low throughput, relative complexity, and postprocessing limitations. Here, we address these issues simultaneously by decoupling and independently optimizing the nano-, micro-, and macro-scales of an enrichment device that is simple to fabricate and operate. Unlike other affinity-based devices, our scalable mesh approach enables optimum capture conditions at any flow rate, as demonstrated with constant capture efficiencies, above 75% between 50 and 200 μL min–1. The device achieved 96% sensitivity and 100% specificity when used to detect CTCs in the blood of 79 cancer patients and 20 healthy controls. We demonstrate its postprocessing capacity with the identification of potential responders to immune checkpoint inhibition (ICI) therapy and the detection of HER2 positive breast cancer. The results compare well with other assays, including clinical standards. This suggests that our approach, which overcomes major limitations associated with affinity-based liquid biopsies, could help improve cancer management