25 results found
Chatzimichail S, Supramaniam P, Salehi-Reyhani SA, 2021, Absolute quantification of protein copy number in single cells with immunofluorescence microscopy calibrated using single molecule microarrays, Analytical Chemistry, Vol: 93, Pages: 6656-6664, ISSN: 0003-2700
Great strides toward routine single cell analyses have been made over the last decade, particularly in the field of transcriptomics. For proteomics, amplification is not currently possible and has necessitated the development of ultra-sensitive platforms capable of performing such analyses on single cells. These platforms are improving in terms of throughput and multiplexability but still fall short in relation to more established methods such as fluorescence microscopy. However, microscopy methods rely on fluorescence intensity as a proxy for protein abundance and are not currently capable of reporting this in terms of absolute copy number. Here, a microfluidic implementation of single molecule microarrays for single cell analysis is assessed in its ability to calibrate fluorescence microscopy data. We show that the equivalence of measurements of the steady-state distribution of protein abundance to single molecule microarray data can be exploited to pave the way for absolute quantitation by fluorescence and immunofluorescence microscopy. The methods presented have been developed using GFP but are extendable to other proteins and other biomolecules of interest.
Chatzimichail S, Rahimi F, Saifuddin A, et al., 2021, Hand-portable HPLC with broadband spectral detection enables analysis of complex polycyclic aromatic hydrocarbon mixtures, Communications Chemistry, Vol: 4, ISSN: 2399-3669
Polycyclic aromatic hydrocarbons (PAHs) are considered priority hazardous substances due to their carcinogenic activity and risk to public health. Strict regulations are in place limiting their release into the environment, but enforcement is hampered by a lack of adequate field-testing procedure, instead relying on sending samples to centralised analytical facilities. Reliably monitoring levels of PAHs in the field is a challenge, owing to the lack of field-deployable analytical methods able to separate, identify, and quantify the complex mixtures in which PAHs are typically observed. Here, we report the development of a hand-portable system based on high-performance liquid chromatography incorporating a spectrally wide absorption detector, capable of fingerprinting PAHs based on their characteristic spectral absorption profiles: identifying 100% of the 24 PAHs tested, including full coverage of the United States Environmental Protection Agency priority pollutant list. We report unsupervised methods to exploit these new capabilities for feature detection and identification, robust enough to detect and classify co-eluting and hidden peaks. Identification is fully independent of their characteristic retention times, mitigating matrix effects which can preclude reliable determination of these analytes in challenging samples. We anticipate the platform to enable more sophisticated analytical measurements, supporting real-time decision making in the field.
Rahimi F, Chatzimichail S, Saifuddin A, et al., 2020, A review of portable high-performance liquid chromatography: the future of the field?, Chromatographia, Vol: 83, Pages: 1165-1195, ISSN: 0009-5893
There is a growing need for chemical analyses to be performed in the field, at the point of need. Tools and techniques often found in analytical chemistry laboratories are necessary in performing these analyses, yet have, historically, been unable to do so owing to their size, cost and complexity. Technical advances in miniaturisation and liquid chromatography are enabling the translation of these techniques out of the laboratory, and into the field. Here we examine the advances that are enabling portable liquid chromatography (LC). We explore the evolution of portable instrumentation from its inception to the most recent advances, highlighting the trends in the field and discussing the necessary criteria for developing in-field solutions. While instrumentation is becoming more capable it has yet to find adoption outside of research.
Mickleburgh TG, Salehi-Reyhani A, Magness AJ, et al., 2020, A miniaturized microfluidic assay for single plant cell protein quantitation, Pages: 183-184
We present a method based on a miniaturized antibody capture chip to perform protein quantitation in plants with single cell single molecule resolution. We demonstrate the versatility of the method in capturing and analyzing membrane, cytosolic, and chloroplast stroma located proteins, specifically demonstrated by SNARE 12: YFP, 35S: GFP, and Ribulose-1, 5-bisphosphate carboxylase/oxygenase, respectively. Single cell protein expression quantification is achieved through a bi or tri-molecular antibody microarray. We have also reported single cell experimental evidence that a reduction in chamber volume enhances assay sensitivity.
Chatzimichail S, Casey D, Salehi-Reyhani A, 2019, Zero electrical power pump for portable high-performance liquid chromatography, The Analyst, Vol: 144, Pages: 6207-6213, ISSN: 0003-2654
A major trend in analytical chemistry is the miniaturization of laboratory instrumentation. We report a pump requiring no power to operate based on the controlled expansion of a pre-pressurised gas for use in portable applications of high-performance liquid chromatography. The performance of the gas pump is characterised and integrated into a compact liquid chromatography system capable of isocratic separations integrating an LED-based UV-absorption detector. The system weighed 6.7 kg when the mobile phase reservoir was fully charged with 150 mL solvent and included an on-board computer to control the system and analyse data. We characterise the flow-rate through chromatography columns with a variety of geometries and packing materials for a range of pressures up to 150 bar. The maximum variation in flow rate was measured to be 6.5 nL min−1, limited by the resolution of the flow detector. All tests were made on battery power and results are a mixture of those made in the laboratory and in the field. Additionally, we performed a series of 1 m drop tests on the device and show the system's high tolerance to mechanical shocks during operation in the field.
Supramaniam P, Ces O, Salehi-Reyhani A, 2019, Microfluidics for artificial life: techniques for bottom-up synthetic biology, Micromachines, Vol: 10, Pages: 1-27, ISSN: 2072-666X
Synthetic biology is a rapidly growing multidisciplinary branch of science that exploits the advancement of molecular and cellular biology. Conventional modification of pre-existing cells is referred to as the top-down approach. Bottom-up synthetic biology is an emerging complementary branch that seeks to construct artificial cells from natural or synthetic components. One of the aims in bottom-up synthetic biology is to construct or mimic the complex pathways present in living cells. The recent, and rapidly growing, application of microfluidics in the field is driven by the central tenet of the bottom-up approach—the pursuit of controllably generating artificial cells with precisely defined parameters, in terms of molecular and geometrical composition. In this review we survey conventional methods of artificial cell synthesis and their limitations. We proceed to show how microfluidic approaches have been pivotal in overcoming these limitations and ushering in a new generation of complexity that may be imbued in artificial cells and the milieu of applications that result.
Friddin M, Bolognesi G, Salehi-Reyhani A, et al., 2019, Direct manipulation of liquid ordered lipid membrane domains using optical traps, Communications Chemistry, Vol: 2, Pages: 1-7, ISSN: 2399-3669
Multicomponent lipid bilayers can give rise to coexisting liquid domains that are thought to influence a host of cellular activities. There currently exists no method to directly manipulate such domains, hampering our understanding of their significance. Here we report a system that allows individual liquid ordered domains that exist in a liquid disordered matrix to be directly manipulated using optical tweezers. This allows us to drag domains across the membrane surface of giant vesicles that are adhered to a glass surface, enabling domain location to be defined with spatiotemporal control. We can also use the laser to select individual vesicles in a population to undergo mixing/demixing by locally heating the membrane through the miscibility transition, demonstrating a further layer of control. This technology has potential as a tool to shed light on domain biophysics, on their role in biology, and in sculpting membrane assemblies with user-defined membrane patterning.
Barlow N, Kusumaatmaja H, Salehi-Reyhani A, et al., 2018, Measuring bilayer surface energy and curvature in asymmetric droplet interface bilayers, Journal of the Royal Society Interface, Vol: 15, ISSN: 1742-5662
For the past decade, droplet interface bilayers (DIBs) have had an increased prevalence in biomolecular and biophysical literature. However, much of the underlying physics of these platforms is poorly characterized. To further our understanding of these structures, lipid membrane tension on DIB membranes is measured by analysing the equilibrium shape of asymmetric DIBs. To this end, the morphology of DIBs is explored for the first time using confocal laser scanning fluorescence microscopy. The experimental results confirm that, in accordance with theory, the bilayer interface of a volume-asymmetric DIB is curved towards the smaller droplet and a lipid-asymmetric DIB is curved towards the droplet with the higher monolayer surface tension. Moreover, the DIB shape can be exploited to measure complex bilayer surface energies. In this study, the bilayer surface energy of DIBs composed of lipid mixtures of phosphatidylgylcerol (PG) and phosphatidylcholine are shown to increase linearly with PG concentrations up to 25%. The assumption that DIB bilayer area can be geometrically approximated as a spherical cap base is also tested, and it is discovered that the bilayer curvature is negligible for most practical symmetric or asymmetric DIB systems with respect to bilayer area.
Chatzimichail S, Supramaniam P, Ces O, et al., 2018, Micropatterning of planar metal electrodes by vacuum filling microfluidic channel geometries, Scientific Reports, Vol: 8, ISSN: 2045-2322
We present a simple, facile method to micropattern planar metal electrodes defined by the geometry of a microfluidic channel network template. By introducing aqueous solutions of metal into reversibly adhered PDMS devices by desiccation instead of flow, we are able to produce difficult to pattern “dead end” or discontinuous features with ease. We characterize electrodes fabricated using this method and perform electrical lysis of mammalian cancer cells and demonstrate their use as part of an antibody capture assay for GFP. Cell lysis in microwell arrays is achieved using the electrodes and the protein released is detected using an antibody microarray. We show how the template channels used as part of the workflow for patterning the electrodes may be produced using photolithography-free methods, such as laser micromachining and PDMS master moulding, and demonstrate how the use of an immiscible phase may be employed to create electrode spacings on the order of 25 – 50 μm, that overcome the current resolution limits of such methods. This work demonstrates how the rapid prototyping of electrodes for use in total analysis systems can be achieved on the bench with little or no need for centralized facilities.
Trantidou T, Friddin M, Salehi-Reyhani S, et al., 2018, Droplet microfluidics for the construction of compartmentalised model membranes, Lab on a Chip, Vol: 18, Pages: 2488-2509, ISSN: 1473-0189
The design of membrane-based constructs with multiple compartments is of increasing importance given their potential applications as microreactors, as artificial cells in synthetic-biology, as simplified cell models, and as drug delivery vehicles. The emergence of droplet microfluidics as a tool for their construction has allowed rapid scale-up in generation throughput, scale-down of size, and control over gross membrane architecture. This is true on several levels: size, level of compartmentalisation and connectivity of compartments can all be programmed to various degrees. This tutorial review explains and explores the reasons behind this. We discuss microfluidic strategies for the generation of a family of compartmentalised systems that have lipid membranes as the basic structural motifs, where droplets are either the fundamental building blocks, or are precursors to the membrane-bound compartments. We examine the key properties associated with these systems (including stability, yield, encapsulation efficiency), discuss relevant device fabrication technologies, and outline the technical challenges. In doing so, we critically review the state-of-play in this rapidly advancing field.
Chatzimichail S, Supramaniam P, Ces O, et al., 2018, Counting proteins in single cells with addressable droplet microarrays, Jove-Journal of Visualized Experiments, Vol: 137, ISSN: 1940-087X
Often cellular behaviour and cellular responses are analysed at the population level where the responses of many cells are pooled together as an average result masking the rich single cell behaviour within a complex population. Single cell protein detection and quantification technologies have made a remarkable impact in recent years. Here we describe a practical and flexible single cell analysis platform based on addressable droplet microarrays. This study describes how the absolute copy numbers of target proteins may be measured with single cell resolution. The tumour suppressor p53 is the most commonly mutated gene in human cancer, with more than 50% of total cancer cases exhibiting a non-healthy p53 expression pattern. The protocol describes steps to create 10nL droplets within which single human cancer cells are isolated and the copy number of p53 protein is measured with single molecule resolution to precisely determine the variability in expression. The method may be applied to any cell type including primary material to determine the absolute copy number of any target proteins of interest.
Bolognesi G, Friddin MS, Salehi-Reyhani S, et al., 2018, Sculpting and fusing biomimetic vesicle networks using optical tweezers, Nature Communications, Vol: 9, Pages: 1-11, ISSN: 2041-1723
Constructing higher-order vesicle assemblies has discipline-spanning potential from responsive soft-matter materials to artificial cell networks in synthetic biology. This potential is ultimately derived from the ability to compartmentalise and order chemical species in space. To unlock such applications, spatial organisation of vesicles in relation to one another must be controlled, and techniques to deliver cargo to compartments developed. Herein, we use optical tweezers to assemble, reconfigure and dismantle networks of cell-sized vesicles that, in different experimental scenarios, we engineer to exhibit several interesting properties. Vesicles are connected through double-bilayer junctions formed via electrostatically controlled adhesion. Chemically distinct vesicles are linked across length scales, from several nanometres to hundreds of micrometres, by axon-like tethers. In the former regime, patterning membranes with proteins and nanoparticles facilitates material exchange between compartments and enables laser-triggered vesicle merging. This allows us to mix and dilute content, and to initiate protein expression by delivering biomolecular reaction components.
Salehi-Reyhani S, 2017, Evaluating single molecule detection methods for microarrays with high dynamic range for quantitative single cell analysis, Scientific Reports, Vol: 7, ISSN: 2045-2322
Single molecule microarrays have been used in quantitative proteomics, in particular, single cell analysis requiring high sensitivity and ultra-low limits of detection. In this paper, several image analysis methods are evaluated for their ability to accurately enumerate single molecules bound to a microarray spot. Crucially, protein abundance in single cells can vary significantly and may span several orders of magnitude. This poses a challenge to single molecule image analysis. In order to quantitatively assess the performance of each method, synthetic image datasets are generated with known ground truth whereby the number of single molecules varies over 5 orders of magnitude with a range of signal to noise ratios. Experiments were performed on synthetic datasets whereby the number of single molecules per spot corresponds to realistic single cell distributions whose ground truth summary statistics are known. The methods of image analysis are assessed in their ability to accurately estimate the distribution parameters. It is shown that super-resolution image analysis methods can significantly improve counting accuracy and better cope with single molecule congestion. The results highlight the challenge posed by quantitative single cell analysis and the implications to performing such analyses using microarray based approaches are discussed.
Salehi-Reyhani A, Ces O, Elani Y, 2017, Artificial cell mimics as simplified models for the study of cell biology, Experimental Biology and Medicine, Vol: 242, Pages: 1309-1317, ISSN: 1535-3702
Living cells are hugely complex chemical systems composed of a milieu of distinct chemical species (including DNA, proteins, lipids, and metabolites) interconnected with one another through a vast web of interactions: this complexity renders the study of cell biology in a quantitative and systematic manner a difficult task. There has been an increasing drive towards the utilization of artificial cells as cell mimics to alleviate this, a development that has been aided by recent advances in artificial cell construction. Cell mimics are simplified cell-like structures, composed from the bottom-up with precisely defined and tunable compositions. They allow specific facets of cell biology to be studied in isolation, in a simplified environment where control of variables can be achieved without interference from a living and responsive cell. This mini-review outlines the core principles of this approach and surveys recent key investigations that use cell mimics to address a wide range of biological questions. It will also place the field in the context of emerging trends, discuss the associated limitations, and outline future directions of the field.
Lakatos E, Salehi-Reyhani S, Barclay M, et al., 2017, Protein degradation rate is the dominant mechanism accounting for the differences in protein abundance of basal p53 in a human breast and colorectal cancer cell line, PLOS One, Vol: 12, ISSN: 1932-6203
We determine p53 protein abundances and cell to cell variation in two human cancer cell lines with single cell resolution, and show that the fractional width of the distributions is the same in both cases despite a large difference in average protein copy number. We developed a computational framework to identify dominant mechanisms controlling the variation of protein abundance in a simple model of gene expression from the summary statistics of single cell steady state protein expression distributions. Our results, based on single cell data analysed in a Bayesian framework, lends strong support to a model in which variation in the basal p53 protein abundance may be best explained by variations in the rate of p53 protein degradation. This is supported by measurements of the relative average levels of mRNA which are very similar despite large variation in the level of protein.
Willison KR, Salehi-Reyhani A, Burgin E, et al., 2015, Absolute quantification of protein copy number in single cells using single molecule microarrays, EUROPEAN BIOPHYSICS JOURNAL WITH BIOPHYSICS LETTERS, Vol: 44, Pages: S179-S179, ISSN: 0175-7571
Salehi-Reyhani A, Gesellchen F, Mampallil D, et al., 2015, Chemical-Free Lysis and Fractionation of Cells by Use of Surface Acoustic Waves for Sensitive Protein Assays, ANALYTICAL CHEMISTRY, Vol: 87, Pages: 2161-2169, ISSN: 0003-2700
Casey D, Wylie D, Gallo J, et al., 2015, A novel, all-optical tool for controllable and non-destructive poration of cells with single-micron resolution, Bio-Optics: Design and Application 2015, Publisher: Optical Society of America
We demonstrate controllable poration within ≈1 µm regions of individual cells, mediated by a near-IR laser interacting with thin-layer amorphous silicon substrates. This technique will allow new experiments in single-cell biology, particularly in neuroscience.
Salehi-Reyhani A, Burgin E, Ces O, et al., 2014, Addressable droplet microarrays for single cell protein analysis, ANALYST, Vol: 139, Pages: 5367-5374, ISSN: 0003-2654
Burgin E, Salehi-Reyhani A, Barclay M, et al., 2014, Absolute quantification of protein copy number using a single-molecule-sensitive microarray, ANALYST, Vol: 139, Pages: 3235-3244, ISSN: 0003-2654
Salehi-Reyhani A, Sharma S, Burgin E, et al., 2014, Scaling advantages and constraints in miniaturized capture assays for single cell protein analysis (vol 13, pg 2066, 2013), LAB ON A CHIP, Vol: 14, Pages: 3430-3430, ISSN: 1473-0197
Salehi-Reyhani A, Sharma S, Burgin E, et al., 2013, Scaling Advantages and Constraints in Miniaturized Capture Assays for Single Cell Protein Analysis, Lab on A Chip, Vol: 13, Pages: 2066-2074, ISSN: 1473-0197
Measuring protein expression in single cells is the basis of single cell proteomics. The sensitivity and dynamic range of a single cell immunoassay should ideally be such that proteins that are expressed between 1 – 106 copies per cell can be detected and counted. We have investigated the effect of miniaturizing antibody microarrays by reducing capture spot sizes from 100 μm to 15 μm using dip pen nanolithography. We demonstrate that protocols developed for printing and passivating antibody capture spots using conventional pin based contact printing can be directly transferred to dip-pen lithography whilst retaining the capture activity per unit area. Using a simple kinetic model, we highlight how the limit of detection and dynamic range of a sandwich immunoassay, respectively, increase and decrease when spot size is reduced. However, we show that reducing spot size is more effective than increasing assay chamber volume when seeking to multiplex such a microfluidic immunoassay. Although, we make particular reference to single cell microfluidic immunoassays, the topics discussed here are applicable to capture assays in general.
Sharma S, Reyhani AS, Bahrami A, et al., 2012, A novel method for fabricating nanostructures via nanotemplates using dip pen nanolithography, Micro & Nano Letters, Vol: 7, Pages: 1038-1040
Dip-pen nanolithography (DPN) relies on the compatibility between the ink used and the substrate it is written on. This has lead to a significant reliance for DPN on thiol-gold chemistries for the fabrication of nanostructures. This reported work demonstrates a method for creating nanostructures through nanotemplates that allows a wider range of substrates and inks to be used. The substrate was spin-coated with a thin layer of polymer which is selectively removed using a scanning probe microscopy tip coated with a solvent. This produces nanotemplates that are subsequently used to create nanostructures by metallisation. Compared with directly written templates these nanotemplates facilitate longer interaction between inking material and the substrate. They also allow a controlled spatial resolution along the z-axis and eliminate the need for any blocking agents to prevent non-specific adsorption. This method allows DPN to be expanded to applications beyond thiol-gold chemistries.
Salehi-Reyhani A, Barclay M, Ces O, et al., 2012, Towards Practical Single Cell Proteomics: A Microfluidic Antibody Capture Chip with TIRF Detection, FREE RADICAL BIOLOGY AND MEDICINE, Vol: 53, Pages: S128-S128, ISSN: 0891-5849
Salehi-Reyhani A, Kaplinsky J, Burgin E, et al., 2011, A first step towards practical single cell proteomics: a microfluidic antibodycapture chip with TIRF detection, Lab Chip, Vol: 11, Pages: 1256-1261
We have developed a generic platform to undertake the analysis of protein copy number from singlecells. The approach described here is ‘all-optical’ whereby single cells are manipulated into separate analysis chambers using an optical trap; single cells are lysed by a shock wave caused by laser-induced microcavitation, and the protein released from a single cell is measured by total internal reflection microscopy as it is bound to micro-printed antibody spots within the device. The platform was tested using GFP transfected cells and the relative precision of the measurement method was determined to be 88%. Single cell measurements were also made on a breast cancer cell line to measure the relative levels of unlabelled human tumour suppressor protein p53 using a chip incorporating an antibody sandwich assay format. These results suggest that this is a viable method for measuring relative protein levels in single cells.
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