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Functional integration of proteins with carbon-based nanomaterials such as nanotubes holds great promise in emerging electronic and optoelectronic applications. Control over protein attachment poses a major challenge for consistent and useful device fabrication, especially when utilizing single/few molecule properties. Here, we exploit genetically encoded phenyl azide photochemistry to define the direct covalent attachment of four different proteins, including the fluorescent protein GFP and a β-lactamase binding protein (BBP), to carbon nanotube side walls. AFM showed that on attachment BBP could still recognize and bind additional protein components. Single molecule fluorescence revealed that on attachment to SWCNTs function was retained and there was feedback to GFP in terms of fluorescence intensity and improved resistance to photobleaching; GFP is fluorescent for much longer on attachment. The site of attachment proved important in terms of electronic impact on GFP function, with the attachment site furthest from the chromophore having the larger effect on fluorescence. Our approach provides a versatile and general method for generating intimate protein–CNT hybrid bioconjugates. It can be potentially applied to any protein of choice; the attachment position and thus interface characteristics with the CNT can easily be changed by simply placing the phenyl azide chemistry at different residues by gene mutagenesis. Thus, our approach will allow consistent construction and modulate functional coupling through changing the protein attachment position.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Fluorescent proteins (FPs) are commonly used in pairs to monitor dynamic biomolecular events through changes in proximity via distance dependent processes such as Förster resonance energy transfer (FRET). The impact of FP association is assessed by predicting dimerization sites in silico and stabilizing the dimers by bio‐orthogonal covalent linkages. In each tested case dimerization changes inherent fluorescence, including FRET. GFP homodimers demonstrate synergistic behavior with the dimer being brighter than the sum of the monomers. The homodimer structure reveals the chromophores are close with favorable transition dipole alignments and a highly solvated interface. Heterodimerization (GFP with Venus) results in a complex with ≈87% FRET efficiency, significantly below the 99.7% efficiency predicted. A similar efficiency is observed when the wild‐type FPs are fused to a naturally occurring protein–protein interface system. GFP complexation with mCherry results in loss of mCherry fluorescence. Thus, simple assumptions used when monitoring interactions between proteins via FP FRET may not always hold true, especially under conditions whereby the protein–protein interactions promote FP interaction., Fluorescent proteins (FP) are an important tool in the life sciences, including monitoring dynamic processes such as protein–protein interactions. Here, it is shown that when FPs associate their core functional parameters can change. Thus, simple assumptions used when monitoring interactions between proteins via FRET may not always hold true, especially under conditions whereby the protein–protein interactions promote FP interaction.

Microfluidic droplet generation affords precise, low volume, high throughput opportunities for molecular diagnostics. Isothermal DNA amplification with bioluminescent detection is a fast, low-cost, highly specific molecular diagnostic technique that is triggerable by temperature. Combining loop-mediated isothermal nucleic acid amplification (LAMP) and bioluminescent assay in real time (BART), with droplet microfluidics, should enable high-throughput, low copy, sequence-specific DNA detection by simple light emission. Stable, uniform LAMP–BART droplets are generated with low cost equipment. The composition and scale of these droplets are controllable and the bioluminescent output during DNA amplification can be imaged and quantified. Furthermore these droplets are readily incorporated into encapsulated droplet interface bilayers (eDIBs), or artificial cells, and the bioluminescence tracked in real time for accurate quantification off chip. Microfluidic LAMP–BART droplets with high stability and uniformity of scale coupled with high throughput and low cost generation are suited to digital DNA quantification at low template concentrations and volumes, where multiple measurement partitions are required. The triggerable reaction in the core of eDIBs can be used to study the interrelationship of the droplets with the environment and also used for more complex chemical processing via a self-contained network of droplets, paving the way for smart soft-matter diagnostics.

The bottom‐up construction of synthetic cells with user‐defined chemical organization holds considerable promise in the creation of bioinspired materials. Complex emulsions, droplet networks, and nested vesicles all represent platforms for the engineering of segregated chemistries with controlled communication, analogous to biological cells. Microfluidic manufacture of such droplet‐based materials typically results in radial or axisymmetric structures. In contrast, biological cells frequently display chemical polarity or gradients, which enable the determination of directionality, and inform higher‐order interactions. Here, a dual‐material, 3D‐printing methodology to produce microfluidic architectures that enable the construction of functional, asymmetric, hierarchical, emulsion‐based artificial cellular chassis is developed. These materials incorporate droplet networks, lipid membranes, and nanoparticle components. Microfluidic 3D‐channel arrangements enable symmetry‐breaking and the spatial patterning of droplet hierarchies. This approach can produce internal gradients and hemispherically patterned, multilayered shells alongside chemical compartmentalization. Such organization enables incorporation of organic and inorganic components, including lipid bilayers, within the same entity. In this way, functional polarization, that imparts individual and collective directionality on the resulting artificial cells, is demonstrated. This approach enables exploitation of polarity and asymmetry, in conjunction with compartmentalized and networked chemistry, in single and higher‐order organized structures, thereby increasing the palette of functionality in artificial cellular materials., Polarized artificial cells are created by 3D‐microfluidics. Multi‐material 3D‐printed microfluidics is used to create hierarchical droplet assemblies, where 3D‐channel geometries enable asymmetric and polarized patterning in droplet assemblies. These materials contain lipid‐bilayer‐segregated droplet networks and integrate patterned organic and inorganic components. Polarized patterning with magnetic microparticles bestows directional properties and motion in aqueous environments, with constructs exhibiting simple group‐level behaviors.

In article number https://doi.org/10.1002/advs.201901719, David Anthony Barrow, Oliver Kieran Castell, and co‐workers create polarized artificial cells from compartmentalized droplet networks and nanomaterials, using multi‐material, 3D‐printed fluidics. Polarized patterning with magnetic microparticles bestow directional properties and motion in aqueous environments, with artificial cells exhibiting simple group‐level behaviors.

Fluorescent proteins (FPs) are commonly used in pairs to monitor dynamic biomolecular events through changes in their proximity via distance dependent processes such as Förster resonance energy transfer (FRET). Many FPs have a tendency to oligomerise, which is likely to be promoted through attachment to associating proteins through increases in local FP concentration. We show here that on association of FP pairs, the inherent function of the FPs can alter. Artificial dimers were constructed using a bioorthogonal Click chemistry approach that combined a commonly used green fluorescent protein (superfolder GFP) with itself, a yellow FP (Venus) or a red FP (mCherry). In each case dimerisation changes the inherent fluorescent properties, including FRET capability. The GFP homodimer demonstrated synergistic behaviour with the dimer being brighter than the sum of the two monomers. The structure of the GFP homodimer revealed that a water-rich interface is formed between the two monomers, with the chromophores being in close proximity with favourable transition dipole alignments. Dimerisation of GFP with Venus results in a complex displaying ∼86% FRET efficiency, which is significantly below the near 100% efficiency predicted. When GFP is complexed with mCherry, FRET and mCherry fluorescence itself is essentially lost. Thus, the simple assumptions used when monitoring interactions between proteins via FP FRET may not always hold true, especially under conditions whereby the protein-protein interactions promote FP interaction.Abstract Figure

Construction of artificial higher order protein complexes allows sampling of structural architectures and functional features not accessible by classical monomeric proteins. Here, we combine in silico modelling with expanded genetic code facilitated strain promoted azide-alkyne cycloaddition to construct artificial complexes that are structurally integrated protein dimers and demonstrate functional synergy. Using fluorescent proteins sfGFP and Venus as models, homodimers and heterodimers are constructed that switched ON once assembled and display enhanced spectral properties. Symmetrical crosslinks are found to be important for functional enhancement. The determined molecular structure of one artificial dimer shows that a new long-range polar network comprised mostly of organised water molecules links the two chromophores leading to activation and functional enhancement. Single molecule analysis reveals the dimer is more resistant to photobleaching spending longer times in the ON state. Thus, genetically encoded bioorthogonal chemistry can be used to generate truly integrated artificial protein complexes that enhance function.

Functional integration of proteins with carbon-based nanomaterials such as nanotubes holds great promise in emerging electronic and optoelectronic applications. Control over protein attachment poses a major challenge for consistent and useful device fabrication, especially when utilizing single/few molecule properties. Here, we exploit genetically encoded phenyl azide photochemistry to define the direct covalent attachment of three different proteins, including the fluorescent protein GFP, to carbon nanotube side walls. Single molecule fluorescence revealed that on attachment to SWCNTs GFP’s fluorescence changed in terms of intensity and improved resistance to photobleaching; essentially GFP is fluorescent for much longer on attachment. The site of attachment proved important in terms of electronic impact on GFP function, with the attachment site furthest from the functional center having the larger effect on fluorescence. Our approach provides a versatile and general method for generating intimate protein-CNT hybrid bioconjugates. It can be potentially applied easily to any protein of choice; attachment position and thus interface characteristics with the CNT can easily be changed by simply placing the phenyl azide chemistry at different residues by gene mutagenesis. Thus, our approach will allow consistent construction and modulate functional coupling through changing the protein attachment position.

The ability to make artificial lipid bilayers compatible with a wide range of environments, and with sufficient structural rigidity for manual handling, would open up a wealth of opportunities for their more routine use in real‐world applications. Although droplet interface bilayers (DIBs) have been demonstrated in a host of laboratory applications, from chemical logic to biosynthesis reaction vessels, their wider use is hampered by a lack of mechanical stability and the largely manual methods employed in their production. Multiphase microfluidics has enabled us to construct hierarchical triple emulsions with a semipermeable shell, in order to form robust, bilayer‐bound, droplet networks capable of communication with their external surroundings. These constructs are stable in air, water, and oil environments and overcome a critical obstacle of achieving structural rigidity without compromising environmental interaction. This paves the way for practical application of artificial membranes or droplet networks in diverse areas such as medical applications, drug testing, biophysical studies and their use as synthetic cells.

We combined in silicomodelling with fully genetically encoded strain promoted azide-alkyne cycloaddition, to construct bespoke protein dimers. Using fluorescent proteins GFP and Venus as models, homo and heterodimers were constructed that switched ON once assembled and displayed enhanced spectral properties. The determined molecular structure reveals long range polar bond networks involving amino acids and structured water molecules play a key role in activation and functional enhancement by directly linking the two functional centres. Single molecule analysis revealed the dimer is more resistant to photobleaching spending longer times in the ON state with only one CRO likely to be active at any one time. Thus, genetically encoded bioorthogonal chemistry can be used beyond simple passive linkage approaches to generate new and truly integrated protein complexes that form long range bonds networks, which have a profound effect on function and our understanding of fluorescent protein function.

Protein nanopores such as α-haemolysin and Mycobacterium smegmatis porin A (MspA) can be used to sequence long strands of DNA at low cost. To provide high-speed sequencing, large arrays of nanopores are required, but current nanopore sequencing methods rely on ionic current measurements from individually addressed pores and such methods are likely to prove difficult to scale up. Here we show that, by optically encoding the ionic flux through protein nanopores, the discrimination of nucleic acid sequences and the detection of sequence-specific nucleic acid hybridization events can be parallelized. We make optical recordings at a density of ∼10(4) nanopores per mm(2) in a single droplet interface bilayer. Nanopore blockades can discriminate between DNAs with sub-picoampere equivalent resolution, and specific miRNA sequences can be identified by differences in unzipping kinetics. By creating an array of 2,500 bilayers with a micropatterned hydrogel chip, we are also able to load different samples into specific bilayers suitable for high-throughput nanopore recording.

G protein-coupled receptors (GPCRs) are the largest class of membrane receptors, playing a key role in the regulation of processes as varied as neurotransmission and immune response. Evidence for GPCR oligomerisation has been accumulating that challenges the idea that GPCRs function solely as monomeric receptors; however, GPCR oligomerisation remains controversial primarily due to the difficulties in comparing evidence from very different types of structural and dynamic data. Using a combination of single-molecule and ensemble FRET, double electron–electron resonance spectroscopy, and simulations, we show that dimerisation of the GPCR neurotensin receptor 1 is regulated by receptor density and is dynamically tuneable over the physiological range. We propose a “rolling dimer” interface model in which multiple dimer conformations co-exist and interconvert. These findings unite previous seemingly conflicting observations, provide a compelling mechanism for regulating receptor signalling, and act as a guide for future physiological studies., Evidence suggests oligomerisation of G protein-coupled receptors in membranes, but this is controversial. Here, authors use single-molecule and ensemble FRET, and spectroscopy to show that the neurotensin receptor 1 forms multiple dimer conformations that interconvert - “rolling” interfaces.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

We form an artificial lipid bilayer between a nanolitre aqueous droplet and a supporting hydrogel immersed in an oil/lipid solution. Manipulation of the axial position of the droplet relative to the hydrogel controls the size of the bilayer formed at the interface; this enables the surface density of integral membrane proteins to be controlled. We are able to modulate the surface density of the β-barrel pore-forming toxin α-hemolysin over a range of 4 orders of magnitude within a time frame of a few seconds. The concentration changes are fully reversible. Membrane protein function and diffusion are unaltered, as measured by single molecule microscopy and single channel electrical recording.

Optical platforms for assaying membrane protein function offer a promising route to scalable high-throughput screening (see picture). For the first time quantitative measurements of membrane protein inhibition are reported in an optically addressable lipid bilayer array. Wide-field total internal reflection fluorescence (TIRF) imaging of Ca 2+ flux enables the quantification of α-hemolysin inhibition by γ-cyclodextrin. Copyright © 2012 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim.

A contributing factor to the labored advance of molecularly imprinting as a viable commercial solution to molecular recognition needs is the absence of a standard and robust method for assessing and reporting on molecular imprinted polymer (MIP) performance. The diversity and at times inappropriateness of MIP performance indicators means that the usefulness of the literature back-catalogue, for predicting, elucidating or understanding patterns in MIP efficacy, remains largely inaccessible. We hereby put forward the case that the simple binding isotherm is the most versatile and useful method of assessing and reporting MIP function, allowing direct comparison between polymers prepared and evaluated in different studies. In this study we describe how to correctly plot and interpret a bound / free isotherm and show how such plots can be readily used to predict outcomes, retro-analyze data and optimize experimental design. We propose that by adopting the use of correctly constructed isotherms as the primary form of data representation researchers will enable inter-laboratory comparisons, promote good experimental design and encourage a greater collective understanding of molecular imprinting. Copyright © 2011 John Wiley & Sons, Ltd.

We report the first development of a novel, planar, microfluidic, graphitic carbon separations column utilizing an array of graphitic micropillars of diamond cross-section as the chromatographic stationary phase. 795 nm femtosecond laser ablation was employed to subtractively machine fluidic architectures and a micropillared array in a planar, graphitic substrate as a monolithic structure. A sample injector was integrated on-chip, together with fluid-flow distribution architectures to minimize band-broadening and ensure sample equi-distribution across the micro-pillared column width. The separations chip was interfaced directly to the ESI probe of a Thermofisher Surveyor mass spectrometer, enabling the detection of test-mixture analytes following their differential retention on the micro-pillared graphitic column, thus demonstrating the exciting potential of this novel separations format. Importantly, unlike porous, graphitic microspheres, the temperature and pressure resilience of the microfluidic device potentially enables use in subcritical H(2)O chromatography.

Capillary forces on the microscale are exploited to create a continuous flow liquid-liquid phase separator. Segmented flow regimes of immiscible fluids are generated and subsequently separated into their component phases through an array of high aspect ratio, laser machined, separation ducts (36 microm wide, 130 microm deep) in a planar, integrated, polytetrafluoroethylene (PTFE) microdevice. A controlled pressure differential across the phase separator architecture facilitates the selective passage of the wetting, organic, phase through the separator ducts, enabling separation of microfluidic multiphase flow streams. The reported device is demonstrated to separate water and chloroform segmented flow regimes at flow rates up to 0.4 ml min(-1). Separation efficiency is quantified over a range of flow rates and applied pressure differentials, characterising device behaviour and limits of operation. Experimental measurements and observations are supported by theoretical hydrodynamic and capillary pressure modelling. The influence of material properties and geometric design parameters on phase separation is quantified and optimisation strategies proposed. The novel ability of the membrane free device to separate an organic phase containing suspended microparticulates, from an aqueous phase, is also demonstrated.

Molecularly imprinted polymers (MIPs) represent a class of artificial receptors that promise an environmentally robust alternative to naturally occurring biorecognition elements of biosensing devices and systems. However, in general, the performance of conventional MIPs in aqueous environments is poor. In the study reported here, this limitation has been addressed by the novel application of MIPs as a solvent extraction solid phase in a biphasic solvent system. This paper describes a previously unreported use of MIPs as solvent extraction reagents, their successful application to aqueous sample media and the opportunities for utilisation of this unique system in novel biosensing and separation procedures. This study demonstrates the development of a novel biphasic solvent system utilising MIP in the extracting phase to enhance both efficiency and selectivity of a simple two phase liquid extraction. Monodisperse propranolol imprinted polymer microspheres [p(divinylbenzene-co-methacrylic acid)] were prepared by precipitation polymerisation. Initially, the affinity of the polymers for (R,S)-propranolol was assessed by established techniques whereby the MIP demonstrated greater affinity for the template than did the non-imprinted control polymer (NIP). Importantly, MIP performance was also assessed using the novel dual solvent system. The depletion of (R,S)-propranolol from the aqueous phase into the polymer containing organic phase was determined. When compared to control extractions containing no polymer the presence of MIP in the extracting solvent phase resulted in an increased extraction of (R,S)-propranolol from the aqueous phase. Importantly, this extraction was significantly greater in the presence of MIP when compared to NIP. This unique principle generates opportunities for MIP based extractions and chemical enrichments in industrial applications, offering commercial, ecological and practical advantages to traditional solvent extraction techniques. The technique is readily transferable to analytical microsystems utilising MIP recognition elements generating promising opportunities for MIP based sensing of aqueous sample media.

A Droplet Interface Bilayer (DIB) forms when nanolitre aqueous droplets are contacted together in an oil solution in the presence of phospholipids: A lipid monolayer forms at each oil-water interface, and by bringing together two monolayers a bilayer is created.We have recently extended this methodology to modulate both two-dimensional protein concentration[1] and membrane curvature.Manipulation of the axial position of the droplet relative to a hydrogel substrate controls the size of the bilayer formed at the interface; this enables the surface density of integral membrane proteins to be controlled. We are able to modulate the surface density of integral membrane proteins over a range of 4 orders of magnitude within a timeframe of a few seconds. By imaging DIBs on curved substrates we have observed correlation of lipid domain formation with sites of intrinsic curvature. This technique provides a new method for dictating the curvature of artificial bilayers, enabling single molecule measurements on the role of curvature in membrane organization and function.[1] Gross LCM, Castell OK, Wallace MI. Nano Letters (2011) http://dx.doi.org/10.1021/nl201689v

A TIRF imaging platform for the simultaneous interrogation of multiple lipid bilayers in an arrayed format has been developed. Here, a proof of principle demonstration is made with the quantification of Calcium flux through the trans membrane pore, alpha-hemolysin. The effect of pore blocking by cyclodextrins is also demonstrated.Utilising a droplet on hydrogel method of lipid bilayer formation, an array of multiple lipid bilayers are illuminated simultaneously by the evanescent field of laser light undergoing total internal reflection at the surface of the glass hydrogel support layer.The droplet based method of bilayer production is ideally suited to an array platform, with the segregated contents of each droplet affording the opportunity to incorporate various membrane proteins, or simultaneously screen a variety of molecular candidates for their interaction with incorporated membrane components. The system offers the opportunity of asymmetric bilayer generation and the option of rapidly screening environmental dependencies for protein function. The scalable and automatable nature of the approach, together with the ability to tailor droplet contents, creates many exciting opportunities for characterisation of membrane protein behaviour and ligand interactions, the focus of future studies with the platform.View Large Image | View Hi-Res Image | Download PowerPoint Slide

This is the first direct experimental probe, using EXAFS, of the active site within molecularly imprinted polymers and paves the way to a more detailed understanding of the inner workings of molecular imprinting.

Highly efficient molecular extractions in continuous flow microfluidic systems are demonstrated utilising the rapid mixing properties of biphasic segmented flow in conjunction with suspended micro-particulate adsorbents. A continuous flow technique providing potential for continual on-line sample enrichment, purification and clean-up in chemical synthesis, and sample preparation.

Membrane curvature plays a crucial role in lipid and protein sorting within the cell membrane [1] giving rise to spontaneous self-organisation and localisation of complex biological macromolecules. Such spatial organisation in response to, and in generating, membrane curvature contributes to many important biological processes such as neurotransmission, trafficking and cell division [2]. Despite this importance, the majority of in-vitro studies are performed in planar or pseudo-planar (GUV) bilayer systems. As such, there is a need for novel strategies to more closely mimic the curvature of cellular membranes, whilst allowing for correlated measurements of protein function.Here, we create Droplet Interface Bilayers (DIBs) [3] on an underlying curved substrate, resulting in the morphology of the underlying substrate being reflected in the curvature of the bilayer. Total Internal Reflection Fluorescence (TIRF) imaging reveals the correlation of lipid domain formation at sites of intrinsic curvature and the preferential localisation and pore formation by antimicrobial peptides at regions of positive curvature. This technique provides a new method for dictating the curvature of artificial bilayers, enabling single molecule studies on the role of curvature in membrane organisation and function.1. Ramamurthi, K. S. et al. Science 2009 323, 1354.2. McMahon, H. T. et al. Nature 2005 438, 590.3. Thompson, J. R. et al. Nano Letters 2007 7, 3875.

G protein-coupled receptors (GPCRs) are the largest class of eukaryotic membrane proteins. They trigger intracellular signalling cascades by activation of heterotrimeric G proteins. They are of great pharmaceutical interest, with approximately 40% of marketed drugs targeting GPCRs. It has been shown that GPCRs can form oligomers in phospholipid bilayers in vivo and in vitro, affecting both ligand binding and G protein coupling [1].Neurotensin receptor 1 (NTS1) is one of few GPCRs that can be produced in E. coli in an active state, and has been implicated in conditions such as schizophrenia and Parkinson's and postulated as a biomarker for various cancers [2]. NTS1 has been shown to dimerise in lipid bilayers [3], and though a crystal structure of NTS1 in detergent was recently published [4], there is still no structural data on the receptor and its dimer in a membrane environment.We use a range of biophysical techniques to characterize the structure and function of NTS1 in model membrane systems, including ensemble and single molecule Forster resonance energy transfer (FRET), and double electron-electron resonance (DEER, also known as PELDOR). Fluorescence or nitroxide spin probes are attached to engineered cysteines on the transmembrane helices. By measuring intradimer distances between the probes on each monomer, we are studying dimerisation behaviour of NTS1 to produce a model of its dimeric structure in a more native lipid environment.References1. N.J. Smith and G. Milligan (2010), Pharmacol Rev 62, 701-25.2. R. M. Myers et al. (2009), ACS Chem Biol 4, 503-525.3. P.J. Harding et al. (2009), Biophys J 96, 964-73.4. J.F. White et al. (2012), Nature 490, 508-13.