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Advancements in understanding and engineering of virus-based nanomaterials (VBNs) for biomedical applications motivate a need to explore the interfaces between VBNs and other biomedically-relevant chemistries and materials. While several strategies have been used to investigate some of these interfaces with promising initial results, including VBN-containing slow-release implants and VBN-activated bioceramic bone scaffolds, there remains a need to establish VBN-immobilized three dimensional materials that exhibit improved stability and diffusion characteristics for biosensing and other analyte-capture applications. Silica sol-gel chemistries have been researched for biomedical applications over several decades and are well understood; various cellular organisms and biomolecules (e.g., bacteria, algae, enzymes) have been immobilized in silica sol-gels to improve viability, activity, and form factor (i.e., ease of use). Here we present the immobilization of an antibody-binding VBN in silica sol-gel by pore confinement. We have shown that the resulting system is sufficiently diffuse to allow antibodies to migrate in and out of the matrix. We also show that the immobilized VBN is capable of antibody binding and elution functionality under different buffer conditions for multiple use cycles. The promising results of the VBN and silica sol-gel interface indicate a general applicability for VBN-based bioseparations and biosensing applications.

Cell-free protein synthesis can enable the combinatorial screening of many different components and concentrations. However, manual pipetting methods are unfit to handle many cell-free reactions. Here, we describe a microfluidic method that can generate hundreds of unique submicroliter scale reactions. The method is coupled with a high yield cell-free system that can be applied for broad protein screening assays.

The fluidity and polar environment of ~100 nm hybrid vesicles combining dipalmitoylphosphatidylcholine (DPPC) and poly(1,2-butadiene)-block-polyethylene oxide (PBd-PEO, average molecular weight 950 g/mol) were studied upon vesicle heating using the fluorescence spectroscopy techniques of DPH anisotropy and laurdan generalized polarization (GP). These techniques indicated PBd-PEO membranes are less ordered than solid DPPC, but slightly more ordered than fluid DPPC or dioleoylphosphatidylcholine (DOPC) membranes. We find the DPH anisotropy values are less than expected from additivity of the components' anisotropies in the fluid phase mixture of DPPC and PBd-PEO, inferring that DPPC strongly fluidizes the PBd-PEO. We use transitions in DPH anisotropy and laurdan GP to create a temperature/composition phase diagram for DPPC/PBd-PEO which we find displays a significantly broader solid/fluid phase coexistence region than DPPC/DOPC, showing that DPPC partitions less readily into fluid PBd-PEO than into fluid DOPC. The existence of a broad solid/fluid phase coexistence region in DPPC/PBd-PEO vesicles is verified by Förster resonance energy transfer results and the visualization of phase separation in giant unilamellar vesicles containing up to 95% PBd-PEO and a single phase in 100% PBd-PEO vesicles at room temperature. These results add to the limited knowledge of phase behavior and phase diagrams of hybrid vesicles, and should be useful in understanding and tailoring membrane surface architecture toward biomedical applications such as drug delivery or membrane protein reconstitution.

Synthetic biology has focused on engineering genetic modules that operate orthogonally from the host cells. A synthetic biological module, however, can be designed to reprogram the host proteome, which in turn enhances the function of the synthetic module. Here, we apply this holistic synthetic biology concept to the engineering of cell-free systems by exploiting the crosstalk between metabolic networks in cells, leading to a protein environment more favorable for protein synthesis. Specifically, we show that local modules expressing translation machinery can reprogram the bacterial proteome, changing the expression levels of more than 700 proteins. The resultant feedback generates a cell-free system that can synthesize fluorescent reporters, protein nanocages, and the gene-editing nuclease Cas9, with up to 5-fold higher expression level than classical cell-free systems. Our work demonstrates a holistic approach that integrates synthetic and systems biology concepts to achieve outcomes not possible by only local, orthogonal circuits.

Here we examine how the force on an atomic force microscope (AFM) tip varies as it approaches micellar surfactant films, and use this information to discern the film's surface structure and Young's modulus. Rows of wormlike hemimicelles were created at a graphite interface using 10 mM sodium dodecyl sulfate (SDS). We found that the repulsive force on a silicon nitride tip as it approached the surface was exponential, with a decay length of 2.0 ± 0.1 nm. The addition of Na2SO4 was found to cause a change in this behavior, with a clear split into two exponential regions at concentrations above 1 mM. We also observed that the range of these forces increased with added salt from ∼15 nm in pure SDS to ∼20 nm at a Na2SO4 concentration of 1.34 mM. These forces were inconsistent with electrostatic repulsion, and were determined to be steric in nature. We show that the behavior at higher salt concentrations is consistent with the theory of polyelectrolyte brushes in the osmotic regime. From this, we hypothesize the presence of micellar brushes at the surface that behave similarly to adsorbed polymer chains. In addition, the Young's modulus of the film was taken from data near the interface using Sneddon's model, and found to be 80 ± 40 MPa. Similar experiments were performed with 10 mM dodecylamine hydrochloride (DAH) solutions in the presence of added magnesium chloride. The decay length for the pure DAH solution was found to be 2.6 ± 0.3 nm, and the addition of 1.34 mM of MgCl2 caused this to increase to 3.7 ± 0.3 nm. No decay length splitting was observed for DAH. We conclude that the behavior at the surface resembles that of an uncharged polymer brush, as the ionic and surface charge densities are much lower for DAH than for SDS.

Titanium dioxide gel monoliths were synthesized using an organic precursor and 0-30 vol % ethanol in water. The visible-light-activated proton pump, bacteriorhodopsin, in its native purple membrane form, was successfully encapsulated within the titanium dioxide gels. Absorption spectra showed that the folded functional state of the protein remained intact within gels made with 0 and 15 vol % ethanol and retained the ability to make reversible conformational changes associated with the photocycle within the gel made with 0 vol % ethanol. The photocatalytic activity of gels made with no ethanol was significantly detectable and gels made with 0-30 vol % ethanol were comparable to commercial crystalline nanoparticles in similar solution conditions when irradiated with UV light. Our results show that sol-gel-derived photocatalytic titanium dioxide can be made biocompatible for a membrane-associated protein by minimizing the amount of ethanol and maximizing the amount of water in the synthesis procedure. The entrapment of the membrane protein, bacteriorhodopsin, in sol-gel-derived titanium dioxide provides the first step in future explorations of this bionanocomposite for visible light photocatalysis, including hydrogen production.

In this work, we study the mechanical properties of sodium dodecyl sulfate (SDS) and dodecylamine hydrochloride (DAH) micellar films at a graphite surface via atomic force microscopy (AFM). Breakthrough forces for these films were measured using silicon nitride cantilevers and were found to be 1.1 ± 0.1 nN for a 10 mM DAH film and 3.0 ± 0.3 nN for a 10 mM SDS film. For 10 mM SDS films, it was found that the addition of 1.5 mM of NaCl, Na2SO4, or MgCl2 produced a 50-70% increase in the measured breakthrough force. Similar results were found for 10 mM DAH films when NaCl and MgCl2 were added. A model was developed on the basis of previous work on lipid films and CMC data gathered via spectrofluorometry measurements to predict the change in normalized breakthrough forces with added salt concentrations for SDS and DAH films. Using this model, it was found that the activation volume required to initiate the breakthrough was roughly 0.4 nm3 for SDS and 0.3 nm3 for DAH, roughly the volume of a single molecule. Normalized breakthrough force data for SDS films with added MgCl2 showed an unexpected dip at low added salt concentrations. The model was adapted to account for changing activation volumes, and a curve of activation volume versus magnesium concentration was obtained, showing a minimum volume of 0.21 nm3. The addition of 0.2 mM SDS to a 10 mM DAH solution was found to double the measured breakthrough force of the film. Images taken of the surface showed a phase change from cylindrical hemimicelles to a planar film that may have produced the observed differences. The pH of the bulk solution was varied for both 10 mM SDS and DAH films and was found to have little effect on the breakthrough force.

We use fluorescence microscopy to examine the dynamics of the crowding-induced mixing transition of liquid ordered (Lo)-liquid disordered (Ld) phase separated lipid bilayers when the following particles of increasing size bind to either the Lo or Ld phase: Ubiquitin, green fluorescent protein (GFP), and nanolipoprotein particles (NLPs) of two diameters. These proteinaceous particles contained histidine-tags, which were phase targeted by binding to iminodiacetic acid (IDA) head groups, via a Cu2+ chelating mechanism, of lipids that specifically partition into either the Lo phase or Ld phase. The degree of steric pressure was controlled by varying the size of the bound particle (10-240 kDa) and the amount of binding sites present (i.e., DPIDA concentrations of 9 and 12 mol%) in the supported lipid multibilayer platform used here. We develop a mass transfer-based diffusional model to analyze the observed Lo phase domain dissolution that, along with visual observations and activation energy calculations, provides insight into the sequence of events in crowding-induced mixing. Our results suggest that the degree of steric pressure and target phase influence not only the efficacy of steric-pressure induced mixing, but the rate and controlling mechanism for which it occurs.

In an effort to develop a general thermodynamic model from first-principles to describe the mixing behavior of lipid membranes, we examined lipid mixing induced by targeted binding of small (Green Fluorescent Protein (GFP)) and large (nanolipoprotein particles (NLPs)) structures to specific phases of phase-separated lipid bilayers. Phases were targeted by incorporation of phase-partitioning iminodiacetic acid (IDA)-functionalized lipids into ternary lipid mixtures consisting of DPPC, DOPC, and cholesterol. GFP and NLPs, containing histidine tags, bound the IDA portion of these lipids via a metal, Cu(2+), chelating mechanism. In giant unilamellar vesicles (GUVs), GFP and NLPs bound to the Lo domains of bilayers containing DPIDA, and bound to the Ld region of bilayers containing DOIDA. At sufficiently large concentrations of DPIDA or DOIDA, lipid mixing was induced by bound GFP and NLPs. The validity of the thermodynamic model was confirmed when it was found that the statistical mixing distribution as a function of crowding energy for smaller GFP and larger NLPs collapsed to the same trend line for each GUV composition. Moreover, results of this analysis show that the free energy of mixing for a ternary lipid bilayer consisting of DOPC, DPPC, and cholesterol varied from 7.9 × 10(-22) to 1.5 × 10(-20) J/lipid at the compositions observed, decreasing as the relative cholesterol concentration was increased. It was discovered that there appears to be a maximum packing density, and associated maximum crowding pressure, of the NLPs, suggestive of circular packing. A similarity in mixing induced by NLP1 and NLP3 despite large difference in projected areas was analytically consistent with monovalent (one histidine tag) versus divalent (two histidine tags) surface interactions, respectively. In addition to GUVs, binding and induced mixing behavior of NLPs was also observed on planar, supported lipid multibilayers. The mixing process was reversible, with Lo domains reappearing after addition of EDTA for NLP removal.

The changes in the orientation and conformation of three different membrane scaffold proteins (MSPs) upon entrapment in sol-gel-derived mesoporous silica monoliths were investigated. MSPs were examined in either a lipid-free or a lipid-bound conformation, where the proteins were associated with lipids to form nanolipoprotein particles (NLPs). NLPs are water-soluble, disk-shaped patches of a lipid bilayer that have amphiphilic MSPs shielding the hydrophobic lipid tails. The NLPs in this work had an average thickness of 5 nm and diameters of 9.2, 9.7, and 14.8 nm. We have previously demonstrated that NLPs are more suitable lipid-based structures for silica gel entrapment than liposomes because of their size compatibility with the mesoporous network (2-50 nm) and minimally altered structure after encapsulation. Here we further elaborate on that work by using a variety of spectroscopic techniques to elucidate whether or not different MSPs maintain their protein-lipid interactions after encapsulation. Fluorescence spectroscopy and quenching of the tryptophan residues with acrylamide, 5-DOXYL-stearic acid, and 16-DOXYL-stearic acid were used to determine the MSP orientation. We also utilized fluorescence anisotropy of tryptophans to measure the relative size of the NLPs and MSP aggregates after entrapment. Finally, circular dichroism spectroscopy was used to examine the secondary structure of the MSPs. Our results showed that, after entrapment, all of the lipid-bound MSPs maintained orientations that were minimally changed and indicative of association with lipids in NLPs. The tryptophan residues appeared to remain buried within the hydrophobic core of the lipid tails in the NLPs and appropriately spaced from the bilayer center. Also, after entrapment, lipid-bound MSPs maintained a high degree of α-helical content, a secondary structure associated with protein-lipid interactions. These findings demonstrate that NLPs are capable of serving as viable hosts for functional integral membrane proteins in the synthesis of sol-gel-derived bioinorganic hybrid nanomaterials.

The entrapment of nanolipoprotein particles (NLPs) and liposomes in transparent, nanoporous silica gel derived from the precursor tetramethylorthosilicate was investigated. NLPs are discoidal patches of lipid bilayer that are belted by amphiphilic scaffold proteins and have an average thickness of 5 nm. The NLPs in this work had a diameter of roughly 15 nm and utilized membrane scaffold protein (MSP), a genetically altered variant of apolipoprotein A-I. Liposomes have previously been examined inside of silica sol-gels and have been shown to exhibit instability. This is attributed to their size (∼150 nm) and altered structure and constrained lipid dynamics upon entrapment within the nanometer-scale pores (5-50 nm) of the silica gel. By contrast, the dimensional match of NLPs with the intrinsic pore sizes of silica gel opens the possibility for their entrapment without disruption. Here we demonstrate that NLPs are more compatible with the nanometer-scale size of the porous environment by analysis of lipid phase behavior via fluorescence anisotropy and analysis of scaffold protein secondary structure via circular dichroism spectroscopy. Our results showed that the lipid phase behavior of NLPs entrapped inside of silica gel display closer resemblance to its solution behavior, more so than liposomes, and that the MSP in the NLPs maintain the high degree of α-helix secondary structure associated with functional protein-lipid interactions after entrapment. We also examined the effects of residual methanol on lipid phase behavior and the size of NLPs and found that it exerts different influences in solution and in silica gel; unlike in free solution, silica entrapment may be inhibiting NLP size increase and/or aggregation. These findings set precedence for a bioinorganic hybrid nanomaterial that could incorporate functional integral membrane proteins.

The oxygen permeation resistance of highly condensed monolayers composed of a homologous series of saturated diacyl phosphatidylcholine lipids was measured using a novel technique that combined micromanipulation and electrochemical techniques. The use of lipid monolayer-coated air microbubbles allowed measurement of the oxygen permeation resistance of fully condensed lipid monolayers that were previously unavailable using classical film balance techniques.