Sliding Contact Dynamic Force Spectroscopy Method for Interrogating Slowly Forming Polymer Cross-Links

Dynamic Single Molecule Force Spectroscopy (SMFS), conducted most commonly using AFM, has become a widespread and valuable tool for understanding the kinetics and thermodynamics of fundamental molecular processes such as ligand-receptor interactions and protein unfolding. Where slowly forming bonds are responsible for the primary characteristics of a material, as is the case in crosslinks in some polymer gels, care must be taken to ensure that a fully equilibrated bond has first formed before its rupture can be interpreted. Here we introduce a method, sliding contact force spectroscopy (SCFS), which effectively eliminates the kinetics of bond formation from the measurement of bond rupture. In addition it permits bond rupture measurements in systems where one of the binding partners may be introduced into solution prior to binding without tethering to a surface. Taking as an exemplar of a slowly forming bond the ‘eggbox’ junction crosslinks between oligoguluronic acid chains (oligoGs) in the commercially important polysaccharide alginate, we show that SCFS measures accurately the equilibrated bond strength of the crosslink when one chain is introduced into the sample solution without tethering to a surface. The results validate the SCFS technique for performing single molecule force spectroscopy experiments, and show that it has advantages in cases where the bond to be studied forms slowly and where tethering of one of the binding partners is impractical

A Titratable Cell Lysis-on-Demand System for Droplet-Compartmentalized Ultrahigh-Throughput Screening in Functional Metagenomics and Directed Evolution.

Water-in-oil emulsion droplets are an attractive format for ultrahigh-throughput screening in functional metagenomics and directed evolution applications that allow libraries with more than 107 members to be characterized in a day. Single library members are compartmentalized in droplets that are generated in microfluidic devices and tested for the presence of target biocatalysts. The target proteins can be produced intracellularly, for example, in bacterial hosts in-droplet cell lysis is therefore necessary to allow the enzymes to encounter the substrate to initiate an activity assay. Here, we present a titratable lysis-on-demand (LoD) system enabling the control of the cell lysis rate in Escherichia coli. We demonstrate that the rate of cell lysis can be controlled by adjusting the externally added inducer concentration. This LoD system is evaluated both at the population level (by optical density measurements) and at the single-cell level (on single-cell arrays and in alginate microbeads). Additionally, we validate the LoD system by droplet screening of a phosphotriesterase expressed from E. coli, with cell lysis triggered by inducer concentrations in the μM range. The LoD system yields sufficient release of the intracellularly produced enzymes to bring about a detectable quantity of product (measured by fluorescence in flow cytometry of double emulsions), while leaving viable cells for the downstream recovery of the genetic material.H202

Nanoscopic and Photonic Ultrastructural Characterization of Two Distinct Insulin Amyloid States

Two different conformational isoforms or amyloid strains of insulin with different cytotoxic capacity have been described previously. Herein these filamentous and fibrillar amyloid states of insulin were investigated using biophysical and spectroscopic techniques in combination with luminescent conjugated oligothiophenes (LCO). This new class of fluorescent probes has a well defined molecular structure with a distinct number of thiophene units that can adopt different dihedral angles depending on its binding site to an amyloid structure. Based on data from surface charge, hydrophobicity, fluorescence spectroscopy and imaging, along with atomic force microscopy (AFM), we deduce the ultrastructure and fluorescent properties of LCO stained insulin fibrils and filaments. Combined total internal reflection fluorescence microscopy (TIRFM) and AFM revealed rigid linear fibrous assemblies of fibrils whereas filaments showed a short curvilinear morphology which assemble into cloudy deposits. All studied LCOs bound to the filaments afforded more blue-shifted excitation and emission spectra in contrast to those corresponding to the fibril indicating a different LCO binding site, which was also supported by less efficient hydrophobic probe binding. Taken together, the multi-tool approach used here indicates the power of ultrastructure identification applying AFM together with LCO fluorescence interrogation, including TIRFM, to resolve structural differences between amyloid states

Single molecule investigation of the onset and minimum size of the calcium-mediated junction zone in alginate

One of the principal roles of alginate, both natively and in commercial applications, is gelation via Ca2+-mediated crosslinks between blocks of guluronic acid. In this work, single molecule measurements were carried out between well-characterised series of nearly monodisperse guluronic acid blocks (‘oligoGs’) using dynamic force spectroscopy. The measurements provide evidence that for interaction times on the order of tens of milliseconds the maximum crosslink strength is achieved by pairs of oligoGs long enough to allow the coordination of 4 Ca2+ ions, with both shorter and longer oligomers forming weaker links. Extending the interaction time from tens to hundreds of milliseconds allows longer oligoGs to achieve much stronger crosslinks but does not change the strength of individual links between shorter oligoGs. These results are considered in light of extant models for the onset of cooperative crosslinking in polyelectrolytes and an anisotropic distribution of oligoGs on interacting surfaces and provide a timescale for the formation and relaxation of alginate gels at the single crosslink level

Responsive Hydrogels for Label-Free Signal Transduction within Biosensors

Hydrogels have found wide application in biosensors due to their versatile nature. This family of materials is applied in biosensing either to increase the loading capacity compared to two-dimensional surfaces, or to support biospecific hydrogel swelling occurring subsequent to specific recognition of an analyte. This review focuses on various principles underpinning the design of biospecific hydrogels acting through various molecular mechanisms in transducing the recognition event of label-free analytes. Towards this end, we describe several promising hydrogel systems that when combined with the appropriate readout platform and quantitative approach could lead to future real-life applications

Polysaccharide Hydrogels

Polysaccharides are a unique source of organic materials in terms of abundance, structural diversity and functionalities [...

DNA aptamer functionalized hydrogels for interferometric fiber-optic based continuous monitoring of potassium ions

In the present paper, we describe a potassium sensor based on DNA-aptamer functionalized hydrogel, that is capable of continuous label-free potassium ion (K+) monitoring with potential for in situ application. A hydrogel attached to the end of an optical fiber is designed with di-oligonucleotides grafted to the polymer network that may serve as network junctions in addition to the covalent crosslinks. Specific affinity toward K+ is based on exploiting a particular aptamer that exhibits conformational transition from single-stranded DNA to G-quadruplex formed by the di-oligonucleotide in the presence of K+. Integration of this aptamer into the hydrogel transforms the K+ specific conformational transition to a K+ concentration dependent deswelling of the hydrogel. High-resolution interferometry monitors changes in extent of swelling at 1 Hz and 2 nm resolution for the hydrogel matrix of 50 µm. The developed hydrogel-based biosensor displayed high selectivity for K+ ions in the concentration range up to 10 mM, in the presence of physiological concentrations of Na+. Additionally, the concentration dependent and selective K+ detection demonstrated in the artificial blood buffer environment, both at room and physiological temperatures, suggests substantial potential for practical applications such as monitoring of potassium ion concentration in blood levels in intensive care medicine

Morpholino target molecular properties affect the swelling process of oligomorpholino-functionalized responsive hydrogels

Responsive hydrogels featuring DNA as a functional unit are attracting increasing interest due to combination of versatility and numerous applications. The possibility to use nucleic acid analogues opens for further customization of the hydrogels. In the present work, the commonly employed DNA oligonucleotides in DNA-co-acrylamide responsive hydrogels are replaced by Morpholino oligonucleotides. The uncharged backbone of this nucleic acid analogue makes it less susceptible to possible enzymatic degradation. In this work we address fundamental issues related to key processes in the hydrogel response; such as partitioning of the free oligonucleotides and the strand displacement process. The hydrogels were prepared at the end of optical fibers for interferometric size monitoring and imaged using confocal laser scanning microscopy of the fluorescently labeled free oligonucleotides to observe their apparent diffusion and accumulation within the hydrogels. Morpholino-based hydrogels’ response to Morpholino targets was compared to DNA hydrogels’ response to DNA targets of the same base-pair sequence. Non-binding targets were observed to be less depleted in Morpholino hydrogels than in DNA hydrogels, due to their electroneutrality, resulting in faster kinetics for Morpholinos. The electroneutrality, however, also led to the total swelling response of the Morpholino hydrogels being smaller than that of DNA, since their lack of charges eliminates swelling resulting from the influx of counter-ions upon oligonucleotide binding. We have shown that employing nucleic acid analogues instead of DNA in hydrogels has a profound effect on the hydrogel response

Novel imaging technologies for characterization of microbial extracellular polysaccharides

Understanding of biology is underpinned by the ability to observe structures at various length scales. This is so in a historical context and is also valid today. Evolution of novel insight often emerges from technological advancement. Recent developments in imaging technologies that is relevant for characterization of extraceullar microbiological polysaccharides are summarized. Emphasis is on scanning probe and optical based techniques since these tools offers imaging capabilities under aqueous conditions more closely resembling the physiological state than other ultramicroscopy imaging techniques. Following the demonstration of the scanning probe microscopy principle, novel operation modes to increase data capture speed toward video rate, exploitation of several cantilever frequencies, and advancement of utilization of specimen mechanical properties as contrast, also including their mode of operation in liquid, have been developed on this platform. Combined with steps in advancing light microscopy with resolution beyond the far field diffraction limit, non-linear methods, and combinations of the various imaging modalities, the potential ultramicroscopy toolbox available for characterization of exopolysaccharides (EPS) are richer than ever. Examples of application of such ultramicroscopy strategies range from imaging of isolated microbial polysaccharides, structures being observed when they are involved in polyelectrolyte complexes, aspects of their enzymatic degradation, and cell surface localization of secreted polysaccharides. These, and other examples, illustrate that the advancement in imaging technologies relevant for EPS characterization supports characterization of structural aspects

Morpholino Target Molecular Properties Affect the Swelling Process of Oligomorpholino-Functionalized Responsive Hydrogels

Responsive hydrogels featuring DNA as a functional unit are attracting increasing interest due to combination of versatility and numerous applications. The possibility to use nucleic acid analogues opens for further customization of the hydrogels. In the present work, the commonly employed DNA oligonucleotides in DNA-co-acrylamide responsive hydrogels are replaced by Morpholino oligonucleotides. The uncharged backbone of this nucleic acid analogue makes it less susceptible to possible enzymatic degradation. In this work we address fundamental issues related to key processes in the hydrogel response; such as partitioning of the free oligonucleotides and the strand displacement process. The hydrogels were prepared at the end of optical fibers for interferometric size monitoring and imaged using confocal laser scanning microscopy of the fluorescently labeled free oligonucleotides to observe their apparent diffusion and accumulation within the hydrogels. Morpholino-based hydrogels’ response to Morpholino targets was compared to DNA hydrogels’ response to DNA targets of the same base-pair sequence. Non-binding targets were observed to be less depleted in Morpholino hydrogels than in DNA hydrogels, due to their electroneutrality, resulting in faster kinetics for Morpholinos. The electroneutrality, however, also led to the total swelling response of the Morpholino hydrogels being smaller than that of DNA, since their lack of charges eliminates swelling resulting from the influx of counter-ions upon oligonucleotide binding. We have shown that employing nucleic acid analogues instead of DNA in hydrogels has a profound effect on the hydrogel response