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

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

Exploring the effects of approach velocity on depletion force and coalescence in oil-in-water emulsions

An emulsion is a thermodynamically unstable system consisting of at least two immiscible liquid phases, one of which is dispersed in the other in the form of droplets of varying size. Most studies on emulsions have focused on the behaviour of emulsion droplets with diameter from ∼50 μm and upwards. However, the properties of smaller droplets may be highly relevant in order to understand the behaviour of emulsions, including their performance in numerous applications within the fields of food, industry, and medical science. The relatively long life-time and small size of these droplets compared to other emulsion droplets, make them suited for optical trapping and micromanipulation technologies. Optical tweezers have previously shown potential in the study of stabilized emulsions. Here we employ optical tweezers to examine unstable oil-in-water emulsions to determine the effects of system parameters on depletion force and coalescence times

Tn and STn are members of a family of carbohydrate tumour antigens that possess carbohydrate-carbohydrate interactions

The mucin-type O-glycome in cancer aberrantly expresses the truncated glycans Tn (GalNAcα1-Ser/Thr) and STn (Neu5Acα2,6GalNAcα1-Ser/Thr). However, the role of Tn and STn in cancer and other diseases is not well understood. Our recent discovery of the self-binding properties (carbohydrate–carbohydrate interactions, CCIs) of Tn (Tn–Tn) and STn (STn–STn) provides a model for their possible roles in cellular transformation. We also review evidence that Tn and STn are members of a larger family of glycan tumor antigens that possess CCIs, which may participate in oncogenesis

Thin film breakage in oil–in–water emulsions, a multidisciplinary study

Coalescence is a critical phenomenon in separation and transport processes. An improved understanding of coalescence can enhance current models predicting emulsion stability and separation. We here report a combined experimental and simulation study to investigate the thin film prior to its rupture in a coalescence event. Optical tweezers measured the influence of ions and of surface-active agents on coalescence time and the forces acting between colliding oil droplets. Molecular simulation described the composition and constituent distribution of the thin films in systems comparable with the ones investigated via optical tweezers. We identify a potential relationship between the disruption of the electrical double layer and the formation of nanocrystals with the thin film breakage times and depletion forces

The Design of Simple Bacterial Microarrays: Development towards Immobilizing Single Living Bacteria on Predefined Micro-Sized Spots on Patterned Surfaces.

In this paper we demonstrate a procedure for preparing bacterial arrays that is fast, easy, and applicable in a standard molecular biology laboratory. Microcontact printing is used to deposit chemicals promoting bacterial adherence in predefined positions on glass surfaces coated with polymers known for their resistance to bacterial adhesion. Highly ordered arrays of immobilized bacteria were obtained using microcontact printed islands of polydopamine (PD) on glass surfaces coated with the antiadhesive polymer polyethylene glycol (PEG). On such PEG-coated glass surfaces, bacteria were attached to 97 to 100% of the PD islands, 21 to 62% of which were occupied by a single bacterium. A viability test revealed that 99% of the bacteria were alive following immobilization onto patterned surfaces. Time series imaging of bacteria on such arrays revealed that the attached bacteria both divided and expressed green fluorescent protein, both of which indicates that this method of patterning of bacteria is a suitable method for single-cell analysis

Thin film breakage in oil–in–water emulsions, a multidisciplinary study

Coalescence is a critical phenomenon in separation and transport processes. An improved understanding of coalescence can enhance current models predicting emulsion stability and separation. We here report a combined experimental and simulation study to investigate the thin film prior to its rupture in a coalescence event. Optical tweezers measured the influence of ions and of surface-active agents on coalescence time and the forces acting between colliding oil droplets. Molecular simulation described the composition and constituent distribution of the thin films in systems comparable with the ones investigated via optical tweezers. We identify a potential relationship between the disruption of the electrical double layer and the formation of nanocrystals with the thin film breakage times and depletion forces

Quantum dots as handles for optical manipulation

Individual colloidal quantum dots can be optically trapped and manipulated by a single infrared laser beam operated at low laser powers. If the absorption spectrum and the emission wavelength of the trapping laser are appropriately chosen, the trapping laser light can act as a source for two-photon excitation of the trapped quantum dot. This eliminates the need for an additional excitation laser in experiments where individual quantum dots are used both as force transducers and for visualization of the system. To use quantum dots as handles for quantitative optical force transduction, it is crucial to perform a precise force calibration. Here, we present an Allan variance analysis of individual optically trapped quantum dots and show that the optimal measurement time for experiments involving individual quantum dots is on the order of 0.3 seconds. Due to their small size and strong illumination, quantum dots are optimal for single molecule assays where, optimally, the presence of the tracer particle should not dominate the dynamics of the system. As an example, we investigated the thermal fluctuations of a DNA tether using an individual colloidal quantum dot as marker, this being the smallest tracer for tethered particle method reported