Molecular Beacon Modified Sensor Chips for Oligonucleotide Detection with Optical Readout

Three different surface bound molecular beacons (MBs) were investigated using surface plasmon fluorescence spectroscopy (SPFS) as an optical readout technique. While MB1 and MB2, both consisting of 36 bases, differed only in the length of the linker for surface attachment, the significantly longer MB3, consisting of 56 bases, comprised an entirely different sequence. For sensor chip preparation, the MBs were chemisorbed on gold via thiol anchors together with different thiol spacers. The influence of important parameters, such as the length of the MBs, the length of the linker between the MBs and the gold surface, the length and nature of the thiol spacers, and the ratio between the MBs and the thiol spacers was studied. After hybridization with the target, the fluorophore of the longer MB3 was oriented close to the surface, and the shorter MBs were standing more or less upright, leading to a larger increase in fluorescence intensity. Fluorescence microscopy revealed a homogeneous distribution of the MBs on the surface. The sensor chips could be used for simple and fast detection of target molecules with a limit of detection in the larger picomolar range. The response time was between 5 and 20 min. Furthermore, it was possible to distinguish between fully complementary and singly mismatched targets. While rinsing with buffer solution after hybridization with target did not result in any signal decrease, complete dehybridization could be carried out by intense rinsing with pure water. The MB modified sensor chips could be prepared in a repeatable manner and reused many times without significant decrease in performance

The Effect of Size and Geometry of Poly(acrylamide) Brush-Based Micropatterns on the Behavior of Cells

In this study, the fabrication, detailed characterization, and application of long-term stable micropatterned bio-interfaces of passivating poly­(acrylamide) (PAAm) brushes on transparent gold for application in the study of cell-surface interactions is reported. The micropatterns were fabricated by microcontact printing of an initiator for surface-initiated atom transfer radical polymerization (SI-ATRP), SI-ATRP of acrylamide, and subsequently backfilling of the unfunctionalized areas of 400–2500 μm² size and systematically altered number of corners with octadecanethiol. As verified by surface plasmon resonance spectroscopy, the physisorption of fibronectin (FN) was restricted to the adhesive areas. Exploiting this platform, the effect of micropattern geometry and size of cell-adhesive FN areas surrounded by passivating PAAm brushes on transparent gold substrates on the attachment of cells and cytoskeleton alignment was investigated at the single-cell level. Exceptional long-term stability of the patterned PAAm brushes and arrays of adhesive areas, in which human pancreatic tumor cells (Patu 8988T) and fibroblast cells (NIH 3T3) were confined for more than one week, was observed. Adhesive areas of 1600 μm² or less constrained the cell shape and caused focal adhesions to accumulate in the corners of the pattern. These changes were most obvious for the PatuT cells in adhesive areas of ∼900 μm², in which the actin filaments were aligned, following the boundary of the pattern, and merged in the focal adhesions concentrated in the corners of the pattern. NIH 3T3 cells possessed a larger cell area, which led to an optimal cytoskeleton alignment in adhesive patterns of ∼1600 μm². The alignment of the cytoskeleton was found to be less pronounced in cells on larger adhesive areas, where the PatuT cells spread similarly to cells on unpatterned substrates. By contrast, the NIH 3T3 cells were found to stretch even on larger adhesive areas, spanning from one corner to the other. The long-term stability under cell culture conditions of the patterns introduced here will also be useful for long-term studies of single and multiple cells, cell motility in toxicity assays, and stem cell differentiation

The Effect of Size and Geometry of Poly(acrylamide) Brush-Based Micropatterns on the Behavior of Cells

In this study, the fabrication, detailed characterization, and application of long-term stable micropatterned bio-interfaces of passivating poly­(acrylamide) (PAAm) brushes on transparent gold for application in the study of cell-surface interactions is reported. The micropatterns were fabricated by microcontact printing of an initiator for surface-initiated atom transfer radical polymerization (SI-ATRP), SI-ATRP of acrylamide, and subsequently backfilling of the unfunctionalized areas of 400–2500 μm² size and systematically altered number of corners with octadecanethiol. As verified by surface plasmon resonance spectroscopy, the physisorption of fibronectin (FN) was restricted to the adhesive areas. Exploiting this platform, the effect of micropattern geometry and size of cell-adhesive FN areas surrounded by passivating PAAm brushes on transparent gold substrates on the attachment of cells and cytoskeleton alignment was investigated at the single-cell level. Exceptional long-term stability of the patterned PAAm brushes and arrays of adhesive areas, in which human pancreatic tumor cells (Patu 8988T) and fibroblast cells (NIH 3T3) were confined for more than one week, was observed. Adhesive areas of 1600 μm² or less constrained the cell shape and caused focal adhesions to accumulate in the corners of the pattern. These changes were most obvious for the PatuT cells in adhesive areas of ∼900 μm², in which the actin filaments were aligned, following the boundary of the pattern, and merged in the focal adhesions concentrated in the corners of the pattern. NIH 3T3 cells possessed a larger cell area, which led to an optimal cytoskeleton alignment in adhesive patterns of ∼1600 μm². The alignment of the cytoskeleton was found to be less pronounced in cells on larger adhesive areas, where the PatuT cells spread similarly to cells on unpatterned substrates. By contrast, the NIH 3T3 cells were found to stretch even on larger adhesive areas, spanning from one corner to the other. The long-term stability under cell culture conditions of the patterns introduced here will also be useful for long-term studies of single and multiple cells, cell motility in toxicity assays, and stem cell differentiation

Surface Nanobubbles Studied by Time-Resolved Fluorescence Microscopy Methods Combined with AFM: The Impact of Surface Treatment on Nanobubble Nucleation

The impact of surface treatment and modification on surface nanobubble nucleation in water has been addressed by a new combination of fluorescence lifetime imaging microscopy (FLIM) and atomic force microscopy (AFM). In this study, rhodamine 6G (Rh6G)-labeled surface nanobubbles nucleated by the ethanol–water exchange were studied on differently cleaned borosilicate glass, silanized glass as well as self-assembled monolayers on transparent gold by combined AFM-FLIM. While the AFM data confirmed earlier reports on surface nanobubble nucleation, size, and apparent contact angles in dependence of the underlying substrate, the colocalization of these elevated features with highly fluorescent features observed in confocal intensity images added new information. By analyzing the characteristic contributions to the excited state lifetime of Rh6G in decay curves obtained from time-correlated single photon counting (TCSPC) experiments, the characteristic short-lived (<600 ps) component of could be associated with an emission at the gas–water interface. Its colocalization with nanobubble-like features in the AFM height images provides evidence for the observation of gas-filled surface nanobubbles. While piranha-cleaned glass supported nanobubbles, milder UV-ozone or oxygen plasma treatment afforded glass–water interfaces, where no nanobubbles were observed by combined AFM-FLIM. Finally, the number density of nanobubbles scaled inversely with increasing surface hydrophobicity

Detailed Study of BSA Adsorption on Micro- and Nanocrystalline Diamond/β-SiC Composite Gradient Films by Time-Resolved Fluorescence Microscopy

The adsorption of bovine serum albumin (BSA) on micro- and nanocrystalline diamond/β-SiC composite films synthesized using the hot filament chemical vapor deposition (HFCVD) technique has been investigated by confocal fluorescence lifetime imaging microscopy. BSA labeled with fluorescein isothiocyanate (FITC) was employed as a probe. The BSA^FITC conjugate was found to preferentially adsorb on both O-/OH-terminated microcrystalline and nanocrystalline diamond compared to the OH-terminated β-SiC, resulting in an increasing amount of BSA adsorbed to the gradient surfaces with an increasing diamond/β-SiC ratio. The different strength of adsorption (>30 times for diamond with a grain size of 570 nm) coincides with different surface energy parameters and differing conformational changes upon adsorption. Fluorescence data of the adsorbed BSA^FITC on the gradient film with different diamond coverage show a four-exponential decay with decay times of 3.71, 2.54, 0.66, and 0.13 ns for a grain size of 570 nm. The different decay times are attributed to the fluorescence of thiourea fluorescein residuals of linked FITC distributed in BSA with different dye–dye and dye–surface distances. The longest decay time was found to correlate linearly with the diamond grain size. The fluorescence of BSA^FITC undergoes external dynamic fluorescence quenching on the diamond surface by H- and/or sp²-defects and/or by amorphous carbon or graphite phases. An acceleration of the internal fluorescence concentration quenching in BSA^FITC because of structural changes of albumin due to adsorption, is concluded to be a secondary contributor. These results suggest that the micro- and nanocrystalline diamond/β-SiC composite gradient films can be utilized to spatially control protein adsorption and diamond crystallite size, which facilitates systematic studies at these interesting (bio)­interfaces

Microfluidic-Based Cell-Embedded Microgels Using Nonfluorinated Oil as a Model for the Gastrointestinal Niche

Microfluidic-based cell encapsulation has promising potential in therapeutic applications. It also provides a unique approach for studying cellular dynamics and interactions, though this concept has not yet been fully explored. No in vitro model currently exists that allows us to study the interaction between crypt cells and Peyer’s patch immune cells because of the difficulty in recreating, with sufficient control, the two different microenvironments in the intestine in which these cell types belong. However, we demonstrate that a microfluidic technique is able to provide such precise control and that these cells can proliferate inside microgels. Current microfluidic-based cell microencapsulation techniques primarily use fluorinated oils. Herein, we study the feasibility and biocompatibility of different nonfluorinated oils for application in gastrointestinal cell encapsulation and further introduce a model for studying intercellular chemical interactions with this approach. Our results demonstrate that cell viability is more affected by the solidification and purification processes that occur after droplet formation rather than the oil type used for the carrier phase. Specifically, a shorter polymer cross-linking time and consequently lower cell exposure to the harsh environment (e.g., acidic pH) results in a high cell viability of over 90% within the protected microgels. Using nonfluorinated oils, we propose a model system demonstrating the interplay between crypt and Peyer’s patch cells using this microfluidic approach to separately encapsulate the cells inside distinct alginate/gelatin microgels, which allow for intercellular chemical communication. We observed that the coculture of crypt cells alongside Peyer’s patch immune cells improves the growth of healthy organoids inside these microgels, which contain both differentiated and undifferentiated cells over 21 days of coculture. These results indicate the possibility of using droplet-based microfluidics for culturing organoids to expand their applicability in clinical research

Microfluidic-Based Cell-Embedded Microgels Using Nonfluorinated Oil as a Model for the Gastrointestinal Niche

Microfluidic-based cell encapsulation has promising potential in therapeutic applications. It also provides a unique approach for studying cellular dynamics and interactions, though this concept has not yet been fully explored. No in vitro model currently exists that allows us to study the interaction between crypt cells and Peyer’s patch immune cells because of the difficulty in recreating, with sufficient control, the two different microenvironments in the intestine in which these cell types belong. However, we demonstrate that a microfluidic technique is able to provide such precise control and that these cells can proliferate inside microgels. Current microfluidic-based cell microencapsulation techniques primarily use fluorinated oils. Herein, we study the feasibility and biocompatibility of different nonfluorinated oils for application in gastrointestinal cell encapsulation and further introduce a model for studying intercellular chemical interactions with this approach. Our results demonstrate that cell viability is more affected by the solidification and purification processes that occur after droplet formation rather than the oil type used for the carrier phase. Specifically, a shorter polymer cross-linking time and consequently lower cell exposure to the harsh environment (e.g., acidic pH) results in a high cell viability of over 90% within the protected microgels. Using nonfluorinated oils, we propose a model system demonstrating the interplay between crypt and Peyer’s patch cells using this microfluidic approach to separately encapsulate the cells inside distinct alginate/gelatin microgels, which allow for intercellular chemical communication. We observed that the coculture of crypt cells alongside Peyer’s patch immune cells improves the growth of healthy organoids inside these microgels, which contain both differentiated and undifferentiated cells over 21 days of coculture. These results indicate the possibility of using droplet-based microfluidics for culturing organoids to expand their applicability in clinical research

Microfluidic-Based Cell-Embedded Microgels Using Nonfluorinated Oil as a Model for the Gastrointestinal Niche

Microfluidic-based cell encapsulation has promising potential in therapeutic applications. It also provides a unique approach for studying cellular dynamics and interactions, though this concept has not yet been fully explored. No in vitro model currently exists that allows us to study the interaction between crypt cells and Peyer’s patch immune cells because of the difficulty in recreating, with sufficient control, the two different microenvironments in the intestine in which these cell types belong. However, we demonstrate that a microfluidic technique is able to provide such precise control and that these cells can proliferate inside microgels. Current microfluidic-based cell microencapsulation techniques primarily use fluorinated oils. Herein, we study the feasibility and biocompatibility of different nonfluorinated oils for application in gastrointestinal cell encapsulation and further introduce a model for studying intercellular chemical interactions with this approach. Our results demonstrate that cell viability is more affected by the solidification and purification processes that occur after droplet formation rather than the oil type used for the carrier phase. Specifically, a shorter polymer cross-linking time and consequently lower cell exposure to the harsh environment (e.g., acidic pH) results in a high cell viability of over 90% within the protected microgels. Using nonfluorinated oils, we propose a model system demonstrating the interplay between crypt and Peyer’s patch cells using this microfluidic approach to separately encapsulate the cells inside distinct alginate/gelatin microgels, which allow for intercellular chemical communication. We observed that the coculture of crypt cells alongside Peyer’s patch immune cells improves the growth of healthy organoids inside these microgels, which contain both differentiated and undifferentiated cells over 21 days of coculture. These results indicate the possibility of using droplet-based microfluidics for culturing organoids to expand their applicability in clinical research

Microfluidic-Based Cell-Embedded Microgels Using Nonfluorinated Oil as a Model for the Gastrointestinal Niche

Microfluidic-based cell encapsulation has promising potential in therapeutic applications. It also provides a unique approach for studying cellular dynamics and interactions, though this concept has not yet been fully explored. No in vitro model currently exists that allows us to study the interaction between crypt cells and Peyer’s patch immune cells because of the difficulty in recreating, with sufficient control, the two different microenvironments in the intestine in which these cell types belong. However, we demonstrate that a microfluidic technique is able to provide such precise control and that these cells can proliferate inside microgels. Current microfluidic-based cell microencapsulation techniques primarily use fluorinated oils. Herein, we study the feasibility and biocompatibility of different nonfluorinated oils for application in gastrointestinal cell encapsulation and further introduce a model for studying intercellular chemical interactions with this approach. Our results demonstrate that cell viability is more affected by the solidification and purification processes that occur after droplet formation rather than the oil type used for the carrier phase. Specifically, a shorter polymer cross-linking time and consequently lower cell exposure to the harsh environment (e.g., acidic pH) results in a high cell viability of over 90% within the protected microgels. Using nonfluorinated oils, we propose a model system demonstrating the interplay between crypt and Peyer’s patch cells using this microfluidic approach to separately encapsulate the cells inside distinct alginate/gelatin microgels, which allow for intercellular chemical communication. We observed that the coculture of crypt cells alongside Peyer’s patch immune cells improves the growth of healthy organoids inside these microgels, which contain both differentiated and undifferentiated cells over 21 days of coculture. These results indicate the possibility of using droplet-based microfluidics for culturing organoids to expand their applicability in clinical research

Microfluidic-Based Cell-Embedded Microgels Using Nonfluorinated Oil as a Model for the Gastrointestinal Niche

Microfluidic-based cell encapsulation has promising potential in therapeutic applications. It also provides a unique approach for studying cellular dynamics and interactions, though this concept has not yet been fully explored. No in vitro model currently exists that allows us to study the interaction between crypt cells and Peyer’s patch immune cells because of the difficulty in recreating, with sufficient control, the two different microenvironments in the intestine in which these cell types belong. However, we demonstrate that a microfluidic technique is able to provide such precise control and that these cells can proliferate inside microgels. Current microfluidic-based cell microencapsulation techniques primarily use fluorinated oils. Herein, we study the feasibility and biocompatibility of different nonfluorinated oils for application in gastrointestinal cell encapsulation and further introduce a model for studying intercellular chemical interactions with this approach. Our results demonstrate that cell viability is more affected by the solidification and purification processes that occur after droplet formation rather than the oil type used for the carrier phase. Specifically, a shorter polymer cross-linking time and consequently lower cell exposure to the harsh environment (e.g., acidic pH) results in a high cell viability of over 90% within the protected microgels. Using nonfluorinated oils, we propose a model system demonstrating the interplay between crypt and Peyer’s patch cells using this microfluidic approach to separately encapsulate the cells inside distinct alginate/gelatin microgels, which allow for intercellular chemical communication. We observed that the coculture of crypt cells alongside Peyer’s patch immune cells improves the growth of healthy organoids inside these microgels, which contain both differentiated and undifferentiated cells over 21 days of coculture. These results indicate the possibility of using droplet-based microfluidics for culturing organoids to expand their applicability in clinical research