Characterization of Electrodeposited Chitosan: an Interfacial Layer for Bio-assembly and Sensing

Microfluidics and Lab-on-a-Chip devices have revolutionized the field of analytical biology. To fully optimize the potential of the microfluidic environment it is critical to be able to isolate reactions in specific locations within a channel. One solution is found using chitosan, an amine-rich biopolymer with pH responsive solubility. Induction of hydrolysis at patterned electrodes within the fluidic channel provides a means to spatially control the pH, thus enabling biochemical functionalization that is both spatially and temporally programmable. While chitosan electrodeposition has proven to be reliable at producing films, its growth characteristics are not well understood. In situ optical characterization methods of laser reflectivity, fluorescence microscopy and Raman spectroscopy have been employed to understand the growth rate inter diffusion and lateral resolution of the deposition process. These techniques have also been implemented in determining where a molecule bound to an amine site of the polymer is located within the film. Currently, electrodeposited chitosan films are primarily used for tethering of biomolecules in the recreation of metabolic pathways. Beyond just a biomolecular anchor, chitosan provides a way to incorporate inorganic nanoparticles. These composite structures enable site-specific sensors for the identification of small molecules, an important aspect to many Lab-on-a-Chip applications. New methods for creating spatially localized sites for surface enhanced Raman spectroscopy (SERS) has been developed. These methods have been optimized for particle density and SERS enhancement using TEM and Raman spectroscopy. Through optimization, a viable substrate with retained chitosan amine activity capable of integration into microfluidics has been developed

A triangular three-dye DNA switch capable of reconfigurable molecular logic

Structural DNA nanotechnology has developed profoundly in the last several years allowing for the creation of increasingly sophisticated devices capable of discrete sensing, locomotion, and molecular logic. The latter research field is particularly attractive as it provides information processing capabilities that may eventually be applied in situ, for example in cells, with potential for even further coupling to an active response such as drug delivery. Rather than design a new DNA assembly for each intended logic application, it would be useful to have one generalized design that could provide multiple different logic gates or states for a targeted use. In pursuit of this goal, we demonstrate a switchable, triangular dye-labeled three-arm DNA scaffold where the individual arms can be assembled in different combinations and the linkage between each arm can be physically removed using toehold-mediated strand displacement and then replaced by a rapid anneal. Rearranging this core structure alters the rates of Förster resonance energy transfer (FRET) between each of the two or three pendant dyes giving rise to a rich library of unique spectral signatures that ultimately form the basis for molecular photonic logic gates. The DNA scaffold is designed such that different linker lengths joining each arm, and which are used as the inputs here, can also be used independently of one another thus enhancing the range of molecular gates. The functionality of this platform structure is highlighted by easily configuring it into a series of one-, two- and three-input photonic Boolean logic gates such as OR, AND, INHIBIT, etc., along with a photonic keypad lock. Different gates can be realized in the same structure by altering which dyes are interrogated and implementation of toehold-mediated strand displacement and/or annealing allows reconfigurable switching between input states within a single logic gate as well as between two different gating devices

Restriction Enzymes as a Target for DNA-Based Sensing and Structural Rearrangement

DNA nanostructures have been shown viable for the creation of complex logic-enabled sensing motifs. To date, most of these types of devices have been limited to the interaction with strictly DNA-type inputs. Restriction endonuclease represents a class of enzyme with endogenous specificity to DNA, and we hypothesize that these can be integrated with a DNA structure for use as inputs to trigger structural transformation and structural rearrangement. In this work, we reconfigured a three-arm DNA switch, which utilizes a cyclic Förster resonance energy transfer interaction between three dyes to produce complex output for the detection of three separate input regions to respond to restriction endonucleases, and investigated the efficacy of the enzyme targets. We demonstrate the ability to use three enzymes in one switch with no nonspecific interaction between cleavage sites. Further, we show that the enzymatic digestion can be harnessed to expose an active toehold into the DNA structure, allowing for single-pot addition of a small oligo in solution

A triangular three-dye DNA switch capable of reconfigurable molecular logic

Structural DNA nanotechnology has developed profoundly in the last several years allowing for the creation of increasingly sophisticated devices capable of discrete sensing, locomotion, and molecular logic. The latter research field is particularly attractive as it provides information processing capabilities that may eventually be applied in situ, for example in cells, with potential for even further coupling to an active response such as drug delivery. Rather than design a new DNA assembly for each intended logic application, it would be useful to have one generalized design that could provide multiple different logic gates or states for a targeted use. In pursuit of this goal, we demonstrate a switchable, triangular dye-labeled three-arm DNA scaffold where the individual arms can be assembled in different combinations and the linkage between each arm can be physically removed using toehold-mediated strand displacement and then replaced by a rapid anneal. Rearranging this core structure alters the rates of Förster resonance energy transfer (FRET) between each of the two or three pendant dyes giving rise to a rich library of unique spectral signatures that ultimately form the basis for molecular photonic logic gates. The DNA scaffold is designed such that different linker lengths joining each arm, and which are used as the inputs here, can also be used independently of one another thus enhancing the range of molecular gates. The functionality of this platform structure is highlighted by easily configuring it into a series of one-, two- and three-input photonic Boolean logic gates such as OR, AND, INHIBIT, etc., along with a photonic keypad lock. Different gates can be realized in the same structure by altering which dyes are interrogated and implementation of toehold-mediated strand displacement and/or annealing allows reconfigurable switching between input states within a single logic gate as well as between two different gating devices.This is an article from RSC Advances 4 (2014): 48860, doi: 10.1039/c4ra10580j. Posted with permission.</p

Design optimization for bioMEMS studies of enzyme-controlled metabolic pathways

Abstract Biological microelectromechanical systems (bio-MEMS) provide an attractive approach to understanding and modifying enzymatic pathways by separating and interrogating individual reaction steps at localized sites in a microfluidic network. We have previously shown that electrodeposited chitosan enables immobilization of an enzyme at a specific site while maintaining its catalytic activity. While promising as a methodology to replicate metabolic pathways and search for inhibitors as drug candidates, these investigations also revealed unintended (or parasitic) effects, including products generated by the enzyme either (1) in the homogeneous phase (in the liquid), or (2) nonspecifically bound to microchannel surfaces. Here we report on bioMEMS designs which significantly suppress these parasitic effects. To reduce homogeneous reactions we have developed a new packaging and assembly strategy which eliminates fluid reservoirs that are commonly used for fluidic interconnects with external tubing. To suppress reactions by nonspecifically bound enzyme on microchannel walls we have implemented a cross-flow microfluidic network design so that enzyme flow for assembly and substrate/product for reaction share only the region where the enzyme is immobilized at the intended reaction site. Our results show that the signal-tobackground ratio of sequential enzymatic reactions increases from 0.72 to 1.28 by eliminating the packaging reservoirs, and increases to 2.43 by separating the flow direction of enzymatic reaction from that of enzyme assembly step. These techniques can be easily applied to versatile microfluidic devices to minimize parasitic reactions in sequential biochemical reactions

Bridging Lanthanide to Quantum Dot Energy Transfer with a Short-Lifetime Organic Dye

International audienceSemiconductor nanocrystals or quantum dots (QDs) should act as excellent Förster resonance energy transfer (FRET) acceptors due to their large absorption cross section, tunable emission, and high quantum yields. Engaging this type of FRET can be complicated due to direct excitation of the QD acceptor along with its longer excited-state lifetime. Many cases of QDs acting as energy transfer acceptors are within time-gated FRET from long-lifetime lanthanides, which allow the QDs to decay before observing FRET. Efficient QD sensitization requires the lanthanide to be in close proximity to the QD. To overcome the lifetime mismatch issues and limited transfer range, we utilized a Cy3 dye to bridge the energy transfer from an extremely long lived terbium emitter to the QD. We demonstrated that short-lifetime dyes can be used as energy transfer relays between extended lifetime components and in this way increased the distance of terbium-QD FRET to ∼14 nm

Resonance Energy Transfer in DNA Duplexes Labeled with Localized Dyes

The growing maturity of DNA-based architectures has raised considerable interest in applying them to create photoactive light harvesting and sensing devices. Toward optimizing efficiency in such structures, resonant energy transfer was systematically examined in a series of dye-labeled DNA duplexes where donor–acceptor separation was incrementally changed from 0 to 16 base pairs. Cyanine dyes were localized on the DNA using double phosphoramidite attachment chemistry. Steady state spectroscopy, single-pair fluorescence, time-resolved fluorescence, and ultrafast two-color pump–probe methods were utilized to examine the energy transfer processes. Energy transfer rates were found to be more sensitive to the distance between the Cy3 donor and Cy5 acceptor dye molecules than efficiency measurements. Picosecond energy transfer and near-unity efficiencies were observed for the closest separations. Comparison between our measurements and the predictions of Förster theory based on structural modeling of the dye-labeled DNA duplex suggest that the double phosphoramidite linkage leads to a distribution of intercalated and nonintercalated dye orientations. Deviations from the predictions of Förster theory point to a failure of the point dipole approximation for separations of less than 10 base pairs. Interactions between the dyes that alter their optical properties and violate the weak-coupling assumption of Förster theory were observed for separations of less than four base pairs, suggesting the removal of nucleobases causes DNA deformation and leads to enhanced dye–dye interaction

Evaluating Dye-Labeled DNA Dendrimers for Potential Applications in Molecular Biosensing

DNA nanostructures provide a reliable and predictable scaffold for precisely positioning fluorescent dyes to form energy transfer cascades. Furthermore, these structures and their attendant dye networks can be dynamically manipulated by biochemical inputs, with the changes reflected in the spectral response. However, the complexity of DNA structures that have undergone such types of manipulation for direct biosensing applications is quite limited. Here, we investigate four different modification strategies to effect such dynamic manipulations using a DNA dendrimer scaffold as a testbed, and with applications to biosensing in mind. The dendrimer has a 2:1 branching ratio that organizes the dyes into a FRET-based antenna in which excitonic energy generated on multiple initial Cy3 dyes displayed at the periphery is then transferred inward through Cy3.5 and/or Cy5 relay dyes to a Cy5.5 final acceptor at the focus. Advantages of this design included good transfer efficiency, large spectral separation between the initial donor and final acceptor emissions for signal transduction, and an inherent tolerance to defects. Of the approaches to structural rearrangement, the first two mechanisms we consider employed either toehold-mediated strand displacement or strand replacement and their impact was mainly via direct transfer efficiency, while the other two were more global in their effect using either a belting mechanism or an 8-arm star nanostructure to compress the nanostructure and thereby modulate its spectral response through an enhancement in parallelism. The performance of these mechanisms, their ability to reset, and how they might be utilized in biosensing applications are discussed