DNA Origami Vesicle Sensors with Triggered Single-Molecule Cargo Transfer.

Interacting with living systems typically involves the ability to address lipid membranes of cellular systems. The first step of interaction of a nanorobot with a cell will thus be the detection of binding to a lipid membrane. Utilizing DNA origami, we engineered a biosensor with single-molecule Fluorescence Resonance Energy Transfer (smFRET) as transduction mechanism for precise lipid vesicle detection and cargo delivery. The system hinges on a hydrophobic ATTO647N modified single-stranded DNA (ssDNA) leash, protruding from a DNA origami nanostructure. In a vesicle-free environment, the ssDNA coils, ensuring high FRET efficiency. Upon vesicle binding to cholesterol anchors on the DNA origami, hydrophobic ATTO647N induces the ssDNA to stretch towards the lipid bilayer, reducing FRET efficiency. As the next step, the sensing strand serves as molecular cargo that can be transferred to the vesicle through a triggered strand displacement reaction. Depending on the number of cholesterols on the displacer strands, we either induce a diffusive release of the fluorescent load towards neighboring vesicles or a stoichiometric release of a single cargo-unit to the vesicle on the nanosensor. Ultimately, our multi-functional liposome interaction and detection platform opens up pathways for innovative biosensing applications and controllable stoichiometric loading of vesicles with single-molecule control.

DNA Origami Voltage Sensors for Transmembrane Potentials with Single-Molecule Sensitivity.

Signal transmission in neurons goes along with changes in the transmembrane potential. To report them, different approaches, including optical voltage-sensing dyes and genetically encoded voltage indicators, have evolved. Here, we present a DNA nanotechnology-based system and demonstrated its functionality on liposomes. Using DNA origami, we incorporated and optimized different properties such as membrane targeting and voltage sensing modularly. As a sensing unit, we used a hydrophobic red dye anchored to the membrane and an anionic green dye at the DNA to connect the nanostructure and the membrane dye anchor. Voltage-induced displacement of the anionic donor unit was read out by fluorescence resonance energy transfer (FRET) changes of single sensors attached to liposomes. A FRET change of ∼5% for ΔΨ = 100 mV was observed. The working mechanism of the sensor was rationalized by molecular dynamics simulations. Our approach holds potential for an application as nongenetically encoded membrane sensors.

DNA Origami Curvature Sensors for Nanoparticle and Vesicle Size Determination with Single-Molecule FRET Readout.

Particle size is an important characteristic of materials with a direct effect on their physicochemical features. Besides nanoparticles, particle size and surface curvature are particularly important in the world of lipids and cellular membranes as the cell membrane undergoes conformational changes in many biological processes which leads to diverging local curvature values. On account of that, it is important to develop cost-effective, rapid and sufficiently precise systems that can measure the surface curvature on the nanoscale that can be translated to size for spherical particles. As an alternative approach for particle characterization, we present flexible DNA nanodevices that can adapt to the curvature of the structure they are bound to. The curvature sensors use Fluorescence Resonance Energy Transfer (FRET) as the transduction mechanism on the single-molecule level. The curvature sensors consist of segmented DNA origami structures connected via flexible DNA linkers incorporating a FRET pair. The activity of the sensors was first demonstrated with defined binding to different DNA origami geometries used as templates. Then the DNA origami curvature sensors were applied to measure spherical silica beads having different size, and subsequently on lipid vesicles. With the designed sensors, we could reliably distinguish different sized nanoparticles within a size range of 50-300 nm as well as the bending angle range of 50-180°. This study helps with the development of more advanced modular-curvature sensing devices that are capable of determining the sizes of nanoparticles and biological complexes.

Fabrication of a promising immobilization platform based on electrochemical synthesis of a conjugated polymer.

Since conjugated polymers are an important class of materials with remarkable properties in biosensor applications, in this study, a novel glucose biosensor based on a conjugated polymer was fabricated via the electropolymerization of the monomer 10,13-bis(4-hexylthiophen-2-yl)dipyridol[3,2-a:2',3'-c]phenazine onto a graphite electrode surface. Glucose oxidase (GOx) was used as the model biological recognition element. As a result of the enzymatic reaction between GOx and glucose, the glucose amount was determined by monitoring the change in the oxygen level associated with substrate concentration via the amperometric detection technique. The proposed system possessed superior properties with KMapp value of 0.262 mM, 2.88 × 10-3 mM limit of detection and 105.12 µA mM-1 cm-2 sensitivity. These results show that conjugated polymer film provides an effective and stable immobilization matrix for the enzyme. Finally, the biosensor was applied successfully to several commercially available beverage samples for glucose determination proving an inexpensive and highly sensitive system applicable for real time analyses.

Construction and amperometric biosensing performance of a novel platform containing carbon nanotubes-zinc phthalocyanine and a conducting polymer.

A novel glucose oxidase (GOx) based amperometric biosensor utilizing a conducting polymer (CP), multi walled carbon nanotubes (MWCNTs) and a novel water soluble zinc phthalocyanine (ZnPc) was constructed. For this purpose, a novel ZnPc was synthesized to examine the role of being a part of support material for enzyme deposition. High water solubility was achieved with the introduction of tetra quaternized imidazolyl moieties at the peripheral positions of phthalocyanine. In order to fabricate the proposed biosensor, a graphite electrode was firstly modified with poly[9,9-di-(2-ethylhexyl)- fluorenyl-2,7-diyl] end capped with N,N-Bis(4- methylphenyl)-4-aniline (PFLA) and MWCNTs. Then, GOx was co-immobilized with ZnPc onto the modified surface. To the best our knowledge, a sensor design which combines conjugated polymer/MWCNTs/ZnPc was attempted for the first time and this approach resulted in improved biosensor characteristics. The constructed biosensor showed a linear response for glucose between 0.025-1.0mM with a detection limit of 0.018mM. KMapp and sensitivity values were calculated as 0.53mM and 82.18µAmm-1cm-2, respectively. Moreover, scanning electron microscopy (SEM) and cyclic voltammetry (CV) techniques were used to investigate the surface modifications. Finally, fabricated biosensor was tested on beverages for glucose detection successfully.

Polymerization and biosensor application of water soluble peptide-SNS type monomer conjugates.

A simple and efficient approach for the preparation of a biosensing platform was developed based on newly designed peptide-SNS type monomer conjugates. The approach involves the electrochemical polymerization of the peptide-SNS type monomer on the electrode surface. To synthesize the peptide bearing monomers, the SNS-type monomer having a carboxylic acid functional group was anchored to the C-terminal of the peptide by solid phase peptide synthesis via coupling reagents. Utilization of peptides to increase the solubility of the monomers was first investigated in this report. The obtained monomers, soluble in water, were fully characterized by spectral analyses and utilized as matrices for biomolecule attachment. Polymerization of monomers in water has the potential to provide an alternative process for the electrochemical preparation of the polymers in aqueous media, without using any organic solvent. Under the optimized conditions, the biosensor responded to the target analyte, glucose, in a strikingly selective and sensitive manner, and showed promising feasibility for the quantitative analysis of glucose in beverages.