Manipulation and Confinement of Single Particles Using Fluid Flow

High precision control of micro- and nanoscale objects in aqueous media is an essential technology for nanoscience and engineering. Existing methods for particle trapping primarily depend on optical, magnetic, electrokinetic, and acoustic fields. In this work, we report a new hydrodynamic flow based approach that allows for fine-scale manipulation and positioning of single micro- and nanoscale particles using automated fluid flow. As a proof-of-concept, we demonstrate trapping and two-dimensional (2D) manipulation of 500 nm and 2.2 μm diameter particles with a positioning precision as small as 180 nm during confinement. By adjusting a single flow parameter, we further show that the shape of the effective trap potential can be efficiently controlled. Finally, we demonstrate two distinct features of the flow-based trapping method, including isolation of a single particle from a crowded particle solution and active control over the surrounding medium of a trapped object. The 2D flow-based trapping method described here further expands the micro/nanomanipulation toolbox for small particles and holds strong promise for applications in biology, chemistry, and materials research

Manipulation and Confinement of Single Particles Using Fluid Flow

High precision control of micro- and nanoscale objects in aqueous media is an essential technology for nanoscience and engineering. Existing methods for particle trapping primarily depend on optical, magnetic, electrokinetic, and acoustic fields. In this work, we report a new hydrodynamic flow based approach that allows for fine-scale manipulation and positioning of single micro- and nanoscale particles using automated fluid flow. As a proof-of-concept, we demonstrate trapping and two-dimensional (2D) manipulation of 500 nm and 2.2 μm diameter particles with a positioning precision as small as 180 nm during confinement. By adjusting a single flow parameter, we further show that the shape of the effective trap potential can be efficiently controlled. Finally, we demonstrate two distinct features of the flow-based trapping method, including isolation of a single particle from a crowded particle solution and active control over the surrounding medium of a trapped object. The 2D flow-based trapping method described here further expands the micro/nanomanipulation toolbox for small particles and holds strong promise for applications in biology, chemistry, and materials research

Synthesis and Direct Observation of Thermoresponsive DNA Copolymers

Single-molecule techniques allow for the direct observation of long-chain macromolecules, and these methods can provide a molecular understanding of chemically heterogeneous and stimuli-response polymers. In this work, we report the synthesis and direct observation of thermoresponsive DNA copolymers using single-molecule techniques. DNA-PNIPAM copolymers are synthesized using a two-step strategy based on polymerase chain reaction (PCR) for generating linear DNA backbones containing non-natural nucleotides (dibenzocyclooctyne-dUTP), followed by grafting thermoresponsive side branches (poly­(<i>N</i>-isopropylacrylamide), PNIPAM) onto DNA backbones using copper-free click chemistry. Single-molecule fluorescence microscopy is used to directly observe the stretching and relaxation dynamics of DNA-PNIPAM copolymers both below and above the lower critical solution temperature (LCST) of PNIPAM. Our results show that the intramolecular conformational dynamics of DNA-PNIPAM copolymers are affected by temperature, branch density, and branch molecular weight. Single-molecule experiments reveal an underlying molecular heterogeneity associated with polymer stretching and relaxation behavior, which arises in part due to heterogeneous chemical identity on DNA copolymer dynamics

Topology-Controlled Relaxation Dynamics of Single Branched Polymers

In this work, we report the synthesis and direct observation of branched DNA polymers using single molecule techniques. Polymer topology plays a major role in determining the properties of advanced materials, yet understanding the dynamics of these complex macromolecules has been challenging. Here, we study the conformational relaxation dynamics of single surface-tethered comb polymers from high stretch in a microfluidic device. Our results show that the molecular topology of individual branched polymers plays a direct role on the relaxation dynamics of polymers with complex architectures. Macromolecular DNA combs are first synthesized using a hybrid enzymatic-synthetic approach, wherein chemically modified DNA branches and DNA backbones are generated in separate polymerase chain reactions, followed by a “graft-onto” reaction via strain-promoted [3 + 2] azide–alkyne cycloaddition. This method allows for the synthesis of branched polymers with nearly monodisperse backbone and branch molecular weights. Single molecule fluorescence microscopy is then used to directly visualize branched polymers, such that the backbone and side branches can be tracked independently using single- or dual-color fluorescence labeling. Using this approach, we characterize the molecular properties of branched polymers, including apparent contour length and branch grafting distributions. Finally, we study the relaxation dynamics of single comb polymers from high stretch following the cessation of fluid flow, and we find that polymer relaxation depends on branch grafting density and position of branch point along the main chain backbone. Overall, this work effectively extends single polymer dynamics to branched polymers, which allows for dynamic, molecular-scale observation of polymers with complex topologies

Template-Directed Synthesis of Structurally Defined Branched Polymers

A grand challenge in materials chemistry is the synthesis of macromolecules and polymers with precise shapes and architectures. In this work, we describe a hybrid synthetic strategy to produce structurally defined branched polymer architectures based on chemically modified DNA. Overall, this approach enables precise control over branch placement, grafting density, and chemical identity of side branches. We utilize a two-step scheme based on polymerase chain reaction (PCR) for site-specific incorporation of non-natural nucleotides along the main polymer backbone, followed by copper-free “click” chemistry for grafting side branches at specific locations. In this way, linear DNA backbones are first synthesized via PCR by utilizing the promiscuity of a high yield thermophilic DNA polymerase to incorporate nucleotides containing bioorthogonal dibenzocyclooctyne (DBCO) functional groups at precise locations along one strand of the DNA backbone. Following PCR, copper-free “click” chemistry is used to attach synthetic polymer branches or oligonucleotide branches to the DNA backbone, thereby allowing for the synthesis of a variety of precise polymer architectures, including three-arm stars, H-polymers, and graft block copolymers. Branched polymer architectures are characterized using polyacrylamide gel electrophoresis, denaturing high performance liquid chromatography (HPLC), and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. In a proof-of-principle demonstration, we synthesize miktoarm stars with AB₂ structures via attachment of mPEG-azide branches (1 and 10 kDa) at precise locations along a DNA backbone, thereby expanding the chemical functionality of structurally defined DNA topologies

Topology-Controlled Relaxation Dynamics of Single Branched Polymers

In this work, we report the synthesis and direct observation of branched DNA polymers using single molecule techniques. Polymer topology plays a major role in determining the properties of advanced materials, yet understanding the dynamics of these complex macromolecules has been challenging. Here, we study the conformational relaxation dynamics of single surface-tethered comb polymers from high stretch in a microfluidic device. Our results show that the molecular topology of individual branched polymers plays a direct role on the relaxation dynamics of polymers with complex architectures. Macromolecular DNA combs are first synthesized using a hybrid enzymatic-synthetic approach, wherein chemically modified DNA branches and DNA backbones are generated in separate polymerase chain reactions, followed by a “graft-onto” reaction via strain-promoted [3 + 2] azide–alkyne cycloaddition. This method allows for the synthesis of branched polymers with nearly monodisperse backbone and branch molecular weights. Single molecule fluorescence microscopy is then used to directly visualize branched polymers, such that the backbone and side branches can be tracked independently using single- or dual-color fluorescence labeling. Using this approach, we characterize the molecular properties of branched polymers, including apparent contour length and branch grafting distributions. Finally, we study the relaxation dynamics of single comb polymers from high stretch following the cessation of fluid flow, and we find that polymer relaxation depends on branch grafting density and position of branch point along the main chain backbone. Overall, this work effectively extends single polymer dynamics to branched polymers, which allows for dynamic, molecular-scale observation of polymers with complex topologies

iLOV as a reporter of phage lambda promoter activity in <i>E. </i>coli.

<p>GFP and iLOV were expressed in <i>E. coli</i> MG1655 cells using a constitutive phage lambda promoter. Fluorescence and optical density were recorded over the logarithmic phase of cell growth (typically after ≈2 hours of lag phase following re-inoculation of overnight culture) in M9 medium supplemented with glucose at 20 mM or glycerol at 0.5% as the carbon source. Fluorescence was divided by the optical density at 600 nm and normalized to the maximum value reached over 16 h. of cell growth. Steady state promoter activity was verified using GFP as a reporter. We generally observed good agreement between the iLOV and GFP expression profiles. Promoter activities are shown corresponding only to the logarithmic phase of cell growth (0.4< <i>A_600nm</i> <0.8). As the duration of the logarithmic phase varies for <i>E. coli</i> cells expressing the GFP and iLOV reporter constructs, so does the time frame over which the phage λ promoter activity is depicted in the figures.</p

Effect of pH on FbFP fluorescence.

<p>Histograms depict the fraction of peak fluorescence (measured at 495 nm at pH 7 for PpFbFP and EcFbFP and pH 6 for iLOV) retained by A) PpFbFP, B) EcFbFP, and C) iLOV after incubation in buffers of pH 2, 4, 10, and 11 for 2.5 h. D) FbFPs are readily denatured by incubation at pH 2 and are characterized by the appearance of a flavin-spectrum with a peak at 525 nm, as is depicted for iLOV.</p

FbFPs as transcriptional reporters of T5 promoter activity in <i>E.coli</i> grown in M9-glucose.

<p>FbFPs and YFP were expressed in <i>E. coli</i> MG1655 cells using an IPTG-inducible T5 promoter. Fluorescence and optical density were recorded over the logarithmic phase of cell growth (typically after ≈2 hours of lag phase following re-inoculation of overnight culture) in M9 medium supplemented with glucose at 20 mM concentration as the carbon source. IPTG concentrations were varied to span different levels of transcriptional activity of the T5 promoter. Fluorescence was divided by the optical density at 600 nm. Normalized fluorescence values are depicted for A) PpFbFP, B) EcFbFP, C) iLOV, and D) YFP. Steady state promoter activity was verified using YFP as a reporter. PpFbFP and EcFbFP deviated considerably from the expected steady state promoter dynamics. However, iLOV revealed close agreement with the YFP expression profile over a broad range of IPTG concentrations. Promoter activities are depicted corresponding only to the logarithmic phase of cell growth (0.4< <i>A_600nm</i> <0.8). As the duration of the logarithmic phase varies for <i>E. coli</i> cells expressing distinct transcriptional reporter constructs, so does the time frame over which the promoter activity is depicted in the figures.</p

Characterization of Flavin-Based Fluorescent Proteins: An Emerging Class of Fluorescent Reporters

<div><p>Fluorescent reporter proteins based on flavin-binding photosensors were recently developed as a new class of genetically encoded probes characterized by small size and oxygen-independent maturation of fluorescence. Flavin-based fluorescent proteins (FbFPs) address two major limitations associated with existing fluorescent reporters derived from the green fluorescent protein (GFP)–namely, the overall large size and oxygen-dependent maturation of fluorescence of GFP. However, FbFPs are at a nascent stage of development and have been utilized in only a handful of biological studies. Importantly, a full understanding of the performance and properties of FbFPs as a practical set of biological probes is lacking. In this work, we extensively characterize three FbFPs isolated from <i>Pseudomonas putida</i>, <i>Bacillus subtilis</i>, and <i>Arabidopsis thaliana,</i> using <i>in vitro</i> studies to assess probe brightness, oligomeric state, maturation time, fraction of fluorescent holoprotein, pH tolerance, redox sensitivity, and thermal stability. Furthermore, we validate FbFPs as stable molecular tags using <i>in vivo</i> studies by constructing a series of FbFP-based transcriptional constructs to probe promoter activity in <i>Escherichia coli</i>. Overall, FbFPs show key advantages as broad-spectrum biological reporters including robust pH tolerance (4–11), thermal stability (up to 60°C), and rapid maturation of fluorescence (<3 min.). In addition, the FbFP derived from <i>Arabidopsis thaliana</i> (iLOV) emerged as a stable and nonperturbative reporter of promoter activity in <i>Escherichia coli</i>. Our results demonstrate that FbFP-based reporters have the potential to address key limitations associated with the use of GFP, such as pH-sensitive fluorescence and slow kinetics of fluorescence maturation (10–40 minutes for half maximal fluorescence recovery). From this view, FbFPs represent a useful new addition to the fluorescent reporter protein palette, and our results constitute an important framework to enable researchers to implement and further engineer improved FbFP-based reporters with enhanced brightness and tighter flavin binding, which will maximize their potential benefits.</p></div