26 research outputs found
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 AB<sub>2</sub> 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<sub>600nm</sub></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<sub>600nm</sub></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