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1.
Anal Chem ; 95(28): 10492-10497, 2023 07 18.
Article in English | MEDLINE | ID: mdl-37403691

ABSTRACT

Immunophenotyping of vesicles, such as extracellular vesicles (EVs), is essential to understanding their origin and biological role. We previously described a custom-built flow analyzer that utilizes a gravity-driven flow, high numerical aperture objective, and micrometer-sized flow channels to reach the sensitivity needed for fast multidimensional analysis of the surface proteins of EVs, even down to the smallest EVs (e.g., ∼30-40 nm). It is difficult to flow focus small EVs, and thus, the transiting EVs exhibit a distribution in particle velocities due to the laminar flow. This distribution of vesicle velocities leads to potentially incorrect results when immunophenotyping nanometer-sized vesicles using cross-correlation analysis (Xcorr), as the order of appearance of the vesicles might not be the same at different spatially offset laser excitation regions. Here, we describe an alternative cross-correlation analysis strategy (Scorr), which uses information on particle transit time across the laser excitation beam width to improve multicolor colocalization in single-vesicle immunoprofiling. We tested the performance of the algorithm for colocalization analysis of multicolor nanobeads and EVs experimentally and via simulations and found that Scorr improved both the efficiency and accuracy of colocalization versus Xcorr. As shown from Monte Carlo simulations, Scorr provided an ∼1.2-4.7-fold increase in the number of colocalized peaks and ensured negligible colocalization of peaks. In silico results were in good agreement with experimental data, which showed an increase in colocalized peaks of ∼1.3-2.5-fold and ∼1.2-2-fold for multicolor beads and EVs, respectively.


Subject(s)
Extracellular Vesicles , Flow Cytometry/methods , Extracellular Vesicles/metabolism , Light , Immunophenotyping
2.
Anal Chem ; 93(14): 5897-5905, 2021 04 13.
Article in English | MEDLINE | ID: mdl-33784071

ABSTRACT

Extracellular vesicles (EVs) are membranous particles released by most cells in our body, which are involved in many cell-to-cell signaling processes. Given the nanometer sizes and heterogeneity of EVs, highly sensitive methods with single-molecule resolution are fundamental to investigating their biophysical properties. Here, we demonstrate the sizing of EVs using a fluorescence-based flow analyzer with single-molecule sensitivity. Using a dye that selectively partitions into the vesicle's membrane, we show that the fluorescence intensity of a vesicle is proportional to its diameter. We discuss the constraints in sample preparation which are inherent to sizing nanoscale vesicles with a fluorescent membrane dye and propose several guidelines to improve data consistency. After optimizing staining conditions, we were able to measure the size of vesicles in the range ∼35-300 nm, covering the spectrum of EV sizes. Lastly, we developed a method to correct the signal intensity from each vesicle based on its traveling speed inside the microfluidic channel, by operating at a high sampling rate (10 kHz) and measuring the time required for the particle to cross the laser beam. Using this correction, we obtained a threefold greater accuracy in EV sizing, with a precision of ±15-25%.


Subject(s)
Extracellular Vesicles , Flow Cytometry , Fluorescent Dyes , Light , Staining and Labeling
3.
Anal Chem ; 93(6): 3266-3272, 2021 02 16.
Article in English | MEDLINE | ID: mdl-33534543

ABSTRACT

Cervical cancer is the fourth-leading cause of cancer deaths among women worldwide and most cases occur in developing countries. Detection of high-risk (HR) HPV, the etiologic agent of cervical cancer, is a primary screening method for cervical cancer. However, the current gold standard for HPV detection, real-time PCR, is expensive, time-consuming, and instrumentation-intensive. A rapid, low-cost HPV detection method is needed for cervical cancer screening in low-resource settings. We previously developed a digital loop-mediated isothermal amplification (dLAMP) assay for rapid, quantitative detection of nucleic acids without the need for thermocycling. This assay employs a microfluidic self-digitization chip to automatically digitize a sample into an array of nanoliter wells in a simple assay format. Here we evaluate the dLAMP assay and self-digitization chip for detection of the commonly tested 14 high-risk HPVs in clinical samples. The dLAMP platform provided reliable genotyping and quantitative detection of the 14 high-risk HPVs with high sensitivity, demonstrating its potential for simple, rapid, and low-cost diagnosis of HPV infection.


Subject(s)
Alphapapillomavirus , Uterine Cervical Neoplasms , Early Detection of Cancer , Female , Humans , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques
4.
J Am Chem Soc ; 141(4): 1515-1525, 2019 01 30.
Article in English | MEDLINE | ID: mdl-30605325

ABSTRACT

We present a method to determine the concentration of nucleic acids in a sample by partitioning it into droplets with a nonuniform volume distribution. This digital PCR method requires no special equipment for partitioning, unlike other methods that require nearly identical volumes. Droplets are generated by vortexing a sample in an immiscible oil to create an emulsion. PCR is performed, and droplets in the emulsion are imaged. Droplets with one or more copies of a nucleic acid are identified, and the nucleic acid concentration of the sample is determined. Numerical simulations of droplet distributions were used to estimate measurement error and dynamic range and to examine the effects of the total volume of droplets imaged and the shape of the droplet size distribution on measurement accuracy. The ability of the method to resolve 1.5- and 3-fold differences in concentration was assessed by using simulations of statistical power. The method was validated experimentally; droplet shrinkage and fusion during amplification were also assessed experimentally and showed negligible effects on measured concentration.


Subject(s)
Polymerase Chain Reaction/methods , Statistics as Topic , Emulsions , Lab-On-A-Chip Devices , Poisson Distribution , Polymerase Chain Reaction/instrumentation
5.
PLoS One ; 13(5): e0196801, 2018.
Article in English | MEDLINE | ID: mdl-29718986

ABSTRACT

Cancer is a heterogeneous disease, and patient-level genetic assessments can guide therapy choice and impact prognosis. However, little is known about the impact of genetic variability within a tumor, intratumoral heterogeneity (ITH), on disease progression or outcome. Current approaches using bulk tumor specimens can suggest the presence of ITH, but only single-cell genetic methods have the resolution to describe the underlying clonal structures themselves. Current techniques tend to be labor and resource intensive and challenging to characterize with respect to sources of biological and technical variability. We have developed a platform using a microfluidic self-digitization chip to partition cells in stationary volumes for cell imaging and allele-specific PCR. Genotyping data from only confirmed single-cell volumes is obtained and subject to a variety of relevant quality control assessments such as allele dropout, false positive, and false negative rates. We demonstrate single-cell genotyping of the NPM1 type A mutation, an important prognostic indicator in acute myeloid leukemia, on single cells of the cell line OCI-AML3, describing a more complex zygosity distribution than would be predicted via bulk analysis.


Subject(s)
Genotyping Techniques , Neoplasms/genetics , Oligonucleotide Array Sequence Analysis , Mutation/genetics
6.
Anal Chem ; 90(10): 6089-6095, 2018 05 15.
Article in English | MEDLINE | ID: mdl-29672026

ABSTRACT

We describe here a flow platform for quantifying the number of biomolecules on individual fluorescent nanoparticles. The platform combines line-confocal fluorescence detection with near nanoscale channels (1-2 µm in width and height) to achieve high single-molecule detection sensitivity and throughput. The number of biomolecules present on each nanoparticle was determined by deconvolving the fluorescence intensity distribution of single-nanoparticle-biomolecule complexes with the intensity distribution of single biomolecules. We demonstrate this approach by quantifying the number of streptavidins on individual semiconducting polymer dots (Pdots); streptavidin was rendered fluorescent using biotin-Alexa647. This flow platform has high-throughput (hundreds to thousands of nanoparticles detected per second) and requires minute amounts of sample (∼5 µL at a dilute concentration of 10 pM). This measurement method is an additional tool for characterizing synthetic or biological nanoparticles.


Subject(s)
Nanoparticles/chemistry , Polymers/chemistry , Streptavidin/analysis , Biotin/chemistry , Fluorescence , Fluorescent Dyes/chemistry , Microfluidic Analytical Techniques , Microscopy, Confocal , Semiconductors
7.
Curr Opin Chem Biol ; 39: 64-73, 2017 Aug.
Article in English | MEDLINE | ID: mdl-28623730

ABSTRACT

Quantitative microscopy is needed to understand reactions or phenomena carried out by biological molecules such as enzymes, receptors, and membrane-localized proteins. Counting the biomolecules of interest in single organelles or cellular compartments is critical in these approaches. In this brief perspective, we focus on the development of quantitative fluorescence microscopies that measure the precise copy numbers of proteins in cellular organelles or purified samples. We introduce recent improvements in quantitative microscopies to overcome undercounting or overcounting errors in certain conditions. We conclude by discussing biological applications.


Subject(s)
Microscopy, Fluorescence/methods , Animals , DNA/metabolism , Humans , Proteins/metabolism , RNA/metabolism
8.
J Am Chem Soc ; 137(1): 173-8, 2015 Jan 14.
Article in English | MEDLINE | ID: mdl-25494172

ABSTRACT

This article describes the design and development of squaraine-based semiconducting polymer dots (Pdots) that show large Stokes shifts and narrow-band emissions in the near-infrared (NIR) region. Fluorescent copolymers containing fluorene and squaraine units were synthesized and used as precursors for preparing the Pdots, where exciton diffusion and likely through-bond energy transfer led to highly bright and narrow-band NIR emissions. The resulting Pdots exhibit the emission full width at half-maximum of ∼36 nm, which is ∼2 times narrower than those of inorganic quantum dots in the same wavelength region (∼66 nm for Qdot705). The squaraine-based Pdots show a high fluorescence quantum yield (QY) of 0.30 and a large Stokes shift of ∼340 nm. Single-particle analysis indicates that the average per-particle brightness of the Pdots is ∼6 times higher than that of Qdot705. We demonstrate bioconjugation of the squaraine Pdots and employ the Pdot bioconjugates in flow cytometry and cellular imaging applications. Our results suggest that the narrow bandwidth, high QY, and large Stokes shift are promising for multiplexed biological detections.


Subject(s)
Cyclobutanes/chemistry , Fluorescence , Neoplasms/pathology , Phenols/chemistry , Polymers/chemistry , Quantum Dots , Cyclobutanes/chemical synthesis , Flow Cytometry , Humans , Infrared Rays , MCF-7 Cells , Molecular Structure , Particle Size , Phenols/chemical synthesis , Polymers/chemical synthesis , Semiconductors , Surface Properties
9.
Biopolymers ; 99(12): 1046-69, 2013 Dec.
Article in English | MEDLINE | ID: mdl-23640759

ABSTRACT

Extension versus twist data of Koster et al. (Nature 2005, 434, 671-674) are analyzed to obtain C for the main-chain segments and the twist energy parameter (ET ) for the supercoiled pseudocircular (sp) domain(s) from which C is estimated via simulations. The torsional rigidity in the tension-free sp domain(s) (C = 163 fJ fm) is typical of the unstrained DNA and is less than half the value in the main-chain segments under tension (C = 350-410 fJ fm). Tension is suggested to induce a structural transition to a torsionally stiffer state. Data of Koster et al. for the rate of extension owing to unwinding of a covalent complex of DNA with human Topoisomerase Ib (H Topo I) are analyzed to determine the torque and rate of rotation from which an effective friction coefficient is obtained. A Langevin equation for the unwinding motion in a supercoiled DNA:H Topo I complex is solved to obtain the temporal trajectory of the average winding angle and the time-dependent distribution of winding angles. The mean rate constant for the religation reaction is estimated from the measured probability of reaction per turn. We predict that unwinding proceeds rather far during a single-cleavage and religation cycle, and is effectively completely equilibrated during the 3.2 cleavage and religation cycles that occur during each noncovalent binding and dissociation event. H Topo I is predicted to be completely processive as in accord with observations on calf-thymus Topo I (Brewood et al., Biochemistry 2010, 49, 3367-3380).


Subject(s)
DNA, Superhelical , DNA , Animals , DNA/chemistry , Humans
10.
J Phys Chem B ; 117(25): 7626-52, 2013 Jun 27.
Article in English | MEDLINE | ID: mdl-23656252

ABSTRACT

A macromolecule in a gradient of a cosolute that is preferentially (relative to the solvent) either attracted to or excluded from the domain of the macromolecule should experience a thermodynamic force and move, respectively, up or down the gradient. A theory of chemotactic forces arising from such preferential interactions, especially short-range ligand binding and excluded volume interactions, is developed via an extension of Kirkwood-Buff theory. The ligand binding result is confirmed for both non-ionic and ionic cosolutes by standard solution thermodynamics. The effect of increasing the electrolyte concentration to diminish the electrostatic free energy of a charged macromolecule is also treated formally via an electrostatic macromolecule-electrolyte preferential interaction coefficient. For short-range interactions, the induced chemotactic velocity is attributed entirely to tangential tractions at the interface between the macromolecule and its surrounding solution. The velocity of a spherical macromolecule driven by such tractions is derived by a hydrodynamic calculation for steady-state creepy flow with a partial slip boundary condition. Qualitative comparisons of theoretical predictions with experimental observations of Zheng and Pollack pertaining to charged microspheres near the surfaces of non-ionic gels suggest that the reported exclusion zones are due to chemotaxis induced by gradients of base (NaOH) (or acid (HCl)) and salt. With a single adjustable parameter, namely, the ratio of slip length to area per surface carboxyl (or amidine) group, this theory yields nearly quantitative agreement with many observations. The estimated slip length for the microspheres is comparable to that obtained for bovine serum albumen by fitting the chemotactic theory to two reported cross-diffusion coefficients. When a solution with a gradient of NaOH is placed in contact with a smooth glass wall, chemotactic surface tractions are predicted to cause convection of the solution toward the acidic end of the gradient, as observed in preliminary experiments.


Subject(s)
Chemotaxis , Macromolecular Substances/chemistry , Models, Theoretical , Electrolytes/chemistry , Hydrodynamics , Ions/chemistry , Solutions/chemistry , Static Electricity , Thermodynamics
11.
Anal Chem ; 84(24): 10522-5, 2012 Dec 18.
Article in English | MEDLINE | ID: mdl-23210507

ABSTRACT

In cellular and molecular biology, fluorophores are employed to aid in tracking and quantifying molecules involved in cellular function. We previously developed a sensitive single-molecule quantification technique to count the number of proteins and the variation of the protein number over the population of individual subcellular organelles. However, environmental effects on the fluorescent intensity of fluorophores can make it difficult to accurately quantify proteins using these sensitive techniques. In this letter, we demonstrate the use of photobleaching to extract an accurate single-molecule calibration intensity distribution from the sample directly to avoid any differences in environment that may alter the count. Using this technique, we were able to show that goat antimouse IgG antibody labeled with Alexa Fluor 488, an environmentally insensitive fluorophore, exhibited an average fluorescence equivalent to 4.6 single fluorophores. SynaptopHluorin vesicles, which contain the environmentally sensitive green fluorescent protein, exhibited an average of 4.4 single green fluorescent proteins per vesicle.


Subject(s)
Fluorescent Dyes/analysis , Fluorescent Dyes/chemistry , Photobleaching , Animals , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/chemistry , Hydrazines/analysis , Hydrazines/chemistry , Mice , Mice, Transgenic , Synaptic Vesicles/chemistry
12.
Nat Protoc ; 6(12): 1953-68, 2011 Nov 17.
Article in English | MEDLINE | ID: mdl-22094731

ABSTRACT

This protocol describes a method for determining both the average number and variance of proteins, in the few to tens of copies, in isolated cellular compartments such as organelles and protein complexes. Other currently available protein quantification techniques either provide an average number, but lack information on the variance, or they are not suitable for reliably counting proteins present in the few to tens of copies. This protocol entails labeling of the cellular compartment with fluorescent primary-secondary antibody complexes, total internal reflection fluorescence microscopic imaging of the cellular compartment, digital image analysis and deconvolution of the fluorescence intensity data. A minimum of 2.5 d is required to complete the labeling, imaging and analysis of a set of samples. As an illustrative example, we describe in detail the procedure used to determine the copy number of proteins in synaptic vesicles. The same procedure can be applied to other organelles or signaling complexes.


Subject(s)
Fluorescent Antibody Technique , Proteins/analysis , Algorithms , Cell Compartmentation , Cytoplasmic Structures/metabolism , Image Processing, Computer-Assisted/methods , Microfluidics/methods , Microscopy, Fluorescence/methods , Proteins/chemistry , Software
13.
Biophys J ; 101(7): 1580-9, 2011 Oct 05.
Article in English | MEDLINE | ID: mdl-21961583

ABSTRACT

Uptake of neurotransmitters into synaptic vesicles is driven by the proton gradient established across the vesicle membrane. The acidification of synaptic vesicles, therefore, is a crucial component of vesicle function. Here we present measurements of acidification rate constants from isolated, single synaptic vesicles. Vesicles were purified from mice expressing a fusion protein termed SynaptopHluorin created by the fusion of VAMP/synaptobrevin to the pH-sensitive super-ecliptic green fluorescent protein. We calibrated SynaptopHluorin fluorescence to determine the relationship between fluorescence intensity and internal vesicle pH, and used these values to measure the rate constant of vesicle acidification. We also measured the effects of ATP, glutamate, and chloride on acidification. We report acidification time constants of 500 ms to 1 s. The rate of acidification increased with increasing extravesicular concentrations of ATP and glutamate. These data provide an upper and a lower bound for vesicle acidification and indicate that vesicle readiness can be regulated by changes in energy and transmitter availability.


Subject(s)
Green Fluorescent Proteins/metabolism , Recombinant Fusion Proteins/metabolism , Synaptic Vesicles/chemistry , Synaptic Vesicles/metabolism , Animals , Brain/cytology , Chlorides/metabolism , Endocytosis , Glutamates/metabolism , Green Fluorescent Proteins/genetics , Hydrogen-Ion Concentration , Kinetics , Mice , Neurotransmitter Agents/metabolism , Permeability , Protons , Recombinant Fusion Proteins/genetics , Spectrometry, Fluorescence , Transgenes/genetics
14.
ACS Chem Neurosci ; 2(5): 236-241, 2011 May 18.
Article in English | MEDLINE | ID: mdl-21666849

ABSTRACT

Synaptosomes are intact, isolated nerve terminals that contain the necessary machinery to recycle synaptic vesicles via endocytosis and exocytosis upon stimulation. Here we use this property of synaptosomes to load quantum dots into synaptic vesicles. Vesicles are then isolated from the synaptosomes, providing a method to probe isolated, individual synaptic vesicles where each vesicle contains a single, encapsulated nanoparticle. This technique provided an encapsulation efficiency of ~16%, that is, ~16% of the vesicles contained a single quantum dot while the remaining vesicles were empty. The ability to load single nanoparticles into synaptic vesicles opens new opportunity for employing various nanoparticle-based sensors to study the dynamics of vesicular transporters.

15.
J Neurosci ; 31(4): 1461-70, 2011 Jan 26.
Article in English | MEDLINE | ID: mdl-21273430

ABSTRACT

Protein sorting represents a potential point of regulation in neurotransmission because it dictates the protein composition of synaptic vesicles, the organelle that mediates transmitter release. Although the average number of most vesicle proteins has been estimated using bulk biochemical approaches (Takamori et al., 2006), no information exists on the intervesicle variability of protein number, and thus on the precision with which proteins are sorted to vesicles. To address this, we adapted a single molecule quantification approach (Mutch et al., 2007) and used it to quantify both the average number and variance of seven integral membrane proteins in brain synaptic vesicles. We report that four vesicle proteins, SV2, the proton ATPase, Vglut1, and synaptotagmin 1, showed little intervesicle variation in number, indicating they are sorted to vesicles with high precision. In contrast, the apparent number of VAMP2/synaptobrevin 2, synaptophysin, and synaptogyrin demonstrated significant intervesicle variability. These findings place constraints on models of protein function at the synapse and raise the possibility that changes in vesicle protein expression affect vesicle composition and functioning.


Subject(s)
Membrane Proteins/metabolism , Synaptic Vesicles/metabolism , Animals , Brain/metabolism , In Vitro Techniques , Protein Transport , Rats , Rats, Sprague-Dawley
16.
Lab Chip ; 11(5): 974-7, 2011 Mar 07.
Article in English | MEDLINE | ID: mdl-21229183

ABSTRACT

Quality control is an important and integral part to any microfabrication process. While the widths of features often can be easily assessed by light microscopy, the heights of the fabricated structures are more difficult to determine. Here, we present a rapid, accurate, and low-cost method to measure the heights of microfabricated structures during and after the fabrication process. This technique is based on white-light interferometry, which offers accuracy on the submicrometre scale.

17.
ACS Nano ; 4(12): 7603-11, 2010 Dec 28.
Article in English | MEDLINE | ID: mdl-21117640

ABSTRACT

This paper describes a method by which molecules that are impermeable to cells are encapsulated in dye-sensitized lipid nanocapsules for delivery into cells via endocytosis. Once inside the cells, the molecules are released from the lipid nanocapsules into the cytoplasm with a single nanosecond pulse from a laser in the far red (645 nm). We demonstrate this method with the intracellular release of the second messenger IP(3) in CHO-M1 cells and report that calcium responses from the cells changed from a sustained increase to a transient spike when the average number of IP(3) released is decreased below 50 molecules per nanocapsule. We also demonstrate the delivery of a 23 kDa O(6)-alkylguanine-DNA alkyltransferase (AGT) fusion protein into Ba/F3 cells to inhibit a key player BCR-ABL in the apoptotic pathway. We show that an average of ∼8 molecules of the inhibitor is sufficient to induce apoptosis in the majority of Ba/F3 cells.


Subject(s)
Intracellular Space/metabolism , Intracellular Space/radiation effects , Light , Nanocapsules , Animals , Biological Transport , CHO Cells , Calcium/metabolism , Cricetinae , Cricetulus , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Fusion Proteins, bcr-abl/antagonists & inhibitors , Fusion Proteins, bcr-abl/metabolism , HEK293 Cells , Humans , Inositol 1,4,5-Trisphosphate/chemistry , Inositol 1,4,5-Trisphosphate/metabolism , Inositol 1,4,5-Trisphosphate Receptors/metabolism , Lasers , Lipids/chemistry , Nanocapsules/chemistry , O(6)-Methylguanine-DNA Methyltransferase/antagonists & inhibitors , O(6)-Methylguanine-DNA Methyltransferase/metabolism , Photolysis , Time Factors
18.
Biophys J ; 97(9): 2577-84, 2009 Nov 04.
Article in English | MEDLINE | ID: mdl-19883601

ABSTRACT

The size of a synaptic vesicle (SV) is generally thought to be determined by the amount of lipid and membrane protein it contains. Once formed, it is thought to remain constant in size. Using fluorescence correlation spectroscopy and cryogenic electron microscopy, we show that glutamatergic vesicles reversibly increase their size upon filling with glutamate. The increase ( approximately 25% in diameter) corresponds to an increase in surface area of approximately 50% and in volume of approximately 100%. This large size increase implies a large structural change in the SV upon loading with neurotransmitters. Vesicles lacking SV protein 2A (SV2A) did not manifest a change in size after loading with glutamate, indicating that SV2A is required for this phenomenon.


Subject(s)
Lipids/chemistry , Neurotransmitter Agents/metabolism , Synaptic Vesicles/chemistry , Animals , Biophysics/methods , Brain/metabolism , Calcium/chemistry , Cryoelectron Microscopy/methods , Glutamic Acid/chemistry , Membrane Proteins/chemistry , Mice , Protein Isoforms , Rats , Spectrometry, Fluorescence/methods , Synaptic Vesicles/metabolism
19.
J Phys Chem B ; 112(42): 13359-66, 2008 Oct 23.
Article in English | MEDLINE | ID: mdl-18717543

ABSTRACT

Two different methods are proposed to estimate the persistence length ( P) of DNA from the measured torsion elastic constant (alpha) and the twist energy parameter ( E T ) that governs the supercoiling free energy. The first method involves Monte Carlo simulations and reversible-work calculations of E T for model DNAs that possess the measured alpha and selected trial values of P. Comparison of the computed E T values with the experimental value allows estimation of P (or equivalently the bending elastic constant (kappa beta)) by interpolation. A far simpler, though less accurate, alternative is to solve a previously conjectured analytical relation connecting E T , alpha, kappa beta (or P), and an unknown "constant" ( B). The present simulations are used to ascertain the optimum value of B and to assess the validity and accuracy of that relation. Within the simulation errors, P values obtained from the measured alpha and E T via this analytical expression agree with those determined from the simulations and E T values reckoned from the input alpha and kappa beta by this analytical expression agree with the corresponding simulated values. Although B is found to be insensitive to variation in alpha, it appears to decline slightly with increasing kappa beta. The original analytical expression is modified to take this apparent variation of B with kappa beta into account. By using this modified analytical relation to estimate P (from the measured alpha and E T ) or E T (from the input alpha and kappa beta), much closer agreement is obtained respectively with the values of P or E T obtained from the simulations. As specific examples, these methods are applied to determine P in 0 and 20 w/v % ethylene glycol, which has been shown to induce a structural transition in duplex DNA.


Subject(s)
DNA, Superhelical/chemistry , Elasticity/drug effects , Ethylene Glycol/pharmacology , Torsion, Mechanical , Models, Molecular , Monte Carlo Method , Nucleic Acid Conformation/drug effects , Reproducibility of Results , Thermodynamics
20.
Anal Chem ; 80(9): 3450-7, 2008 May 01.
Article in English | MEDLINE | ID: mdl-18363409

ABSTRACT

This article describes two complementary techniques, single-particle tracking and correlation spectroscopy, for accurately sizing nanoparticles confined within picoliter volume aqueous droplets. Single-particle tracking works well with bright particles that can be continuously illuminated and imaged, and we demonstrated this approach for sizing single fluorescent beads. Fluorescence correlation spectroscopy detects small intensity bursts from particles or molecules diffusing through the confocal probe volume, which works well with dim and rapidly diffusing particles or molecules; we demonstrated FCS for sizing synaptic vesicles confined in aqueous droplets. In combination with recent advances in droplet manipulations and analysis, we anticipate this capability to size single nanoparticles and molecules in free solution will complement existing tools for probing cellular systems, subcellular organelles, and nanoparticles.


Subject(s)
Microfluidics/methods , Nanoparticles/analysis , Organelles/chemistry , Water/chemistry , Algorithms , Animals , Diffusion , Microscopy, Fluorescence , Nanoparticles/chemistry , Rats , Spectrometry, Fluorescence , Surface Properties , Synaptic Vesicles/chemistry
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