Rani Kishore

Optically controlled collisions of biological objects to evaluate potent polyvalent inhibitors of virus-cell adhesion

BACKGROUND The biochemical and biomechanical determinants of adhesion between two biological objects following a collision are complex, and may vary from one system to another. We wished to develop an assay in which all the relevant factors, including the components of the solution, the relative orientation and the relative collision velocity, are under the users control. RESULTS A new assay is described in which two mesoscale particles are caused to collide using two independently controlled optical tweezers (optically controlled collision, OPTCOL). This assay enables precise examination of the probability of adhesion under biologically relevant conditions. The OPTCOL assay was used to evaluate the probability of adhesion of a single erythrocyte to a single virus-coated microsphere, in the absence and presence of a sialic acidbearing inhibitor. Inhibition constants for the most effective inhibitors could not be measured using other types of assays. The best inhibitor prevented attachment 50 % of the time at a sialic acid concentration of 35 pmol l-1; it is the most potent known inhibitor of attachment of influenza virus to erythrocytes. CONCLUSIONS OPTCOL is a versatile new bioassay for studying dynamic interactions in biochemistry. It offers an approach to investigating interactions between moving biological objects that is both quantitative and interpretable. The simplicity of the OPTCOL technique suggests broad applicability to the study of adhesion of mesoscale (1-100 microm) objects in the areas of cell biology, microbiology, medicinal chemistry, and biophysics.

Real-time measurement of spontaneous antigen-antibody dissociation.

We report observations in real time of thermally driven adhesion and dissociation between a monoclonal IgE antibody and its specific antigen N-epsilon-2,4-dinitrophenyl-L-lysine. Both molecules were attached to the surfaces of different polystyrene microspheres trapped by optical tweezers. Monitoring spontaneous successive attachment and detachment events allowed a direct determination of the reaction-limited detachment rate k(off) for a single bond and also for multiple bonds. We observed both positive and negative cooperativity between multiple bonds depending on whether the antigen was linked to the microsphere with or without a tether, respectively.

Optically trapped aqueous droplets for single molecule studies

We demonstrate a technique for creating, manipulating, and combining femtoliter volume chemical containers. The containers are surfactant-stabilized aqueous droplets in a low index-of-refraction fluorocarbon medium. The index-of-refraction mismatch between the container and fluorocarbon is such that individual droplets can be optically trapped by single focus laser beams, i.e., optical tweezers. Here, we trap and manipulate individual droplets, detect the fluorescence from single dye and red fluorescent protein molecules encapsulated in droplets, and observe fluorescence resonance energy transfer from a single dye pair on a deoxyribonucleic acid molecule encapsulated in a droplet.

Stable and robust polymer nanotubes stretched from polymersomes.

We create long polymer nanotubes by directly pulling on the membrane of polymersomes using either optical tweezers or a micropipette. The polymersomes are composed of amphiphilic diblock copolymers, and the nanotubes formed have an aqueous core connected to the aqueous interior of the polymersome. We stabilize the pulled nanotubes by subsequent chemical cross-linking. The cross-linked nanotubes are extremely robust and can be moved to another medium for use elsewhere. We demonstrate the ability to form networks of polymer nanotubes and polymersomes by optical manipulation. The aqueous core of the polymer nanotubes together with their robust character makes them interesting candidates for nanofluidics and other applications in biotechnology.

Generation and mixing of subfemtoliter aqueous droplets on demand.

We describe a novel method of generating monodisperse subfemtoliter aqueous droplets on demand by means of piezoelectric injection. Droplets with volumes down to 200 aL are generated by this technique. The droplets are injected into a low refractive index perfluorocarbon so that they can be optically trapped. We demonstrate the use of optical tweezers to manipulate and mix droplets. For example, using optical tweezers we bring two droplets, one containing a calcium sensitive dye and the other calcium chloride, into contact. The droplets coalesce with a resulting reaction time of about 1 ms. The monodispersity, manipulability, repeatability, small size, and fast mixing afforded by this system offer many opportunities for nanochemistry and observation of chemical reactions on a molecule-by-molecule basis.

Green fluorescent protein in inertially injected aqueous nanodroplets.

We inertially inject and study the contents of optically trappable aqueous nanodroplets (hydrosomes) emulsified in a perfluorinated matrix. A new piezoelectric actuated device for production of single hydrosomes on demand is introduced. Hydrosomes containing enhanced green fluorescent protein (EGFP) were injected, optically trapped, and held at the focus of an excitation laser in a confocal microscope, and single-molecule photobleaching events were observed. The rotational diffusion time of EGFP in trapped hydrosomes was measured using time-resolved fluorescence anisotropy. In free solution, the mean rotational diffusion time was determined to be 13.8 +/- 0.1 ns at 3 microM and 14.0 +/- 0.2 ns at 10 microM. In hydrosomes, the mean rotational diffusion time was similar and determined to be 12.6 +/- 1.0 ns at 3 microM and 15.5 +/- 1.6 ns at 10 microM. We conclude that the rotational motion inside the nanodroplets is consistent with rotation in free solution and that the protein therefore does not aggregate at the water-oil interface. Protein can be confined in hydrosomes with high efficiency using this technique, which provides an alternative to surface attachment or lipid encapsulation and opens up new avenues of research using single molecules contained in fluid nanovolumes.

Detection of heteroplasmic mitochondrial DNA in single mitochondria.

Background Mitochondrial DNA (mtDNA) genome mutations can lead to energy and respiratory-related disorders like myoclonic epilepsy with ragged red fiber disease (MERRF), mitochondrial myopathy, encephalopathy, lactic acidosis and stroke (MELAS) syndrome, and Lebers hereditary optic neuropathy (LHON). It is not well understood what effect the distribution of mutated mtDNA throughout the mitochondrial matrix has on the development of mitochondrial-based disorders. Insight into this complex sub-cellular heterogeneity may further our understanding of the development of mitochondria-related diseases. Methodology This work describes a method for isolating individual mitochondria from single cells and performing molecular analysis on that single mitochondrions DNA. An optical tweezer extracts a single mitochondrion from a lysed human HL-60 cell. Then a micron-sized femtopipette tip captures the mitochondrion for subsequent analysis. Multiple rounds of conventional DNA amplification and standard sequencing methods enable the detection of a heteroplasmic mixture in the mtDNA from a single mitochondrion. Significance Molecular analysis of mtDNA from the individually extracted mitochondrion demonstrates that a heteroplasmy is present in single mitochondria at various ratios consistent with the 50/50 heteroplasmy ratio found in single cells that contain multiple mitochondria.

Optically controlled collisions of biological objects

We have developed a new assay in which two mesoscale particles are caused to collide using two independently controlled optical tweezers. This assay involves the measurement of the adhesion probability following a collision. Since the relative orientation, impact parameter (i.e., distance of closest approach), and collision velocity of the particles, as well as the components of the solution, are all under the users control, this assay can mimic a wide range of biologically relevant collisions. We illustrate the utility of our assay by evaluating the adhesion probability of a single erythrocyte (red blood cell) to an influenza virus-coated microsphere, in the presence of sialic acid-bearing inhibitors of adhesion. This probability as a function of inhibitor concentration yields a measure of the effectiveness of the inhibitor for blocking viral adhesion. Most of the inhibition constants obtained using the tweezers agree well with those obtained from other techniques, although the inhibition constants for the best of the inhibitors were beyond the limited resolution of conventional assays. They were readily evaluated using our tweezers-based assay, however, and prove to be the most potent inhibitors of adhesion between influenza virus and erythrocytes ever measured. Further studies are underway to investigate the effect of collision velocity on the adhesion probability, with the eventual goal of understanding the various mechanisms of inhibition (direct competition for viral binding sites versus steric stabilization). Analysis of these data also provide evidence that the density of binding sites may be a crucial parameter in the application of this assay and polymeric inhibition in general.

An optical tweezers-based immunosensor for detection of femtomoles-per-liter concentrations of antigens.

We used optical tweezers—optical trapping with focused laser beams—to pull microspheres coated with antigens off of an antibody-coated surface. Using this technique, we could quantify the force required to separate antigen to antibody bonds. At very low surface density of antigen, we were able to detect the single antigen to antibody binding. The force required to break the antigen-antibody bonds and pull the microsphere off the surface was shown to increase monotonically with increasing surface density of antigens. Using the force determination as a transducer, we were able to detect concentrations of free antigens in solution as small as 10−15 mol/L in a competitive binding assay.

Hydrosomes: femtoliter containers for fluorescence spectroscopy studies

We report on improvements and innovations in the use of hydrosomes to encapsulate and study single molecules. Hydrosomes are optically-trappable aqueous nanodroplets. The droplets are suspended in a fluorocarbon medium that is immiscible with water and has an index of refraction lower than water, so hydrosomes are stable and optically trapped by a focused laser beam (optical tweezers). Using optical tweezers, we hold the hydrosomes within a confocal observation volume and interrogate the encapsulated molecule by fluorescence excitation. This method allows for long observation times of a molecule without the need for surface immobilization or liposome encapsulation. We have developed a new way for creating hydrosomes on demand by inertially launching them into the fluorocarbon matrix using a piezo-activated micropipette. Time-resolved fluorescence anisotropy studies are carried out to characterize the effects of the hydrosome interface boundary on biological molecules and to determine whether molecules encapsulated within hydrosomes diffuse freely throughout the available volume. We measured the fluorescence anisotropy decay of 20mer DNA duplexes, and enhanced green fluorescent protein (GFP). We conclude that the molecules rotate freely inside the nanodroplets and do not stick or aggregate at the boundary.