1. "Polymerosomes," polymer bilayer vesicles for micro-reaction purposes

2. Liposomes, optical manipulation and fusion

3. Antibody-antigen binding, real-time measurements

4. OPTCOL, interactions between red blood cells and virus coated microspheres

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"Polymerosomes," polymer bilayer vesicles for micro-reaction purposes

Giant vesicles, made out of polymer bilayers are being investigated for use as microreaction containers. The advantage of such nanovials over liposomes is a much reduced leakage rate of encapsulated small molecules, they are more robust and not easily deformable. Polymersomes can be prepared by reverse evaporation or electroformation and are more easily trappable than liposomes.

Cartoon of a vesicle fusion experiment a) Two vesicles, one containing reagent A and the other containing reagent B, are identified in the sample. b) The two vesicles are trapped in separate optical tweezers and translated such that their membranes come into contact. c) Fusion is initiated by a pulsed UV laser, which disrupts the membranes of both vesicles at the contact point. d) The membranes repair spontaneously by forming one larger vesicle in which reagents A and B mix and react.

The experiment is done in collaboration with Laurie Locascio, from the analytical chemistry division at NIST. The goal is to combine the UV laser fusion technique with a microfluidic device as part of a laboratory-on-a-chip.

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Liposomes, optical manipulation and fusion

Giant liposomes (10-50 microns in diameter) are promising candidates to perform chemical reactions in closed nanovials using only picoliters of reagent. We have developed an all optical method to manipulate and fuse giant liposomes. Optical tweezers are used to trap two individual liposomes, which are then brought into contact. A single pulse of ultraviolet laser light induces the fusion between liposomes.

Quicktime animation - (1.2 MB) Real time fusion of two liposomes containing only buffer solution as recorded with video microscopy. The UV-laser pulse which induces the fusion is applied at the beginning of the video clip.

As a consequence of the fusion reagents that are encapsulated in the liposomes mix and react. We found that the fused liposome assumes a spherical shape and that volume is conserved during fusion. This considerably reduces the possibility of leakage of small encapsulated molecules and may open the way for quantitative studies of mixing of chemicals, which is importantfor combinatorial chemistry with picoliters of reagents.

Fusion of two liposomes, one containing fluo-3 dye and the other one containing calcium ions. The bright field video microscopy images and the fluorescent images were recorded before and after the fusion was initiated (upper and lower images, respectively). After fusion the fluorescence increases as a consequence of the reaction between the two reaction in which fluo-3 chelates the calcium ions. The experiment is done in collaboration with Laurie Locascio, from the Analytical Chemistry Division at NIST. Read more about this work in: Optical manipulation of liposomes as microreactors (1.3 MB ) Simone Kulin, Rani Kishore,Kristian Helmerson and Laurie Locascio (submitted to Langmuir, March 2003).

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Antibody-antigen binding, real-time measurements

Adhesion is an ubiquitous process in biological systems. We have developed a new technique to study the adhesion of biomolecules in real time under biologically relevant conditions. By studyingadhesion events that occur spontaneously, i.e., without being induced by an externally applied force, our aim is to better understand the initial adhesion mechanisms when only a single bond is formed or ruptured, as well as the nature of the cooperative behaviour between multiple bonds and their effect on existing bonds. We have measured the spontaneous dissociation between monoclonal antibody IgE and its specific antigen DNP.

Brownian motion of microsphere trapped in the fixed optical trap as monitored using optical trapping interferometry. The trace shows a typical time evolution of the successive association and dissociation events. The length of the association intervals varies stochastically.

Using optical tweezers, we trap a pair of microspheres, one coated with antigen and the other coated with antibody, and bring them close enough to each other that they repeatedly collide due to to thermally driven motion. By monitoring the position of the trapped, antigen-coated microsphere, we can observe antigen-to-antibody binding events in real time.

Histogram of the time intervals during which the antigen and antibody molecules are attached. a) Case when only a single, non-tethered bond can be formed during an adhesion event: The characteristic decay rate is 1.6 s-1. b) Case of multiple bonds and no tether: The decay time is 3.4 s-1. c) Case of multiple bonds with tether: The decay time is 1.05 s-1. Read more about this work in: Real-time measurement of spontaneous antigen-antibody dissociation (184 kB ) Simone Kulin, Rani Kishore, Joseph Hubbard and Kristian Helmerson Biophysical Journal, 83, 1965-1973 (2002).

By measuring the time interval between attachment and subsequent detachment events, we determine a characteristic dissociation rate k_off. We vary the surface density of antigens such that we can study the detachment of either single or multiple bonds. For single bonds we thus directly infer the spontaneous detachment rate. In the multiple bond regime, when the antigen molecule is directly (and rigidly) attached to the surface, we observe a negative cooperativity between bonds, an effect that is rare in nature. Dissociation occurs at higher rates compared with the single bond rupture rate. We attribute this effect to bond strain induced by the presence of competing bonds that may prevent sufficient penetration of the antigen into the receptor binding pocket. When attaching the antigen molecule to the surface via a short, but flexible tether, we observe the opposite, more intuitive effect of positive cooperativity amongst bonds. In this case, increasing the number of bonds increases the tenacity of the adhesion and accordingly the detachment rate decreases.

The analysis of this experiment is done in collaboration with Joseph Hubbard, from the Biotechnology division at NIST.

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OPTCOL, interactions between red blood cells and virus coated microspheres

In collaboration with Whitesides group in the Chemistry Department at Harvard University, we have used a new assay in which two mesoscale particles (1-100 mm) undergo a collision under the precise control of two independently controlled optical tweezers (optically controlled collision, OPTCOL). This assay, which uses a dual optical tweezers, enables precise examination of the probability of adhesion under biologically relevant conditions.

We translate a virus coated microsphere, which is held in a moving trap, to create a collision with a red blood cell that is held in a fixed, relatively weak trap. We record whether the virus coated sphere sticks to the cell and pulls it out of trapas we continue to translate the mobile trap.We thus measure the sticking probability for the virus coated microsphere and the red blood cell as a function of the concentration of inhibitors added to solution.

Quicktime animation (360 kB) comparing collision with and without adhesion... Violet: (top) Collision followed by adhesion of a virus coated microsphere and a red blood cell in the absence of inhibitors. Green: (bottom) Collision without adhesion of a virus coated microsphere and a red blood cell in the presence of a high concentration of inhibitor.

Using the OPTCOL method, we were able to measure the effectiveness of the highly potent inhibitors (polymers bearing sialic acid on side chains), which were too effective to be measured by the hemaglutinin inhibition (HAI) assay. We measured, with the OPTCOL assay, an inhibition concentration of 35 picomoles (of sialic acid) per liter for the most effective inhibitor. The effectiveness of this inhibitor had previously been measured with the HAI assay to be about 0.6 nanomoles (of sialic acid) per liter, which is the limit of sensitivity of the HAI technique.

Read more about this work in: Optically controlled collisions of biological objects to evaluate potent polyvalent inhibitors of virus-cell adhesion (488 kB ) Mathai Mammen, Kristian Helmerson, Rani Kishore, Seok-Ki Choi, William D. Phillips and George M. Whitesides Chemistry & Biology 3, 757-763 (1996).

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Inquiries or comments: Kristian.Helmerson@nist.gov
Online: May 2003