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Clinical Chemistry 43: 251-253, 1997;
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(Clinical Chemistry. 1999;43:251-253.)
© 1999 American Association for Clinical Chemistry, Inc.

Editorial

Optical Tweezers and Immunoassay

Larry J. Kricka

Department of Pathology and Laboratory Medicine, University of Pennsylvania, 3400 Spruce St., Philadelphia, PA 19104, fax 215-662-7529, e-mail larry kricka{at}path1a.med.upenn.edu

Our scientific ancestors who advanced the atomic and molecular theories of matter and the cellular basis of biological organisms would be astounded by our current ability to visualize and manipulate atoms, molecules, and cells. The new techniques of atomic force microscopy (AFM; or scanning force microscopy) (1)(2)(3)(4) and optical trapping (optical or laser tweezers) (5)(6) have allowed us to locate individual atoms and molecules on surfaces and to manipulate cells directly. In this issue Helmerson et al. (7) report one of the first studies that uses laser tweezers to detect the binding of an antigen to an immobilized antibody and applies this technique in a competitive immunoassay to determine antigen concentration.

In immunoassay, attempts to detect individual or a few antigen or antibody molecules have relied on labeling one component of the reaction with a high-specific-activity label or a label that can participate in an amplification reaction (e.g., enzymes, DNA) (8). Now the presence of a component of a ligand:binder pair can be sensed directly by using the laser-based technique of optical tweezers.

The principle of optical tweezers (optical trapping) is the use of radiation pressure from light incident on an object. This pressure arises from the transfer of photon momentum when light is reflected or scattered by the object (6). Nd:YAG lasers (neodymium in yttrium aluminum garnet crystals) commonly used as the light source operate in the infrared (1.06 µm), and power as great as 150 mW can be used with objects such as cells without causing serious damage. Various trapping schemes are possible. Objects floating in a medium can be transported in the direction of propagation of a parallel laser beam, and objects in transit along the first beam can be selected and removed by a second laser beam that intersects the first beam at 90° (transport trapping). In optical tweezer trapping, a lens of high numerical aperture focuses a laser beam; the resulting gradient force can trap objects near the focus of the lens. By making the light incident on the object at large angles, the transfer of momentum from the refracted light is great enough to trap the object at the focal point. A third trapping scheme, levitation trapping, traps objects by means of two counter-propagating laser beams. One laser beam transports objects to the focus of a second laser beam, and the objects become trapped at the focal point.

Optical tweezers can be used in an elegant immunoassay strategy, as described in this issue (7). Various concentrations of bovine serum albumin (BSA) were covalently coupled to 4.5-µm-diameter latex beads ("microspheres") that were then dispensed onto the surface of a glass cover slip coated with mouse monoclonal anti-BSA antibodies. BSA immobilized on the beads bound specifically to anti-BSA antibodies on the surface of the cover slip. Next, using a microscope, Helmerson et al. selected a bead and then focused the laser tweezers onto the bead. The bead serves as a "handle" for the optical tweezers to grab. The laser power was increased until the bead was seen to jump away from the surface, indicating that the antibody-binding force had been overcome. The power required to free the bead was found to be directly related to the concentration of BSA on the bead (detection limit 1.45 x 10-12 mol/L). Helmerson et al. applied this principle to a competitive immunoassay for BSA in which free BSA competed with BSA immobilized on a latex bead for a fixed concentration of antibody immobilized on the surface of a glass cover slip; after a 2-h incubation, BSA could be detected in the range 1.45 x 10-12 to 1.45 x 10-15 mol/L.

Optical tweezers are just one example of the new wave of analytical techniques finding application in the biological and analytical sciences. Another technique that can detect single antigen–antibody recognition events is AFM, invented in 1986 (9). In this technique a surface of interest is moved past a very small tip (10 nm in diameter, microfabricated from silicon or silicon nitride) attached to a flexible cantilever. Bending of the cantilever as the tip rises and falls in response to the topology of the surface is registered with a laser beam. Moreover, the tip can be coated with specific molecules to provide direct molecular recognition of substances on a surface. For example, anti-human serum albumin antibody immobilized via a flexible spacer on the tip of an AFM microscope has been used to sense the unbinding of the individual Fab fragments to albumin on a surface; the force of this binding was estimated at 244 pN (10). Sensitized AFM tips have also been used to image functional groups on surfaces, a technique termed chemical force microscopy (11). AFM and its variants have also proved useful in the study of cells and in surface mapping. Some recent applications of AFM and optical tweezers (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37) are listed in Table 1 .


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  		Table 1. Applications of optical tweezers and atomic force microscopy.

The future role of such techniques as optical tweezers and AFM in quantitative analysis has not been firmly established. Issues of sensitivity, reliability, throughput, and cost effectiveness have yet to be answered. Nevertheless, the ability to manipulate molecules of biological interest and individual cells (e.g., to select specific cells for PCR analysis (38)) is an important development that will inevitably have a major impact on both research and routine clinical laboratory medicine.


References

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