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Heating up chemiluminescence experiments

BioPhotonics
Sep 2007
Lynn M. Savage

For the past several years, researchers at the University of Maryland Biotechnology Institute in Baltimore have been investigating ways to enhance chemiluminescence detection techniques. They have developed, for example, surface plasmon-coupled chemiluminescence, in which a sample is placed on a substrate with silver nanoparticles that enhance the luminescence signal. They also have found that pulses of microwave energy can enhance the natural signal of chemiluminescent chromophores.

Now professor Chris D. Geddes and Michael J.R. Previte of the institute have found that the techniques can be combined to multiply their benefits.

They started by coating 5 × 5-mm glass substrates with a 40-nm-thick silver film (they also used plain glass substrates as controls). According to Geddes, 40 nm is the optimal thickness; any thicker or thinner would not permit the level of directional light transmission that they required.

Using aluminum masks, they vapor-deposited 2.5-mm equilateral triangles that were 50 nm thick onto the coated and uncoated substrates. On some, they arranged two triangles together to form bow-tie shapes. They placed each substrate into a microwave cavity — a commercial 700-W, 2.45-GHz oven made by General Electric Co. — through the bottom of which they had cut a 1-in.-diameter hole to grant access by light-collection optics.

BNSurface_microwave.jpg

These images show surface plasmon-coupled chemiluminescence generated from the interface of a solution taken from a green glow stick and either a silver-coated glass substrate (a, b) or a silver-coated substrate further modified with a 2.5-mm silver triangle deposited on top (c, d). Images were acquired before (a, c) and during (b, d) a 5-s pulse of microwave energy. The images were captured in real time from within the microwave cavity. Reprinted with permission of the American Chemical Society.

In each experiment, they filled the chamber with 20 μl of green chemiluminescent solution obtained from over-the-counter glow sticks, then irradiated the solution and substrate with 5-s pulses of microwave energy. The microwaves heat the solution, but more so at the tip of each silver triangle or at the center of each bow-tie configuration. Heating the solution increased the reaction rate and subsequent chemiluminescence.

“Due to the focusing effect induced by the triangle structures, rapid localized heating of the solutions occur,” Geddes said. Combined with the high thermal conductivity of the continuous silver films, the solutions also cool rapidly. As a result, the likelihood of protein denaturing is reduced, which would be important for using the technique in immunoassay experiments.

They also measured the s- and p-polarization intensities, both from free-space and from plasmon-coupled light emissions. They found that the microwave technique aligned nearly all of the chemiluminescence emissions into p-polarization, which again is important for increasing the signal-to-noise ratios for luminescence-based immunoassays.

The collecting optics sent the resulting emissions either to a CCD camera made by QImaging Corp. of Surrey, British Columbia, Canada, or to a spectrometer made by Ocean Optics Inc. of Dunedin, Fla. According to the researchers, the technique is unlike expensive fluorometers because it provides high-sensitivity detection with a standard, inexpensive microwave cavity and basic, inexpensive optical elements.

Journal of the American Chemical Society, Aug. 15, 2007, pp. 9850-9851.


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