Photobleaching Offers Coded Microspheres

Daniel S. Burgess

Using a technique they call spatial selective photobleaching, scientists at Ghent University and at Tibotec in Mechlin, both in Belgium, are producing optically encoded polymer microspheres that may find use in biomedicine, chemistry and anticounterfeiting applications. In contrast to other proposed color-coding schemes, they say, the new method yields massive numbers of unique patterns that are easily read for high throughput.

Researchers typically employ 96-well plates in drug-screening applications, said Stefaan C. De Smedt of Ghent University, but these systems face physical constraints to miniaturization that limit their throughput.

Chris Roelant of Tibotec said that bead assays approach screening from another direction by introducing numerous screening agents to the solution to be analyzed, but they read the coded beads by spectroscopy, making them complex and expensive.

The new method is based on the deliberate and controlled bleaching of the fluorescence in a dyed sample to form patterns that subsequently are read optically as localized variations in the intensity of the response. The encoding light may be raster-scanned over an immobilized bead to produce a two- or three-dimensional pattern, or the light may be modulated as the particles pass by the focus of the laser to create quasi-one-dimensional "dot codes."

Researchers in Belgium have developed a technique based on the photobleaching of a fluorophore to encode polymer microbeads with optically readable patterns. Depending on the application, users can produce complex logos (A), alphanumeric codes (B) or millions of unique bar codes (C). Courtesy of Stefaan C. De Smedt. © Nature Materials.

The researchers exposed dye-loaded 28-, 38- and 45-µm-diameter beads to 1 to 30 mW of 488-nm light from an Ar-ion laser. A confocal scanning microscope, an optical switch and an acousto-optic modulator formed the codes by bleaching the fluorophore at different intensities and in different widths. Writing patterns with two bleaching levels and three widths in 45-µm-diameter beads offers more than 4 million combinations.

Reading the codes is similarly straightforward, and the researchers in their demonstration again employed the confocal scanning microscope to illuminate the microspheres with 488-nm light at intensities of 1 to 30 µW and to collect the fluorescence response for analysis. They have established that they can reorient a bead for readout by including a ferromagnetic layer such as chromium dioxide in the sphere and exposing it to a weak magnetic field at the write and read steps.

Spatially selective photobleaching is suitable for use with any material that can bind a fluorescent dye physically or chemically and that is sufficiently transparent at the write/read laser wavelength. The researchers have employed polystyrene, ArgoGel polyethylene glycol/polystyrene copolymer, and dextran with green fluorescent dyes, but they suggest that other common polymers also should be compatible with the technique.

The ease of the approach is promising for applications beyond drug screening, Roelant said. Physicians, for example, could employ a few hundred beads that are tagged to target particular antibodies to diagnose the cause of a patient's illness. Forensic applications might include marking valuable documents with coded beads to prevent counterfeiting.

De Smedt said that the team is continuing its investigations to verify that the technique is suitable for use in biological specimens. It also is optimizing the encoding and decoding process to make it rapid enough for commercial use. Clinical diagnostic applications that employ only 300 to 500 beads would not require extremely high throughputs, but drug screening, for example, might require readout rates of 10,000 encoded beads per second, Roelant said.

A new company, Memobead Technologies, is being incorporated in the Flanders region of Belgium to develop the technique.