Chemists at Indiana University have taught quantum dots to fear water. And that's a good thing. For the past three years, several research groups have developed these nanometer-scale, semiconductor-based crystals as a potential alternative to the organic dyes used as tags in biological assays. Unlike quantum dots, dyes can be toxic and more prone to photobleaching, and require multiple light sources to fluoresce multiple dyes.Most quantum dots developed in the past were water-soluble (hydrophilic), which presented problems in guiding and bonding the dots to target molecules. However, by producing hydrophobic dots, the Indiana group, led by Shuming Nie, embedded precise ratios and volumes of dots within similarly natured pores of polymer microbeads. This produced smart microstructures capable of molecular recognition and equipped with an optical coding mechanism -- what Nie likened to a bar code for nucleic acids or protein sequences.A fluorescence micrograph of quantum dots emitting single-color signals at 484, 508, 575 and 611 nm were imaged using an off-the-shelf Nikon D1 camera. "Because of the large signal intensities, there are no demanding requirements for the CCD or microscope performance," said Shuming Nie, whose team developed the dots for multiplexed optical coding of biomolecules. "Common CCD cameras and microscopes will do." Courtesy of Indiana University.For example, a batch of polymer beads prepared to exhibit a specific code based on color and intensity can be attached to a DNA probe that will recognize a particular sequence or gene or, alternatively, to antibodies that will detect and analyze proteins. By illuminating the sample under ultraviolet light, an immense number of genes or proteins can be identified and analyzed. In an article published in the July issue of Nature Biotechnology, the group reported that beads encoded with five or six colors, each with six intensity levels, could yield approximately 10,000 to 40,000 recognizable codes.The size of quantum dots determines the wavelength at which they fluoresce. Similar to LEDs, quantum dots emit photons with the recombination of electron-hole pairs generated by optical pumping. However, because a quantum dot's physical structure is smaller in relation to the natural radius of the electron-hole pair, it requires additional energy to confine the optical excitation within its structure. This additional energy translates into the fluoresced light as shorter, or bluer, wavelengths.Before the experiment, the group was prepared to witness the dots couple to each other electronically and undergo fluorescence resonance energy transfer. This would mean that fluorescence intensities at each wavelength would not be linear with the number of embedded quantum dots and would muddle the bar-coding fluorescence intensities. To their surprise, however, this did not occur. Nie theorized that this was due to the porous structure of the polymer beads acting as a matrix to spatially separate embedded dots.The format of data readout is single-bead spectroscopy or multiwavelength imaging, although the latter would be less accurate.