| TECHNOLOGY |
Microbeads embedded with precisely controlled ratios of luminescent semiconductor nanocrystals--also known as quantum dots--can be used as "bar codes" to identify molecules in bioassays. The optical codes are based on different sizes (and thus colors) and intensities of quantum dots. The codes can be used for high-throughput analysis of biological molecules. The nanocrystals offer several advantages over organic dyes. A single excitation wavelength can be used for all of the quantum dots, and their emission wavelengths vary according to their size. In addition, the emission profile of the quantum dots is perfectly symmetric, which increases the capacity for multiplexing--that is, identifying many wavelengths at once. For a single color incorporated in microbeads, 10 intensity levels can be clearly distinguished.As more colors are added to the beads, differentiating intensity levels becomes increasingly difficult. As the number of colors is increased, the number of intensity levels should be decreased for optimum performance. "It's much easier to distinguish the frequency or color than to distinguish the absolute intensity. The intensity can be affected by many things, including instrument response and alignment. It's better to use more colors" than more intensities. Authors embed hydrophobic ZnS-capped CdSe quantum dots into polymeric beads. They previously worked with water-soluble quantum dots. Putting the quantum dots into the beads is a partitioning process," Nie says. "For the quantum dot to go into the polystyrene bead, we have to make the quantum dot's surface match the hydrophobicity of the interior of the polymer bead." FOR THE CODES to be easily identifiable, the quantum dots must not aggregate, which could lead to spectral broadening, wavelength shifting, and energy transfer. Under such circumstances, the intensity is no longer a linear function of the number of quantum dots. However, energy transfer has not proved to be a problem. Authors believes that the porous structure of the beads separates the quantum dots. "We synthesize these beads with a large number of very small pores. The quantum dots move into these pores. Then we use a solvent to shrink the pores and trap the quantum dots. To reduce the chances of aggregation even further,load the quantum dots in descending order of size, starting with the largest dots--the ones that emit red light--first. Another possibility, simply that there are many more pores than quantum dots in the beads, leaving the majority of the pores empty. The chemists demonstrate three-color optical bar codes with a DNA oligonucleotide assay. Probe oligonucleotide sequences are conjugated to the quantum-dot-tagged microbeads. Fluorescently labeled target DNA sequences are then allowed to hybridize to the probes. The beads are analyzed by single-bead spectroscopy, in which signals for both the code and the analyte are detected. "In a real assay, the encoded beads are mixed and are incubated with the same sample. Optical readout is achieved by single-bead spectroscopy or single-bead imaging. "It's like a bar code reader. When you check out items in a store, they are scanned one at a time." In an assay, this could be done in a fashion similar to flow cytometry. Another possibility, would be to use magnetic materials to force the beads into a monolayer in a microtiter plate. The monolayer could be read with a laser beam similarly to compact discs. Spectral overlap is a potential hurdle to increasing the number of available optical codes. "If you put too many colors, they will overlap. If you have that overlap, you will need some kind of deconvolution software or algorithms. You will need to do curve fitting to resolve those peaks. These technical hurdles can be overcome, especially by using software to recognize the codes," |
| UPDATE | 07.01 |
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| LITERATURE REF. | This data is not available for free |
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