Fluorescent semiconductor quantum dots that can serve as "on/off" labels for bacteria and other living cells are undergoing development. The "on/off" characterization of these quantum dots refers to the fact that, when properly designed and manufactured, they do not fluoresce until and unless they come into contact with viable cells of biological species that one seeks to detect. In comparison with prior fluorescence-based means of detecting biological species, fluorescent quantum dots show promise for greater speed, less complexity, greater sensitivity, and greater selectivity for species of interest. There are numerous potential applications in medicine, environmental monitoring, and detection of bioterrorism.
- The established method of using fluorescent dyes to label live bacteria has several drawbacks:
- The high autofluorescence of many species renders many common chromophores invisible;
- The anaerobic conditions under which many bacteria live prevent proper folding of fluorescent proteins;
- Typical fluorescent dyes undergo rapid photobleaching and thereby rapidly cease to function as labels;
- Cells can be killed by the ultraviolet light needed to excite fluorescence in typical dyes used heretofore for labeling; and
The addition of labeling dyes to cell cultures often leads to high background fluorescence, and bacteria are difficult to distinguish from debris, even when viewed through high-resolution microscopes.
When conjugated to suitable biological molecules that quench their fluorescence, fluorescent semiconductor quantum dots can be made to stick to the surfaces of, or to be taken up by, specific bacteria. To enable the on/off fluorescent detection of a specific bacterium, one chooses a fluorescence-quenching conjugate molecule that is removed by active enzymes on or in the bacterium.
Unlike conventional labeling dyes, fluorescent semiconductor quantum dots become photobleached very slowly and can be excited by blue light, which does not kill cells. Fluorescent semiconductor quantum dots can be manufactured to emit at wavelengths over a wide range — from blue through infrared. Spectral emission peaks of fluorescent semiconductor quantum dots are narrow — typically 10 nm or less in wavelength. The use of fluorescent semiconductor quantum dots entails the following disadvantages: (1) The dots are large and not always taken up by bacteria and (2) they contain heavy metals, which may prove toxic to organisms over long times.
Feasibility has been demonstrated in experiments on cadmium selenide quantum dots. First, the dots were conjugated to mercaptoacetic acid to render them soluble in water. The dots were then further conjugated to a variety of biological compounds. Conjugation was performed by use of a single-step carbodiimide reagent, which was then removed by dialysis versus pure water.
Conjugation to adenine, guanine, and tryptophan was found to quench all fluorescence from green-emitting quantum dots, and to quench >80 percent of the fluorescence from red-emitting quantum dots. Fluorescence did not return upon (1) exposure to ambient light for one week; (2) exposure to light from a 100-W, full-spectrum Hg lamp for 30 minutes; (3) incubation with a culture medium for 3 hours; or (4) incubation for 3 hours with metabolically inhibited bacterial cells [cells in a medium that contained ethylenediaminetetraacetic acid (EDTA), such that the cells remained intact but did not metabolize]. However, upon incubation for 3 hours in a culture medium with live bacterial cells, fluorescence returned and could be detected visually by color change, spectroscopically, and by fluorescence microscopy of individual cells.
This work was done by Gene McDonald, Jay Nadeau, Kenneth Nealson, Michael Storrie-Lombardi, and Rohit Bhartia of Caltech for NASA's Jet Propulsion Laboratory. For further information, access the Technical Support Package (TSP) free on-line at www.techbriefs.com/tsp under the Bio-Medical category.
NPO-30373
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Flourescent Quantum Dots for Biological Labeling
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Overview
The document discusses the development of fluorescent semiconductor quantum dots for biological labeling, specifically focusing on their application as "on/off" labels for detecting bacteria and other living cells. This innovative technology, developed by researchers at NASA's Jet Propulsion Laboratory, addresses several challenges associated with traditional fluorescence-based detection methods.
Fluorescent quantum dots are designed to remain non-fluorescent until they encounter viable cells. This is achieved through a mechanism where enzymes within the target cells liberate the quantum dots from their quenching agents, resulting in a zero-background signal. This characteristic significantly enhances the specificity and sensitivity of biological labeling, making it easier to distinguish target cells from background noise, which is a common issue with existing techniques.
The document outlines the motivation behind this development, highlighting the limitations of current labeling methods, such as high autofluorescence, photobleaching, and difficulties in distinguishing live bacteria from debris. The quantum dots, particularly cadmium selenide, are noted for their slow photobleaching rates and the ability to be manufactured across a wide range of wavelengths, facilitating multi-labeling applications.
The technical process involves conjugating the quantum dots to mercaptoacetic acid to make them water-soluble, followed by further conjugation to various biological compounds. This process effectively quenches the fluorescence of the quantum dots until they interact with live bacterial cells. The document details experiments demonstrating that fluorescence can be restored after incubation with live cells, allowing for visual detection and analysis through fluorescence microscopy.
The potential applications of this technology are vast, including uses in medical diagnostics, environmental monitoring, and bioterrorism detection. The document emphasizes the advantages of using quantum dots over traditional fluorescent labels, such as greater speed, reduced complexity, and enhanced selectivity for specific biological species.
In summary, the document presents a significant advancement in the field of biological labeling through the use of fluorescent quantum dots, offering a promising solution to existing challenges in detecting and labeling live cells with high specificity and sensitivity. This work represents a collaborative effort by a team of researchers at Caltech for NASA, showcasing the intersection of technology and biological sciences.
