We wish to develop organically-conjugated quantum dots (QDs) into sensors that detect specific bacterial species, metabolism, or growth conditions. QDs show great promise as fluorescent biological labels. Their emission spectra are narrow, allowing for multi-wavelength labeling that is easily distinguished, and all emission wavelengths result from a single excitation line (usually 405 nm). Of particular interest to space applications, they are radiation hard, more so than comparable two-and three-dimensional semiconductor devices. Most importantly, their band structure may allow them to be turned on and off chemically, so that they can be made into zero-background labels that fluoresce only in the presence of a desired compound. They may prove useful for in situ instrumentation, in which sample washing, optical filtration, and background reduction may be impossible. A panel of QDs with different wavelengths, that each turn on in the presence of a specific biomolecule, would be an ideal addition to a wet chemistry suite for planetary exploration.

As yet, QDs have failed to fulfill their promise because of several poorly understood optical properties. Fluorescence quenching and de-quenching do not always occur in a predictable manner after exposure to oxidizing and reducing environments. In addition, while labeling of bacterial cells is almost always successful, the mechanism of QD penetration into the cell is unknown. Most critically, the interaction of the bacteriumÕs internal and excreted enzymes with the organically conjugated surface of the QD have been very difficult to study. Only when these problems have been worked out will be sensors be ready for commercialization and in situ use.

We are performing, or setting up to perform, the following experimentsn on CdSe, CdSe/ZnS and CdSe/CdS quantum dots:

1. Measurement of forward and back-transfer lifetimes for QDs conjugated to a variety of biomolecules. This is performed by exciting the QD-biomolecule conjugate with a fast laser pulse and measuring the lifetime of the fluorescence and/or absorbance spectrum. Resolution needed for our systems is ~100 ps, which is obtainable from currently existing equipment at JPL.
2. In the case where there is no back transfer, measurement of the useful lifetime of the sensors, as well as the dependence of their function on exposure to light and varying temperatures.
3. Development of a theoretical model that predicts the electron-transfer rates between semiconductor QDs and directly conjugated biological molecules. This will be used to expand the repertoire of sensors from the amino acids and DNA bases to other biomolecules.
4. Measurement of Stark effect in order to evaluate the feasibility of voltage-sensitive sensors.
5. Bacterial uptake assays. It is not known how QDs enter bacterial cells. Unlike large eukaryotic cells, bacteria do not endocytose, and their cell walls are rigid. It is thus likely that the conjugated QDs are entering through active transport, using a protein transporter that recognizes and imports the conjugate. Testing this hypothesis requires the development of an uptake assay, in order to distinguish QDs that are on or near the cell from those that are truly inside on a bulk basis (i.e., without using electron microscopy). It may be possible to add a quenching molecule to a solution of bacteria, such as trypan blue or methyl viologen, to quench fluorescence from QDs that are outside cells and thus yield a spectral peak corresponding to internalized QDs only. However, the quencher must itself not enter the cell, and its effect on bacterial autofluorescence must be measured and accounted for. Once the uptake assay is defined, rates of conjugated QD uptake will be compared with known rates of uptake of the conjugate alone. The rates may be slowed by oversaturation with nutrients or by addition of enzyme inhibitors such as EDTA; this will give further data about the relationship between QD uptake and transporter function.

QDs alone and conjugated to bacteria demonstrate unexplained optical properties. (A) Two identically-prepared batches of red-emitting QDs show different electrochemical behavior. Batch 1: Exposure to potentials stepping from Ð1.2 V to +1.0 V has no effect on fluorescence emission. Batch 2: Fluorescence emission decays to ~30% of initial value after stepwise application of negative potentials down to Ð1.2 V. (B) External labeling with lectin-conjugated green QDs (left) is clearly distinguishable from internal labeling due to iron-binding-protein-yellow QDs (right) (the bacteria are identical). While it is likely that the QDs enter the cell through the receptor for the iron-binding protein, this has not been proven or disproven. (C) Left, staphylococci labeled with L-tryptophan-QD show a bright green signal. Right, with D-tryptophan-QD, some cells show green fluorescence, whereas others are redder; all labeling is blue-shifted relative to L-tryptophan-QD. The enzymes involved in differential L- and D-amino acid metabolism are presumably responsible for these differences, but a precise mechanism has not been established. (All data from Nadeau et al., to be submitted).