Uptake of CdSe and CdSe/ZnS Quantum Dots into Bacteria via Purine-Dependent Mechanisms

Quantum dots (QDs) rendered water soluble for biological applications are usually passivated by several inorganic and/or organic layers in order to increase fluorescence yield. However, these coatings greatly increase the size of the particle, making uptake by microorganisms impossible. We find that adenine- and AMP-conjugated QDs are able to label bacteria only if the particles are <5 nm in diameter. Labeling is dependent upon purine-processing mechanisms, as mutants lacking single enzymes demonstrate a qualitatively different signal than do wild-type strains. This is shown for two example species, one gram negative and one gram positive. Wild-type Bacillus subtilis incubated with QDs conjugated to adenine are strongly fluorescent; very weak signal is seen in mutant cells lacking either adenine deaminase or adenosine phosphoribosyltransferase. Conversely, QD-AMP conjugates label mutant strains more efficiently than the wild type. In Escherichia coli, QD conjugates are taken up most strongly by adenine auxotrophs and are extruded from the cells over a time course of hours. No fluorescent labeling is seen in killed bacteria or in the presence of EDTA or an excess of unlabeled adenine, AMP, or hypoxanthine. Spectroscopy and electron microscopy suggest that QDs of <5 nm can enter the cells whole, probably by means of oxidative damage to the cell membrane which is aided by light.     

Conjugation and fluorescence quenching between bovine serum albumin and L-cysteine capped CdSe/CdS quantum dots

Water-soluble, biological-compatible, and excellent fluorescent CdSe/CdS quantum dots (QDs) with L-cysteine as capping agent were synthesized in aqueous medium. Fluorescence (FL) spectra, absorption spectra, and transmission electron microscopy (TEM) were employed to investigate the quality of the products. The interactions between QDs and bovine serum albumin (BSA) were studied by absorption and FL titration experiments. With addition of QDs, the FL intensity of BSA was significantly quenched which can be explained by static mechanism in nature. When BSA was added to the solution of QDs, FL intensity of QDs was faintly quenched. Fluorescent imaging suggests that QDs can be designed as a probe to label the Escherchia coli (E. coli) cells. These results indicate CdSe/CdS/L-cysteine QDs can be used as a probe for labeling biological molecule and bacteria cells.     

Colistin-functionalised CdSe/ZnS quantum dots as fluorescent probe for the rapid detection of Escherichia coli

Intensely fluorescent, colistin-functionalised CdSe/ZnS QDs (Colis-QDs) nanoparticles, are synthesized and used as sensitive probes for the detection of Escherichia coli, a Gram-negative bacteria. Colistin molecules are attached to the terminal carboxyl of the mercaptoacetic acid-capped QDs in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) as amide bond promoters. The TEM analysis of bacteria treated with Colis-QDs conjugates showed the accumulation of Colis-QDs in the cell wall of E. coli. Under the recommended working conditions, the method provides a detection limit as few as 28 E. coli cells per mL, which is competitive which more elaborate detection systems. The simplicity of the method together with short analysis time (< 15 min, without including preparation and photoactivation of the Colis-QDs conjugate) make the proposed approach useful as quick bacteria screening system.     

Luminescent gelatin nanospheres by encapsulating CdSe quantum dots

Quantum dots (QDs) have been encapsulated within gelatin nanoparticles (GNPs), which gives GNPs fluorescent properties and improves the biocompatibility of QDs. Hydrophilic CdSe QDs were produced through thermodecomposition following the ligand‐exchange method, and were then encapsulated in GNPs. The results of high‐resolution transmission electron microscopy and transmission electron microscopy show that CdSe QDs and QDs‐encapsulated GNPs (QDs‐GNPs) have average diameters of 5 ± 1 and 150 ± 10 nm, respectively. Results of both high‐resolution transmission electron microscopy and confocal laser scanning microscopy indicate that CdSe QDs are successfully encapsulated within GNPs. The QDs‐GNPs have distinctive fluorescent properties with maximum emission at 654 nm, with a 24 nm red‐shift comapred with hydrophilic mercaptoundecanoic acid (MUA)‐modified QDs. In addition, an in vitro cytotoxicity test shows that QDs‐GNPs do not have any toxic effect on cells. It is expected that QDs‐GNPs might be an excellent candidate as a contrast agent in bio‐imaging. Copyright © 2013 John Wiley & Sons, Ltd.     

Quantum dot-antibody and aptamer conjugates shift fluorescence upon binding bacteria

CdSe/ZnS quantum dots (QDs) exhibited fluorescence emission blue shifts when conjugated to antibodies or DNA aptamers that are bound to bacteria. The intensity of the shifted emission peak increased with the number of bound bacteria. Curiously, the emission was consistently shifted to approximately 440-460 nm, which is distinctly different from the major component of the natural fluorescence spectrum of these QDs. This minor emission peak can grow upon conjugation to antibodies or aptamers and subsequent binding to bacterial cell surfaces. We hypothesize that the wavelength shift is due to changes in the chemical environment of the QD conjugates when they encounter the bacterial surface and may be due to physical deformation of the QD that changes the quantum confinement state. Regardless of the mechanism, these remarkable emission wavelength shifts of greater than 140 nm in some cases strongly suggest new applications for QD-receptor conjugates.     

Effect of ligand density on the spectral, physical, and biological characteristics of CdSe/ZnS quantum dots

Chemical modification of the surface of CdSe/ZnS quantum dots (QDs) with small molecules or functional ligands often alters the characteristics of these particles. For instance, dopamine conjugation quenches the fluorescence of the QDs, which is a property that can be exploited for sensing applications if the conjugates are taken up into living cells. However, different sizes and/or preparations of mercaptocarboxylic acid solubilized QDs show very different properties when incubated with cells. It is unknown what physical parameters determine a QDs ability to interact with a cell surface, be endocytosed, escape from endosomes, and/or enter the nucleus. In this study, we examine the surface chemistry of QD-dopamine conjugates and present an optimized method for tracking the attachment of small biomolecules to the surface. It is found that the fluorescence intensity, surface charge, colloidal stability, and biological interactions of the QDs vary as a function of the density of dopamine on the surface. Successful targeting of QD-dopamine to dopamine receptor positive PC12 cells correlates with greater homogeneity of particle thiol layer, and a minimum number of ligands required for specific association can be estimated. These results will enable users to develop methods for screening QD conjugates for biological activity before proceeding to experiments with cell lines and animals.     

Solubilization and bio-conjugation of quantum dots and bacterial toxicity assays by growth curve and plate count

Quantum dots (QDs) are fluorescent semiconductor nanoparticles with size-dependent emission spectra that can be excited by a broad choice of wavelengths. QDs have attracted a lot of interest for imaging, diagnostics, and therapy due to their bright, stable fluorescence. QDs can be conjugated to a variety of bio-active molecules for binding to bacteria and mammalian cells. QDs are also being widely investigated as cytotoxic agents for targeted killing of bacteria. The emergence of multiply-resistant bacterial strains is rapidly becoming a public health crisis, particularly in the case of Gram negative pathogens. Because of the well-known antimicrobial effect of certain nanomaterials, especially Ag, there are hundreds of studies examining the toxicity of nanoparticles to bacteria. Bacterial studies have been performed with other types of semiconductor nanoparticles as well, especially TiO(2), but also ZnO and others including CuO. Some comparisons of bacterial strains have been performed in these studies, usually comparing a Gram negative strain with a Gram positive. With all of these particles, mechanisms of toxicity are attributed to oxidation: either the photogeneration of reactive oxygen species (ROS) by the particles or the direct release of metal ions that can cause oxidative toxicity. Even with these materials, results of different studies vary greatly. In some studies the Gram positive test strain is reportedly more sensitive than the Gram negative; in others it is the opposite. These studies have been well reviewed. In all nanoparticle studies, particle composition, size, surface chemistry, sample aging/breakdown, and wavelength, power, and duration of light exposure can all dramatically affect the results. In addition, synthesis byproducts and solvents must be considered. High-throughput screening techniques are needed to be able to develop effective new nanomedicine agents. CdTe QDs have anti-microbial effects alone or in combination with antibiotics. In a previous study, we showed that coupling of antibiotics to CdTe can increase toxicity to bacteria but decrease toxicity to mammalian cells, due to decreased production of reactive oxygen species from the conjugates. Although it is unlikely that cadmium-containing compounds will be approved for use in humans, such preparations could be used for disinfection of surfaces or sterilization of water. In this protocol, we give a straightforward approach to solubilizing CdTe QDs with mercaptopropionic acid (MPA). The QDs are ready to use within an hour. We then demonstrate coupling to an antimicrobial agent. The second part of the protocol demonstrates a 96-well bacterial inhibition assay using the conjugated and unconjugated QDs. The optical density is read over many hours, permitting the effects of QD addition and light exposure to be evaluated immediately as well as after a recovery period. We also illustrate a colony count for quantifying bacterial survival.     

Intracellular delivery of quantum dot-protein cargos mediated by cell penetrating peptides

We utilize cell penetrating peptide functionalized QDs as specific vectors for the intracellular delivery of model fluorescent protein cargos. Multiple copies of two structurally diverse fluorescent proteins, the 27 kDa monomeric yellow fluorescent protein and the 240 kDa multichromophore b-phycoerythrin complex, were attached to QDs using either metal-affinity driven self-assembly or biotin-Streptavidin binding, respectively. Cellular uptake of these complexes was found to depend on the additional presence of cell-penetrating peptides within the QD-protein conjugates. Once inside the cells, the QD conjugates were mostly distributed within endolysosomal compartments, indicating that intracellular delivery of both QD assemblies was primarily driven by endocytotic uptake. Cellular microinjection of QD-fluorescent protein assemblies was also utilized as an alternate delivery strategy that could bypass the endocytic pathway. Simultaneous signals from both the QDs and the fluorescent proteins allowed verification of their colocalization and conjugate integrity upon delivery inside live cells. Due to their intrinsic fluorescence properties, this class of proteins provides a unique tool to test the ability of QDs functionalized with cell penetrating peptides to mediate the intracellular delivery of both small and large size protein cargos. Use of QD-peptide/fluorescent protein vectors may make powerful tools for understanding the mechanisms of nanoparticle-mediated drug delivery.     

Quantum Dots as alternatives to organic fluorophores for Cryptosporidium detection using conventional flow cytometry and specific monoclonal antibodies: lessons learned.

BACKGROUND: Significant developments in biological applications are occurring through the incorporation of Quantum Dots (QDs) as biological labels. The demonstration of QDs unique optical properties may have important implications for the study of environmental samples, where microorganisms of interest need to be isolated away from the background debris. METHODS: Flow cytometric analysis was used to determine the fluorescence intensity of oocysts after mAb staining by QDs or organic fluorophore conjugates. In addition, the level of non-specific binding to detrital particles within a control water concentrate was estimated using the optimal staining concentration determined for each mAb analyzed. RESULTS: Under 488 nm excitation, oocysts stained with QD-conjugates exhibited significantly lower fluorescence intensity than organic conjugates. Moreover, the level of non-specific binding by QD-conjugates to detrital particles present in the water concentrate was significantly higher that of the organic conjugates. CONCLUSIONS: While QDs are noted for their superior spectral characteristics, they have been shown here to be unsuitable for conventional flow cytometric detection of Cryptosporidium. Therefore, we conclude that in their current form, QD's are severely limited for fluorescent detection of pathogens in environmental applications.     

“Use of acidophilic bacteria of the genus Acidithiobacillus to biosynthesize CdS fluorescent nanoparticles (quantum dots) with high tolerance to acidic pH”

The use of bacterial cells to produce fluorescent semiconductor nanoparticles (quantum dots, QDs) represents a green alternative with promising economic potential. In the present work, we report for the first time the biosynthesis of CdS QDs by acidophilic bacteria of the Acidithiobacillus genus. CdS QDs were obtained by exposing A. ferrooxidans, A. thiooxidans and A. caldus cells to sublethal Cd²⁺ concentrations in the presence of cysteine and glutathione. The fluorescence of cadmium-exposed cells moves from green to red with incubation time, a characteristic property of QDs associated with nanocrystals growth. Biosynthesized nanoparticles (NPs) display an absorption peak at 360 nm and a broad emission spectra between 450 and 650 nm when excited at 370 nm, both characteristic of CdS QDs. Average sizes of 6 and 10 nm were determined for green and red NPs, respectively. The importance of cysteine and glutathione on QDs biosynthesis in Acidithiobacillus was related with the generation of H₂S. Interestingly, QDs produced by acidophilic bacteria display high tolerance to acidic pH. Absorbance and fluorescence properties of QDs was not affected at pH 2.0, a condition that totally inhibits the fluorescence of QDs produced chemically or biosynthesized by mesophilic bacteria (stable until pH 4.5–5.0). Results presented here constitute the first report of the generation of QDs with improved properties by using extremophile microorganisms.     

The efflux of a fluorescent probe is catalyzed by an ATP-driven extrusion system in Lactococcus lactis.

Many bacteria, both gram positive and gram negative, extrude in an energy-dependent manner the fluorescent pH indicator 2',7'-bis-(2-carboxyethyl)-5[and -6]-carboxyfluorescein (BCECF) (D. Molenaar, T. Abee, and W. N. Konings, Biochim. Biophys. Acta 1115:75-83, 1991). This efflux was studied in detail in Lactococcus lactis, and several indications that a transport system is involved were found. This transport system is most likely driven by ATP or a related compound. The evidence is that BCECF extrusion (i) occurs against a BCECF gradient, (ii) is strictly correlated with ATP concentration and not with the proton motive force, and (iii) is inhibited by vanadate and to a lesser extent by N,N'-dicyclohexylcarbodiimide. Most convincingly, a UV mutant with a strongly reduced efflux rate was isolated. Such a mutant was isolated from a BCECF-loaded and lactose-energized population by selection of highly fluorescent cells in a flow cytometer-cell sorter. The physiological function of this extrusion system is unknown, but its characteristics classify it among the traffic ATPases.     

Quantum dots as strain- and metabolism-specific microbiological labels

Biologically conjugated quantum dots (QDs) have shown great promise as multiwavelength fluorescent labels for on-chip bioassays and eukaryotic cells. However, use of these photoluminescent nanocrystals in bacteria has not previously been reported, and their large size (3 to 10 nm) makes it unclear whether they inhibit bacterial recognition of attached molecules and whether they are able to pass through bacterial cell walls. Here we describe the use of conjugated CdSe QDs for strain- and metabolism-specific microbial labeling in a wide variety of bacteria and fungi, and our analysis was geared toward using receptors for a conjugated biomolecule that are present and active on the organism's surface. While cell surface molecules, such as glycoproteins, make excellent targets for conjugated QDs, internal labeling is inconsistent and leads to large spectral shifts compared with the original fluorescence, suggesting that there is breakup or dissolution of the QDs. Transmission electron microscopy of whole mounts and thin sections confirmed that bacteria are able to extract Cd and Se from QDs in a fashion dependent upon the QD surface conjugate.     

Quantum Dots as Strain- and Metabolism-Specific Microbiological Labels

Biologically conjugated quantum dots (QDs) have shown great promise as multiwavelength fluorescent labels for on-chip bioassays and eukaryotic cells. However, use of these photoluminescent nanocrystals in bacteria has not previously been reported, and their large size (3 to 10 nm) makes it unclear whether they inhibit bacterial recognition of attached molecules and whether they are able to pass through bacterial cell walls. Here we describe the use of conjugated CdSe QDs for strain- and metabolism-specific microbial labeling in a wide variety of bacteria and fungi, and our analysis was geared toward using receptors for a conjugated biomolecule that are present and active on the organism's surface. While cell surface molecules, such as glycoproteins, make excellent targets for conjugated QDs, internal labeling is inconsistent and leads to large spectral shifts compared with the original fluorescence, suggesting that there is breakup or dissolution of the QDs. Transmission electron microscopy of whole mounts and thin sections confirmed that bacteria are able to extract Cd and Se from QDs in a fashion dependent upon the QD surface conjugate.     

Synchrotron-based X-ray fluorescence imaging of human cells labeled with CdSe quantum dots

Synchrotron-based X-ray fluorescence (S-XRF) is a powerful technique for imaging the distribution of many biologically relevant elements as well as of “artificial” elements deliberately introduced into tissues and cells, for example, through functionalized nanoparticles. In this study, we explored the potential of S-XRF for chemical nanoimaging (100 nm spatial resolution, nanoXRF) of human cells through the use of functionalized CdSe/ZnS quantum dots (QDs). We used a commercially available QD-secondary antibody conjugate to label the cancer marker HER2 (human epidermal growth factor receptor 2) on the surface of SKOV3 cancer cells and β-tubulin, a protein associated with cytoskeleton microtubules. We set up samples with epoxy inclusion and intracellular labeling as well as samples without epoxy inclusion and with surface labeling. Epoxy inclusion, also used in electron microscopy, has the advantage of preserving cell morphology and guaranteeing long-term stability. QDs proved to be suitable probes for nanoXRF due to the Se emission band, which is not in close proximity to any other emission band, and the signal specificity, which is preserved in both types of labeling. Therefore, nanoXRF using QD-based markers can be very effective at colocalizing specific intracellular targets with elements naturally present in the cell and may complement confocal fluorescence microscopy in a synergistic fashion.     

Quantitative characterization of quantum dot‐labeled lambda phage for Escherichia coli detection

We characterize CdSe/ZnS quantum dot (QD) binding to genetically modified bacteriophage as a model for bacterial detection. Interactions among QDs, lambda (λ) phage, and Escherichia coli are examined by several cross‐validated methods. Flow and image‐based cytometry clarify fluorescent labeling of bacteria, with image‐based cytometry additionally reporting the number of decorated phage bound to cells. Transmission electron microscopy, image‐based cytometry, and electrospray differential mobility analysis allow quantization of QDs attached to each phage (4–17 QDs) and show that λ phage used in this study exhibits enhanced QD binding to the capsid by nearly a factor of four compared to bacteriophage T7. Additionally, the characterization methodology presented can be applied to the quantitative characterization of other fluorescent nanocrystal‐biological conjugates. Biotechnol. Bioeng. 2009;104: 1059–1067. Published 2009 Wiley Periodicals, Inc.     

Avoiding false-positive signals with nuclease-vulnerable molecular beacons in single living cells

There have been a growing number of studies where molecular beacons (MBs) are used to image RNA expression in living cells; however, the ability to make accurate measurements can be hampered by the generation of false-positive signals resulting from non-specific interactions and/or nuclease degradation. In the present study, we found that such non-specific signals only arise in the nucleus of living cells. When MBs are retained in the cytoplasmic compartment, by linking them to quantum dots (QDs), false-positive signals are reduced to marginal levels. Consequently, MB–QD conjugates were used to measure the expression of the endogenous proto-oncogene c-myc in MCF-7 breast cancer cells by quantifying the total fluorescent signal emanating from individual cells. Upon the addition of tamoxifen, measurements of MB fluorescence indicated a 71% reduction in c-myc expression, which correlated well with RT-PCR measurements. Variations in MB fluorescence resulting from instrumental fluctuations were accounted for by imaging fluorescent calibration standards on a daily basis. Further, it was established that measurements of the total fluorescent signal were not sensitive to the focal plane. Overall, these results provide evidence that accurate measurements of RNA levels can be made when MBs are retained in the cytoplasm.     

Detection of tumor marker CA125 in ovarian carcinoma using quantum dots

The fluorescent labeling of biological materials usingsmall-molecule organic dyes is widely employed in bio-logical imaging and clinical diagnosis. Organic fluoro-phores, however, have certain characteristics that limittheir advantages in some applications. These limitationsinclude narrow excitation bands and broad emissionbands with red spectral tails, which make the simultaneousevaluation of several light-emitting probes difficult due tospectral overlap. Also, many organic dyes exhibit highp…     

Ultrastructure of Butyrivibrio fibrisolvens: a gram-positive bacterium.

The cells of bacteria of the genus Butyrivibrio are universally described as being gram negative, and they produce an unequivocal gram-negative reaction in the standard staining procedure. However, their cell walls contain derivatives of teichoic acid, which are characteristic of gram-positive cells. In this study, the cell walls of two representative strains of Butyrivibrio were of the gram-positive morphological type, as seen by electron microscopy, but they were very thin (12 to 18 nm). The thinness of these cell walls may account for the tendency of these cells to stain gram negatively in the standard staining procedure. Ruthenium red staining revealed an extracellular structure surrounding cells of Butyrivibio sp. (strain C3). This structure was composed of individual "knobs" that sometimes mediated cell-to-cell adhesion in the culture.     

Imaging Escherichia coli using functionalized core/shell CdSe/CdS quantum dots

The internalization of a series of water-soluble CdSe/CdS quantum dots (QDs) stabilized by citrate, isocitrate, succinate, and malate by Escherichia coli is established by epifluorescence and confocal fluorescence scanning microscopy, fluorimetry, and UV–vis spectroscopy on whole and lysed bacterial cells. The organic-acid-stabilized QDs span a range in size from 3.8±1.1 to 6.0±2.4 nm with emission wavelengths from 540 to 630 nm. QDs of different sizes (i.e., 3.8–6 nm) can enter the bacterium and be detected on different fluorescence channels with little interference from other QDs as a result of the distinct emission profiles (i.e., 540–630 nm, respectively). Costaining QD-labeled E. coli with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) demonstrates that the QDs and DAPI are colocalized within E. coli, whereas costaining QD-labeled E. coli with membrane dye FM4-64 shows that the FM4-64 is localized in the outer bacterial membrane and that the QDs are inside.Electronic Supplementary Material Supplementary material is available to authorized users in the online version of this article at .