Grantee Research Project Results
2006 Progress Report: Transformations of Biologically-Conjugated CdSe Quantum Dots Released Into Water and Biofilms
EPA Grant Number: R831712Title: Transformations of Biologically-Conjugated CdSe Quantum Dots Released Into Water and Biofilms
Investigators: Holden, Patricia , Nadeau, Jay L.
Institution: University of California - Santa Barbara , McGill University
EPA Project Officer: Aja, Hayley
Project Period: October 1, 2004 through September 30, 2007
Project Period Covered by this Report: October 1, 2005 through September 30, 2006
Project Amount: $332,099
RFA: Exploratory Research to Anticipate Future Environmental Issues: Impacts of Manufactured Nanomaterials on Human Health and the Environment (2003) RFA Text | Recipients Lists
Research Category: Nanotechnology , Safer Chemicals , Human Health
Objective:
Semiconductor nanocrystals (quantum dots, QDs) differ in important ways from bulk semiconductor materials. Their increased band gap means that they function as strong oxidizing and/or reducing agents, and their small size allows them to pass into living cells. Conjugation of biomolecules to the crystal surface can alter any or all of these properties. In preliminary experiments, we have observed that nucleobase-conjugated CdSe QDs were actively taken up by soil and aquatic bacteria (for example, Bacillus subtilis and Escherichia coli). Effects on microbial viability attributed to the presence of the QDs included slower doubling times, heavy metal sequestration, and “blebbing” of metals into the environment. The objectives of this research include quantifying these effects using a variety of biologically-conjugated QDs and an assortment of microbial species, monitoring the process of QD uptake and breakdown, and characterizing the breakdown products that result from bacterial metabolism of these particles. Possible hazards to microbial populations with extrapolation to humans through contamination of soil and water with QD breakdown products are to be analyzed and quantified.
Progress Summary:
Overall, bare, core-shell, and biologically-conjugated QDs are being studied. Abiotic and biotic breakdown products in aqueous environments are being determined by inductively coupled plasma (ICP) spectrometry for QDs. Research preceding this project showed that biologically-conjugated QDs were easily taken up by B. subtilis, but that the process was light- and pH-dependent, and some breakdown occurred inside and outside of cells. Working with Pseudomonas aeruginosa, B. subtilis, and E. coli, population growth and fluorescence for pure liquid cultures were studied. Conventional methods (shake flask) are being used to assess the effects of QDs on bacterial growth rates under high and low nutrient conditions. QD fluorescence is monitored, adjusting final results for the dilution effect of growing populations. Concentrations of cadmium and selenium ions are being assessed inside and outside cells, and cellular associations of whole QDs and breakdown products are being quantified. The relationship of QD release and breakdown to cell viability is being assessed. Oxidizing and reducing conditions in cells are being assessed by dopamine-conjugated CdSe QDs to infer electron transfer-mediated changes in nanoparticles both inside and outside of cells. All of the experiments are providing basic insight into cellular interactions with QDs. Unsaturated biofilms are also being cultured on membranes to assess the effects of QD labeling on development under soil-like conditions, and as a function of nutrient and water availability. Transmission electron microscopy (TEM) is being used to visualize ultrastructural QD associations. Environmental scanning electron microscopy (ESEM) is being used to assess surface alterations to biofilms that would be indicative of exopolymeric substances (EPS). For biofilm experiments, QD effects on EPS will be quantified by gas chromatography/mass spectrometry (GC/MS) of derivatized glycosyl residues, and DNA and protein content determined by standard fluorometric and colorimetric methods, respectively.
Work Status and Progress
Research was ongoing throughout the project period in the labs of Holden, Nadeau and an additional collaborator at the University of California at Santa Barbara (UCSB), Galen Stucky (Chemistry and Biochemistry). Three other collaborators were also added: Dr. Jin Ping Zhang (UCSB Materials Research Lab, MRL), whose expertise is in electron microscopy of hard materials and who oversees several key instruments at UCSB including a high resolution scanning TEM (STEM); Randy Mielke (Jet Propulsion Laboratory), whose expertise is in electron microscopy and geomicrobiology; and Dr. Sam Webb (Stanford Synchrotron Radiation Laboratory, SSRL), whose expertise is in synchrotron radiation methods for chemical analysis. The research is active and in progress. Foci in the research are highly consistent with the originally proposed work, but clearly the work is evolving to address both short- term and long -term effects of QDs on cells. The conceptual model for QD fates in cells (that we developed as a guide to our experimentation) evolved into a model that was more definitive, based on our research. We now have, because of the experimental insights gained (see below), a more simplified model.
Principle investigator (PI) Holden attended the annual all-investigator meeting in Washington, DC, and presented a selection of results f rom the past year, including the full understanding of bare QD fate in P. aeruginosa liquid culture. Still not completely understood are the mechanisms that explain our observations, but our observations were compelling, reproducible, and significant. As described below, a number of presentations and proceedings credited this funding in the first year of the project. Also as described below, the third year of the project is quite productive and will result in several publications and presentations at various meetings.
Results for the Second Year
As described in the last report, prior results in the Nadeau lab that occurred before this project, but continued into the first year, produced much insight into the uptake of a variety of QDs by various strains of bacteria. The experiments provided valuable insights into the role of bioconjugates and the potential roles of metabolism in QD uptake. Yet, with the environmental focus of this current project, the current research with bacteria has been more oriented towards longer term phenomena, that is , on the time scale of population growth. Over the course of the last year, many growth experiments were conducted and replicated with P. aeruginosa PG201, a soil isolate and versatile biodegrader that is also highly related to clinically important strains, such as PA01. In the first year, we made much progress and the work produced valuable preliminary results for both planktonic and biofilm culture. In the second year, we focused primarily on finishing the planktonic research of bare QD effects on P. aeruginosa growth. As per last year’s report, bare QDs were chosen because bioconjugates are unlikely to stay intact in the external environment. Yet cells may stabilize or accelerate decomposition of bare CdSe QDs. Working in rich media and at temperature and aeration (i.e., aerobic) conditions similar to the short- term labeling experiments (above), we discovered that:
- Bare QDs are indeed as toxic as dissolved Cd(II) delivered at the same equivalent concentrations of Cd(II). Toxicity is manifested as longer lag times, slower growth rate, and lower cell yield.
- QDs are biotically decomposed into their constituent metals, thus potentially explaining how Cd(II) and QDs can be similarly toxic. We confirmed that breakdown was not happening in the absence of cells.
- The fate of metals is such that Cd(II) liberated from broken- down QDs is sequestered with cells and is not free in solution. We confirmed this by inductively coupled plasma-atomic emission spectroscopy (ICP-AES) and mass balance calculations on the metal.
- The Se(II) liberated from biotic decomposition of QDs is oxidized to Se(0) and remains associated with cells.
- QDs disfigure cell envelopes, similar to what has been observed with reactive oxygen species (ROS)-generating compounds. Yet Cd(II) did not disfigure membranes.
It is worth noting that this past year’s advancements in understanding were significantly enhanced by using synchrotron methods to establish the selenium oxidation state. Also, the use of STEM allowed us to visualize membranes and their integrity, which was also essential to determining QD effects on cells.
In addition to the above work that is related to the fate of QDs within cells, we also learned more about the fates of constituent metals, namely Cd(II) and Se(IV), when added together as solutes under biotic conditions. Our preliminary work suggested that this treatment was causing biotic formation of QDs, as evidenced by the enhanced fluorescence of cells . In this second year, we used synchrotron radiation and X-ray diffraction (XRD) (with Chris Ehrhardt, a UCSB student in Earth Science) to show that the oxidation state of selenium was a combination of Se(0) and Se(II) after growth, and that the crystal structure appeared consistent with CdSe QDs. This work is thus progressing, but it still needs more verification.
Importance to Field and Relevancy to the Science To Achieve Results (STAR)Program
The research is directly relevant to the needs of the U.S. Environmental Protection Agency (EPA) and of society in an increasingly nanotechnologically driven world. Few researchers are focusing on the environmental fates and effects of engineered nanoparticles. Bacterial systems are appropriate for assessing such issues; they also serve as model systems for studying nanoparticles and biological cells generally. The important main findings of the work last year include:
- Demonstrating that QDs are biotic al ly decomposed by bacteria.
- Demonstrating that bare QDs are as toxic as the most toxic constituent metal (i.e., cadmium ions).
- Further gaining evidence that QDs may form in Gram-negative bacteria under specific conditions.
- Developing and characterizing a redox-active, dopamine- conjugated QD system for discovering oxidizing and reducing conditions in cells that indicate electron transfer between nanoparticles and cells.
EPA and society need to know the potential effects of engineered nanoparticles on bacteria that occur in soil and water. Clearly, we are discovering effects over short and long time scales. The work will continue this next period to more completely delineate the observations thus far, and especially to more fully document the effects on biofilms that could occur in soil environments.
Comparison to Stated Goals and Objectives
The original goals of the research included studying abiotic and biotic fates and effects of CdSe QDs. The goals of the work in this second period were still strongly driven by the need to understand the fates of QDs in biotic systems of bacterial cells. The abiotic counterparts were studied via uninoculated controls used in every experiment. Therefore, consistently with the original scope of work, we focused on answering the following questions:
- What are the biotic fates of CdSe QDs?
- What are the effects of QDs on bacteria?
- How do these effects differ from constituent metals’ effects?
- What are the dependencies on growth habit (biofilm or planktonic)?
- How does fluorescence of nanoparticles change in association with bacterial cells?
We still need to pursue the issues of size, conjugate, and other nanoparticle properties on these findings. This is the subject of a proposal that is currently in the award process to our groups. Work in the two labs has centered on different strains of bacteria as described above. It is useful to continue working with the same strains for consistency.
Future Activities:
Over the coming year, the following activities are planned:
- Determine membrane integrity of QD-exposed bacteria using microscopy and relevant, commercially available stains. This is to further shed light on the possibility that ROS-mediated membrane permeabilization is responsible for entry of QDs into cells and/or at least part of the observed toxicity.
- Determine if QDs must be in contact with cells for breakdown to occur.
- Fully study the effects of QDs on biofilms, and compare to planktonic culture.
- Begin new studies on the effects of nanoparticles on P. aeruginosa virulence.
- Submit publications for peer review on both the breakdown and effects of QDs in P. aeruginosa, with one oriented towards planktonic growth and the other oriented towards biofilms with comparisons to planktonic growth.
The other activities this year include submitting an additional proposal for funding, making numerous presentations of this work, and attending the annual all-investigator meeting.
Journal Articles on this Report : 5 Displayed | Download in RIS Format
Other project views: | All 50 publications | 18 publications in selected types | All 17 journal articles |
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Clarke SJ, Hollmann CA, Zhang Z, Suffern D, Bradforth SE, Dimitrijevic NM, Minarik WG, Nadeau JL. Photophysics of dopamine-modified quantum dots and effects on biological systems. Nature Materials 2006;5(5):409-417. |
R831712 (2006) R831712 (Final) |
Exit Exit |
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Nadeau JL, Perreault NN, Niederberger TD, Whyte LG, Sun HJ, Leon R. Fluorescence microscopy as a tool for in situ life detection. Astrobiology 2008;8(4):859-874. |
R831712 (2006) |
Exit |
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Priester JH, Olson SG, Webb SM, Neu MP, Hersman LE, Holden PA. Enhanced exopolymer production and chromium stabilization in Pseudomonas putida unsaturated biofilms. Applied and Environmental Microbiology 2006;72(3):1988-1996. |
R831712 (2006) R831712 (Final) |
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Priester JH, Horst AM, Van De Werfhorst LC, Saleta JL, Mertes LAK, Holden PA. Enhanced visualization of microbial biofilms by staining and environmental scanning electron microscopy. Journal of Microbiological Methods 2007;68(3):577-587. |
R831712 (2006) R831712 (Final) |
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Rochira JA, Gudheti MV, Gould TJ, Laughlin RR, Nadeau JL, Hess ST. Fluorescence intermittency limits brightness in CdSe/ZnS nanoparticles quantified by fluorescence correlation spectroscopy. Journal of Physical Chemistry C2007;111(4):1695-1708. |
R831712 (2006) R831712 (Final) |
Exit Exit |
Supplemental Keywords:
water, soil, chemical transport, effects, ecological effects, bioavailability, metabolism, dose response, organism, population, chemicals, toxics, metals, heavy metals, oxidants, bacteria, terrestrial, aquatic, environmental microbiology, biofilms, chemical analysis, nanotechnology, DNA, RFA, Scientific Discipline, Sustainable Industry/Business, treatment, control, Analytical Chemistry, Biochemistry, Chemistry and Materials Science, Environmental Chemistry, Environmental Engineering, New/Innovative technologies, Sustainable Environment, Technology, Technology for Sustainable Environment, DNA damage, biofilm, engineering, environmental sustainability, environmentally applicable nanoparticles, heavy metal sequestration, innovative technologies, innovative technology, nanotechnology, quantum dots, semiconductor nanocrystals,, RFA, Scientific Discipline, TREATMENT/CONTROL, Sustainable Industry/Business, Sustainable Environment, Environmental Chemistry, Technology, Technology for Sustainable Environment, Biochemistry, New/Innovative technologies, Chemistry and Materials Science, Environmental Engineering, biofilm, quantum dots, DNA damage, heavy metal sequestration, nanotechnology, environmental sustainability, engineering, environmentally applicable nanoparticles, semiconductor nanocrystals, sustainability, innovative technologyProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.