NCER Assistance Agreement Final Project Summary
Date of Final Report: August 31, 2007
EPA Agreement Number: X-83254101-1
Center: Center for Environmental and Energy Research (CEER)
Project Title: Microarray System for Contaminated Water Analysis
Investigator(s): Cardinale, Jean; De Rosa, Rebecca; Thatcher, Ryan
Institution(s) of PI(s): Alfred University
Research Category: Congressionally Mandated Center
Project Period: September 1, 2006 - August 31, 2007
Description and Objective of Project:
Our previous study, on a functionalized glass surface to covalently immobilize antibodies, was conducted to produce a more efficient means of water contamination analysis. The current methods used to detect organisms in water require up to 4 days to produce results that are indicative of only a small number of potential contaminates. Enzyme linked immunosorbent assays (ELISA) can use immunological methods to detect a wide range of biomolecules in less time than the current methods. However, traditional ELISAs produce a significant amount of solid waste and require many high-purity antibodies. The costs incurred and biologically- contaminated waste produced by ELISAs have led to the production of a smaller alternative called a microarray. Microarrays use immobilized biomolecules (proteins, lipids, antibodies, and carbohydrates) to detect conjugate biomolecules in solution. Multiple types of biomolecules can be printed onto a single solid glass or polymer substrate to detect multiple conjugate molecules in a single test. Our previous work focused on modification of a glass substrate to increase the surface area available for immobilization and the means by which antibodies were bound to the surface. Covalent immobilization of the antibodies instead of hydrophobic binding is one method that can optimize the binding concentration of antibodies onto the substrate. Our results indicated that surface modifications we made to glass substrates successfully immobilized antibodies. However, the immobilized antibodies denatured, and did not retain their functionality after the immobilization process. Stabilization of the antibodies structure is a potential solution to prevent the denaturing caused during immobilization.
Two major means of stabilizing protein structures have been developed to prevent denaturing of proteins due to dehydration, elevated temperatures, and long-term storage. Preservation of biological tissue and other materials is commonly performed using an Aldehyde solution. The aldehyde crosslinks proteins, preventing changes in the structure that lead to denaturing. This technique has been applied to microarrays to increase the stability of immobilized proteins with demonstrated success. The second form of protein stabilization modifies the interactions between the protein and solvent solution. Studies suggest that addition of osmolytes to the solvent solution makes the native protein structure more thermodynamically stable than an unfolded or denatured structure. Introduction of osmolytes to the solvent solution has also been shown to allow denatured proteins to return to their native structure after induced denaturing. The experiments completed in this project use both methods of stabilization to retain antibody functionality after immobilization.
The goal of our work is to create a miniaturized ELISA to detect multiple biological contaminates without producing large quantities of solid waste. Development of a microarray with covalently immobilized antibodies can provide a potentially reusable method of detection for biological contaminates. The objective of the work in this study is to stabilize the antibodies structure to prevent denaturing after covalent immobilization on a glass slide. Retaining the functionality of the covalently immobilized antibodies would remedy the denaturing problems faced when using our suggested surface treatment. The functionality of the antibodies can be tested by the capture of green fluorescent protein (GFP) labeled Escherichia coli (E. coli). Successful retention of antibody functionality can lead to the development of a novel immunological method for water quality analysis that will reduce the cost of testing and waste produced by traditional ELISA.
Summary of Findings:
We used the optimum slide treatment as determined by the previous study*: water plasma cleaning, photo-hydrolytic weathering, and silane treatment using 3-aminopropyl triethoxysilane (APS). Anti-E.coli antibodies were printed onto Corning 2947 (soda-lime-silicate) and Corning 1737 (proprietary alkaline earth aluminoborosilicate) glass slides for functionality tests. Fluorescein isothiocynate labeled, goat anti-rabbit antibodies were printed onto slides as a control to confirm the presence of immobilized antibodies. Stabilizers were added to the antibody solutions before printing on the slides. Three stabilizing agents were investigated: gluteraldehyde, trehalose, and 200 molecular weight (MW) polyethylene glycol (PEG). Functionality of the immobilized antibodies was determined by their ability to capture GFP labeled E. coli from a Blotto buffer solution. The results were measured as a positive or negative presence of E. coli based on fluorescent scanning.
None
of the antibodies that had been stabilized by one of the three agents tested
retained their functionality after immobilization. Fluorescent scanning of the slides confirmed the presence of
immobilized antibodies, but did not detect any captured GFP labeled E. coli. XPS
analysis was used to supplement the results from the functionality tests to
determine if the antibodies were remaining in their native structure. The thickness of the antibody layer was
determined using sputter depth profiling.
Antibodies that retained their native structure would create a thicker
layer than the denatured antibodies.
Layer thickness was determined by the sputter time required for the
concentration of carbon to decrease below the concentration of silicon, which
we termed the carbon-silicon crossover.
Longer sputter times indicate a thicker protein layer. The thickness of antibodies stabilized
by trehalose and gluteraldehyde were compared to non-stabilized
antibodies. Results from the
sputter depth profiles can be seen below.
The stabilized antibodies displayed slightly longer time until the
carbon-silicon crossover. Also,
the initial carbon concentration for stabilized antibodies is greater than the
non-stabilized antibodies. Due to
the greater initial carbon concentrations, we concluded the increase in sputter
time was not significant enough to indicate a thicker layer.
* http://ceer.alfred.edu/Research/research_pollutants.html
http://cfpub.epa.gov/ncer_abstracts/index.cfm/fuseaction/outlinks.centers/center/145
Since addition of stabilizers to
the predetermined process did not yield functional antibodies, the conditions
used for stabilization and immobilization were modified to determine if other
variables could be changed to increase the functionality of the
antibodies. A comprehensive list
of the variables changes and the values used is available in the table below.
Variables and Values Changed from
Original Protocol
|
Variables |
Tested values |
|
Stabilization time |
1, 4, and 12 hours |
|
Stabilization temperature |
4¡C and room temperature |
|
Stabilizer concentration |
1M, 1.5M, and 2M solutions |
|
Incubation time |
1, 4, and 12 hours |
|
Incubation temperature |
4¡C and room temperature |
|
Antibody concentration |
1, 5, and 10 µg/mL |
|
APS concentration (slide treatment) |
0.5x, 1x, 5x, 10x (v/v) solutions |
|
PEG concentration (slide treatment) |
1x, 5x, 10x, 20x (v/v) solutions |
*Note: Stabilization is defined as the process of combining a stabilizing agent with antibodies before immobilization onto the substrate. Incubation is defined as the step where antibodies attach to the substrate before a wash is performed.
Variations
of the conditions for stabilization and immobilization were performed changing
only one variable at a time to cover the complete range of possible
combinations. This would pinpoint
individual steps in the process responsible for the antibodies denaturing. All of the tested processes
successfully immobilized antibodies.
However, we were still unable to capture E.coli under any of the new
conditions. The antibodies were
still denatured. Since all
potential variables in the stabilizing and immobilizing processes did not yield
functional antibodies, additional steps are required to prevent the denaturing
of antibodies.
Conclusions:
Two types of Corning Inc. glass compositions were treated according to processes determined in the previous study. To regain functionality of antibodies that denatured due to immobilization, stabilizing agents were added to the antibody solutions. Antibodies were incubated in solutions containing either gluteraldehyde, trehalose, or PEG. None of the stabilized antibodies were able to capture detectable amounts of GFP labeled E. coli in solution. XPS sputter depth profiling was conducted as a secondary means of confirming the denaturing of the antibodies. Results from the depth profiles indicated that both the stabilized and non-stabilized antibodies did not retain their native structure after immobilization onto our treated glass slides. Variables in the stabilizing and immobilizing processes were changed to investigate potential denaturing due to individual steps. All potential changes to the process still yielded non-functional antibodies. Additional means to prevent denaturing of the antibodies are required to create a functional microarray using the predetermined slide treatment.
Selected References:
S.G. Acinas, R. Sarma-Rupavtarm, V. Klepac-Ceraj, and M.F. Polz, "Pcr-Induced Sequence Artifacts and Bias: Insights from Comparison of Two 16s Rrna Clone Libraries Constructed from the Same Sample," Applied and Environmental Microbiology, 71 [12] 8966-9 (2005).
G. Xie and S.N. Timasheff, "The Thermodynamic Mechanism of Protein Stabilization by
Trehalose," Biophysical Chemistry, 64 25-43 (1997).
P. Zancan and M. Sola-Penna, "Trehalose and Glycerol Stabilize and Renature Yeast Inorganic Pyrophosphatase Inactivated by Very High Temperatures," Archives of Biochemistry and Biophysics, 444 52-60 (2005).
T.M. Straub and D.P. Chandler, "Towards a Unified System for Detecting Waterborne
Pathogens," Journal of Microbiological Methods, 53 185-97 (2003).
F. Lopez-Gallego, L. Betancor, C. Mateo, A. Hidalgo, N. Alonso-Morales, G. Dellamora-Ortiz, J.M. Guisan, and R. Fernandez-Lafuente, "Enzyme Stabilization by Glutaraldehyde Crosslinking of Adsorbed Proteins on Aminated Supports," Journal of Biotechnology, 119 70-5 (2005).
K. Blank, T. Mai, I. Gilbert, S. Schiffmann, J. Rankl, R. Zivin, C. Tackney, T. Nicolaus, K.
Spinnler, F. Oesterhelt, M. Benoit, H. Clausen-Schaumann, and H.E. Gaub, "A Force-Based
Protein Biochip," PNAS, 100 11356-60 (2003).
R. Fernandez-Lafuente, C.M. Rosell, V. Rodriguez, and J.M. Guisan, "Strategies for Enzyme
Stabilization by
Intramolecular Crosslinking with Bifunctional Reagents," Enzyme and
Microbial Technology, 17 517-23 (1995).
Publications/Presentations:
A. Cooper, Microarray System for Contaminated Water
Analysis.
M. S. thesis, Alfred University, 2005. Advisors: R. DeRosa & J. Cardinale
R.L. DeRosa, A. Cooper and J.A. Cardinale, ÒFunctionalized Glass Substrate for Microarray
AnalysisÓ, Thin Solid Films, Vol. 515 (2007) pp. 4024-4034.
R.L. DeRosa, A. Cooper, J.A. Cardinale, ÒBioapplications for Photo-hydrolyzed Glass
SurfacesÓ, Ceramic Transactions, v. 173, Advances in Glass and Optical Materials –Proc. 107th Annual Meeting of The American Ceramic Society, 2006, p 47-58.
Supplemental Key Words: ELISA, lab on a chip, water contaminants, glass surface modification, protein stabilization, gluteraldehyde, trehalose
Relevant Web Sites: http://ceer.alfred.edu