Grantee Research Project Results
Final Report: Nanosensors for Detection of Aquatic Toxins
EPA Grant Number: R829599Title: Nanosensors for Detection of Aquatic Toxins
Investigators: Gawley, Robert E.
Institution: University of Miami
EPA Project Officer: Aja, Hayley
Project Period: March 1, 2002 through February 28, 2005
Project Amount: $350,000
RFA: Exploratory Research: Nanotechnology (2001) RFA Text | Recipients Lists
Research Category: Nanotechnology , Water Quality , Safer Chemicals
Objective:
The objective of this research project was to design and prepare nanoscale sensors for the detection of marine toxins.
Summary/Accomplishments (Outputs/Outcomes):
We have investigated several aromatic groups for the fluorescence response, including the anthracene (Gawley, et al., 1999; Gawley, et al., 2002), coumarin (Kele, et al., 2002), and acridine fluorophores shown in Figure 1, for the detection of saxitoxin (STX). In this project, we have investigated the basic science of these types of sensors and have applied them in the nanotechnology arena.
Figure 1. Crown Ether Chemosensors for STX. The buffer used is ammonium phosphate.
We have shown that the coumaryl crown gave no response to sodium, potassium, or calcium ions in aqueous buffer (Kele, et al., 2002). In recently published work (Gawley, et al., 2005a), we have shown that a diaza acridine crown gives no response to tetrodotoxin (TTX), indicating that our sensor system will be selective for STX over TTX, even though they bind to the same protein in vivo, and their clinical symptoms are similar.
There were technical reasons (such as solubility and optical properties) for switching between the various fluorophores, but these selectivities are consistent across all three classes of compounds. Chronologically, we began our work with the anthracene crown, using its 366 nm absorption band. To get water solubility in anticipation of environmental sampling, we switched to coumarins having absorptions at 325 nm. When it became obvious that this wavelength could not be used in a STX assay because interference from trace impurities (see below), we switched to the acridine fluorophore. Although this water-soluble fluorophore also worked well, we later learned that the shellfish extract matrix has an end-absorption that interferes with the assay at wavelengths shorter than approximately 375 nm.
We have obtained samples of both toxic and nontoxic shellfish from the Washington State Department of Health and have done a preliminary evaluation of extracts of these shellfish with our anthracene chemosensor. Absorption spectroscopy (Figure 2) of a number of shellfish extracts, both toxic and nontoxic, indicates that the ultraviolet (UV) absorptions diminish rapidly between 350 and 400 nm. Figure 2 also shows the emission spectra of the same extracts and confirms that there is little emission when irradiated at or beyond 400 nm. To eliminate any other variable, a single nontoxic extract was spiked with STX, and a fluorescence titration was recorded by making successive dilutions with nontoxic (unspiked) extract containing sensor.
Figure 2. Adsorption and Emission Spectra. Upper left: UV-Visible absorption spectra of several shellfish extracts. Upper right: Emission spectra of same shellfish extracts when irradiated at 391 nm, superimposed on the emission spectra of anthracyl crown ether in presence of 100 µM STX. Bottom: “Binding Isotherm” of nontoxic shellfish extract spiked with varying concentrations of STX (unpublished).
In an attempt to improve the sensitivity of our sensor systems, we wondered whether combinatorial chemistry might be used to develop libraries of crowns that have been modified with a short peptide that might wrap around the toxin and improved binding. In 2002, we reported a number of crown ether derivatives, none of which showed significantly improved binding but some of them were amenable to library development. Note, however, that the issues of binding and fluorescence enhancement are separate issues: binding to the crown ether is a necessary, but not sufficient, condition for fluorescence enhancement. To simplify the issue, we prepared a small library of peptides that was designed to envelop the narcotic ohmefentanyl. In the event, a peptide was identified that binds ohmefentanyl with a binding constant of 7 × 104 M-1, as shown in Figure 3 (Gawley, et al., 2005b).
Figure 3. Ohmefentanyl. Upper left: Structure of Ohmefentanyl. Upper right: Structure of Heptapeptide that Binds the Narcotic. Bottom: Fluorescence Titration of Peptide Against Ohmefentanyl and Against Morphine (note selectivity of binding of ohmefentanyl vs. morphine). Also shown is a control in which the three middle residues are replaced by glycine, resulting in loss of binding (Gawley, et al., 2005b).
Two preliminary approaches have been taken to marry the technology described above, all of which was done in solution, to nanotechnology. The first approach incorporates a fluorescence sensor in a self-assembled monolayer on a quartz slide. As shown in Figure 4, we used a coumarin-based crown, tieing the crown to the slide through a tether that was bound covalently. The prototype sensor showed greater than 100 percent fluorescence enhancement at concentrations of greater than or equal to 50 µM STX in aqueous buffer (Kele, et al., 2006).
Figure 4. Self-Assembled Monolayer of Coumaryl Crown Fixed to a Quartz Slide
The second approach was based on the following reasoning. STX binds to the voltage-gated sodium channel with a binding constant of 109 M-1, which corresponds to a 12.5 kcal/mol binding energy. Moreover, the binding site was on the extracellular surface of the cell and binding was specific. It was intriguing to us that, although STX had approximately 1:1 ratio of carbons to heteroatoms and a +2 charge, the channel protein could “extract” the toxin from bulk water with such a high affinity. Using all of the site-directed mutagenesis data that were available at the time, Lipkind and Fozzard devised a detailed model of how STX might bind (Lipkind and Fozzard, 1994). They concluded that “the binding site forms a funnel-like aggregate barrel, consisting of 4 antiparallel hairpins with the beta2 strands facing the [toxin]...” Their model holds the toxins in a bowl without a lid and does not encapsulate the entire toxin. Their model implicates several Glu residues in the bowl, which are considered to bind through both electrostatic and hydrogen bonding forces. We hypothesized that the funnel-like barrel provided a solvent-excluded binding pocket, and that occupation of this pocket by the toxin (perhaps aided entropically by displacement of water from the binding pocket and release of water from the STX solvation sphere) provided a significant part of the binding energy.
To test this hypothesis in the context of nanoscience, we constructed two trifurcated dendrimers with crown sensors at their core. The first is a water-insoluble crown containing an 18-ring crown and a coumarin fluorophore; the second is a water-soluble dendrimer having an 18-ring crown and an anthracene chromophore (Figure 5). Evaluation of these dendritic chemosensors is still underway. An issue that we currently are addressing is the pH response of the aryl crowns in various solvents (Bissell, et al., 1992). Because the hydrophobic environment of aryl crown in the interior of the dendrimer is significantly different from bulk water (solvent), it will be necessary to determine the pKa of the crown ether nitrogen(s) in nonaqueous solvents.
Figure 5. Incorporation of a Crown Ether Fluorescence Chemosensor (Red, or Shaded Portion) Into the Core of a Dendrimer Having a Hydrophobic Interior and Exterior (Left), or a Dendrimer Having a Hydrophilic Periphery with a Hydrophobic Interior (Right) (Unpublished)
References:
Gawley RE, Zhang Q, Higgs PI, Wang S, Leblanc RM. Anthracylmethyl crown ethers as fluorescence sensors of saxitoxin. Tetrahedron Letters 1999;40:5461-5465, corrigendum 6135.
Kele P, Orbulescu J, Calhoun TL, Gawley RE, Leblanc RM. Coumaryl crown ether based chemosensors: selective detection of saxitoxin in the presence of sodium and potassium ions. Tetrahedron Letters 2002;43:4413-4416.
Lipkind GM, Fozzard HA. A structural model of the tetrodotoxin and saxitoxin binding site of the Na+ channel. Biophysical Journal 1994;66:1-13.
Bissell RA, Calle E, Desilva AP, Desilva SA, Gunaratne HQN, Habibjiwan JL, Peiris SLA, Rupasinghe RADD, Samarasinghe TKSD, Sandanayake KRAS, Soumillion JP. Luminescence and Charge-Transfer .2. Aminomethyl Anthracene-Derivatives As Fluorescent Pet (Photoinduced Electron-Transfer) Sensors for Protons. Journal of the Chemical Society-Perkin Transactions 2 1992;(9):1559-1564.
Gawley RE, Shanmugasundaram M, Thorne JB, Tarkka RM. Selective detection of saxitoxin over tetrodotoxin using acridinylmethyl crown ether chemosensor. Toxicon 2005;45(6):783-787.
Gawley RE, Dukh M, Cardona CM, Jannach SH, Greathouse D. Heptapeptide mimic of ohmefentanyl binding in the discontinuous m-opiod receptor. Organic Letters 2005;7(14):2953-2956.
Kele P, Orbulescu J, Gawley RE, Leblanc RM. Spectroscopic detection of saxitoxin: an alternative to mouse bioassay. Chemical Communications 2006;(14):1494-1496.
Journal Articles on this Report : 3 Displayed | Download in RIS Format
Other project views: | All 3 publications | 3 publications in selected types | All 3 journal articles |
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Cardona CM, Gawley RE. An improved synthesis of a trifurcated Newkome-type monomer and orthogonally protected two-generation dendrons. Journal of Organic Chemistry 2002;67(4):1411-1413. |
R829599 (Final) R826655 (Final) |
not available |
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Gawley RE, Pinet S, Cardona CM, Datta PK, Ren T, Guida WC, Nydick J, Leblanc RM. Chemosensors for the marine toxin saxitoxin. Journal of the American Chemical Society 2002;124(45):13448-13453. |
R829599 (Final) R826655 (Final) |
not available |
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Mao H, Thorne JB, Pharr JS, Gawley RE. Effect of crown ether ring size on binding and fluorescence response to saxitoxin in anthracylmethyl monoazacrown ether chemosensors. Canadian Journal of Chemistry 2006;84(10):1273-1279. |
R829599 (Final) GR832382 (2006) GR832382 (Final) |
Exit |
Supplemental Keywords:
saxitoxin, paralytic shellfish poison, PSP, paralytic shellfish toxin, PST, sensors, fluorescence, dendrimers, self assembled monolayer, SAM, peptide host, ohmefentanyl, chemistry, aquatic toxins, environmental monitoring,, RFA, Scientific Discipline, Water, Ecosystem Protection/Environmental Exposure & Risk, Sustainable Industry/Business, Environmental Chemistry, Sustainable Environment, Technology for Sustainable Environment, Monitoring/Modeling, Analytical Chemistry, algal blooms, New/Innovative technologies, Engineering, Chemistry, & Physics, Environmental Engineering, aquatic ecosystem, environmental monitoring, nanosensors, aqautic toxins, marine ecosystem, chemical sensors, marine biotoxins, nanotechnology, environmental sustainability, brevetoxins, chemical composition, aquatic toxins, environmentally applicable nanoparticles, pathogenic quantification, nanoscale sensors, sustainability, nano engineering, innovative technologies, biosensor, nanoengineeringProgress 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.