1. For the past several years, one of the primary focuses of our research group has been the development of novel ion-exchange systems for the purpose of metal remediation from aqueous systems. Expanding on hints from Mother Nature, we chose to explore the metal chelation abilities of proteins and, in particular, their constituent amino acids. In order to simplify these ion-exchange systems, short-chain homopolymers consisting of repeating monomers of a specified amino acid residue have been used. These systems exhibit many of the characteristics for an ideal ion-exchanger – strong binding; fast, efficient release and structural stability. These biologically-based systems also have the added benefit of being environmentally friendly, unlike many traditional exchange systems which require harsh extraction agents. Developing Fluorescence- based Sensors Questions? Email Shelly. slcasciato@mail.utexas.edu M+M+ M+M+ M+M+ M+M+ M+M+ M+M+ M+M+ Support The Bonded-Phase Ion-Exchange System Visit us! On the web: http://research.cm.utexas.edu/jholcombe/ In the Lab: Welch 3.240 and 3.238 http://research.cm.utexas.edu/jholcombe/ Metallomic Studies with PAGE-LA-ICPMS Questions? Email Isaac. isaac.arnquist@mail.utexas.edu Questions? Email Ram. ramk@mail.utexas.edu Questions? Email Haley. hjfinjo@mail.utexas.edu ETV-ICP-MS for Isobars and Isotopes Questions? Email Adam. adamrowland@mail.utexas.edu Creating Chemical-free Remediation Systems Column 3-electrode potentiostat Clean Effluent Stream Metal Recovery Stream valve Reference Electrode E applied Auxiliary Electrode An electrical potential is used to change the binding characteristics of the column. M n+ Oxidation Reduction M n+ Free metal can be bound and released by exposing the ligand to successive reduction and oxidation cycles. Flow Working Electrodes Counter Electrodes Scale up of the electrochemical reactor to practical size requires consideration of materials, geometry, operating conditions, and overall cost. First Vaporization Stage Second Vaporization Stage One problem with ICP-MS is elements of the same nominal mass (isobaric interference). ETV can be used to separate some problematic elements based on their differing volatilities. Rb and Sr can be separated to remove the isobar at mass 87. The time of flight design is able to offer excellent isotope ratio precision as a result of simultaneous ion extraction from the plasma. However, difficulties have been encountered with ratio accuracy. Factors that cause this and possible fixes are actively being researched. ICP-MS is the cutting edge technology for atomic spectrometry. It can offer part per trillion detection limits, over 5 orders of magnitude of linear response, and works for almost all elements in the periodic table. It uses an inductively coupled plasma (~8,000 K) as the ionization source. Our ICP-MS uses a time of flight system for mass analysis. Though many labs rely on solution nebulization for sample introduction, this is not always the best technique. It can be problematic for some matrices (e.g. salty solutions, organic solutions, and solids or slurries). An alternative is electrothermal vaporization (ETV). This uses a carbon tube to vaporize the sample before introduction to the ICP-MS. Vaporization temperatures of up to 3,000 o C can be achieved in a controlled manner. It can handle a wide variety of sample types, and generally has higher sample introduction efficiency than nebulizers. To ICP-MS Sample Internal Standards for ICP- TOF-MS 238 U/(IS) ΔIP %RSD r = 0.0037 Δmass %RSD r = 0.81 238 U/(IS) A B Graphical Illustration of %RSD By definition, a small change in the ratio between two elements as a condition changes, is indicative of a good analyte-IS pair. The %RSD of these ratios is used as a quantitative measure of internal standard compatibility. Each point on the scatter plots illustrated in the example plot above represents a ratio of 238 U and one of approximately 100 IS considered. Analyte-to-IS mass separation typically offered the strongest and most consistent relationship to %RSD for all conditions. Determining the Relationship Between %RSD and Chemical Properties Objective: Determine what metals are associated with what proteins, and how strongly they are bound. First, proteins are separated in their native (metal-containing) state by using native PAGE. This separates the proteins based on molecular weight. We push the protein towards metal complexation by using a metal-loaded buffer during separation (Le Chatelier’s Principle). Cathode - Anode + M+M+ M+M+ ProteinA M+M+ Blotting Membranes Gel Next, the separated proteins/metals are transferred to functionalized quartz membranes through a process known as electroblotting. The metalloproteins are attracted to the anode, while the positively charged free metal ions are attracted to the cathode. The membranes are ablated with a laser and the metals are detected by ICPMS. FdFd Fluorescence resonance energy transfer (FRET) can be used to determine various characteristics of metal binding. FRET involves the transfer of energy between a fluorescent donor and an acceptor molecule. The efficiency of the energy transfer is dependent on the distance between the molecules, which can be related to their spectroscopic properties. This concept can be utilized for a metal ion sensor. FaFa M n+ A FRET pair (a donor, F d, and an acceptor, F a ) lie on opposite ends of a peptide chain. A metal ion (M n+ ) then comes into contact with the peptide chain which has specific binding characteristics. FdFd FaFa M n+ hvhv FRET hvhv When the metal binds, the peptide will wrap around the metal, decreasing the two fluorophores’ distance and causing the likelihood of FRET to increase.