research

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by division

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analytical division

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INTRODUCTION

This brief overview has sections on Bioanalytical Chemistry, Chemical Separations, Magnetic Resonance, Sensors and Molecular Recognition, and Electrochemistry.

BIOANALYTICAL

Several groups are involved in bioanalytical chemistry. For information on the investigation of biomacromolecular structure and function, take a look at the pages on Raman spectroscopy, probe microscopy and magnetic resonance.

The Michael group is developing microanalytical strategies for monitoring chemical events in the extracellular space of brain tissue. Attention is mainly focused on neurotransmitters, substances that carry chemical messages between the cells of the brain, but there is also interest in substances related to metabolism and energy production, e.g. glucose, lactate and oxygen. The group has developed and used various electrochemical microsensors constructed from carbon fibers. The figure at left, for example, is a scanning electron micrograph of a glutamate microsensor. The small fiber, which itself is not visible, is coated with a polymer gel that entraps three different types of enzymes that work together to generate an electrochemical glutamate oxidation current proportional to the concentration of glutamate in the surrounding tissue. The sensor is highly selective for glutamate, which is one of the mostly widely distributed neurotransmitters in the mammalian brain, and has made it possible for the first time to monitor extracellular glutamate in real time in living brain tissue.

The Weber group is developing methods for peptide determinations following separation that can be applied broadly to peptides at low concentrations in biological samples. One approach, applicable to the majority of peptides known in, for example, mammalian brain, is n in the figure. A two-working electrode cell is used to take advantage of the reversibility of the Cu(II)-peptide complex oxidation reaction.

In related work, the Weber group is using fluorogenic oxidants to create a fluorescent signal from redox activity. The application of this principle to the tyrosine- (Y) and tryptophan- (W) containing fragments of dynorphin A is shown in the figure. A fluorogenic oxidant is combined with the effluent from the HPLC to yield fluorescent zones where there was an oxidizable analyte.

CHEMICAL SEPARATIONS

The Weber group is developing selective extractions, which are described on the Sensors page. They are also working on novel ways to separate neutral organic molecules in nonaqueous solvents by capillary electrophoresis. This may have application in the analysis of synthetic mixtures, especially those that result from high throughput approaches in which the amount of material is small and the compounds are similar. The figure shows a separation of several similar neutral organic molecules based on their differential binding to lanthanum. The +3 lanthanum ion migrates in a direction opposite to the electroosmotic flow, so molecules that bind strongly to La³⁺ have longer migration times. Also, the resolution is to some degree controlled by the La³⁺ concentration. A higher concentration increases solute binding, and slows the solute down (compare the left separation at low [La³⁺] to the right separation at high [La³⁺])

ANALYTICAL MICROSCOPY

Analytical Microscopy reveals the workings of cells, the properties of man-made materials, and in some case even aids in the synthesis of next generation devices. Work in this area is pursued by several faculty members. For example, Professor Amemiya uses electrochemical scanning probe microscopy to analyze how polynucleotides transfer across membranes. The Asher group uses resonance Raman microscopy to study of composition of biological tissue and cells. Waldeck applies scanning probe microscopy to examine the formation of self-assembled monolayers. Finally, the Walker group is developing new scanning probe microscope techniques that break the diffraction limit, for polymers analysis and single molecule microscopy.

MAGNETIC RESONANCE

Research in Sunil Saxena’s group involves the development of FT-ESR methods to provide a general framework for identifying global folding patterns in proteins and studying conformational dynamics in proteins. One strategy is to measure the functionally relevant fluctuations in separations between protein-domains. Whereas the measurement of 10-20Å is fairly routine, new methods are required for monitoring large amplitude changes in protein structures. To this end we develop novel instrumentation and methodologies at conventional and at low fields. These include experiments that measure the simultaneous flips (multiple quantum spectroscopy) of coupled electron spins and/or extended pulse sequences that project out the distance containing information onto additional spectral dimensions (two dimensional spectroscopy). The design of such experiments is directly linked to an understanding of the quantum spin dynamics of coupled systems-a parallel interest of the group. These methods promise an approach to the characterization of inter-spin distances in the 5-80Å range.

State-of-the-art two dimensional correlation and exchange experiments are used to further delineate the physical mechanisms and timescales of these biologically important dynamical modes. The strategy is to use additional spectral dimensions to separate out relaxation and spectroscopic information that relates to motion. Quantitative details on the timescales of these processes are obtained by comparing experimental lineshapes with those predicted by theoretical models. These are then used to determine protein structures and provide insights into the relationship of microscopic dynamics to the protein functions.

SENSORS AND MOLECULAR RECOGNITION

The Analytical group has a remarkable variety of intense activity in the sensors area. This work is destined to become part of the fabric of our every day lives as more and more sensors become commercially available. The future will see sensors for medical conditions, both chronic and acute, food freshness, air and water safety, product reliability, soil condition and more.

The Amemiya group studies electrochemical sensors based on molecular recognition at liquid/liquid interfaces. A receptor (an ionophore) molecule selectively facilitates interfacial transfer of an analyte ion from a sample solution into a sensor membrane. This process can be directly detected as an electrical signal, i.e., potential and current, allowing for simple construction of ion sensors, so called potentiometric and voltammetric ion-selective electrodes (ISEs), respectively. The projects are directed toward (1) miniaturization of the sensors for biomembrane studies and (2) improving and understanding their response properties for practical analysis.

One avenue toward better enzyme-based sensors is to tether an enzyme specifically to an electrode surface. The Waldeck group uses electrochemical methods to demonstrate that cytochrome c can be immobilized on gold electrodes that are coated with self-assembled monolayers of 4-pyridinyl-COO-(CH2)n-S (n > 6) through interaction between the pyridine terminal unit and the heme of the cytochrome. Raman and NMR studies have confirmed that the axial ligand (Met-80) of the cytochrome’s heme can be displaced by an extrinsic ligand, such as CN-, imidazole, or pyridine. This opens the avenue to apply this chemistry to other tethers whose terminal functionality can selectively bind a biomolecule.

The discovery of a novel sensor mechanism - a new way to transduce chemical concentration into an observable signal - is a rare and exciting event. Prof. Asher and his group have discovered and elaborated a new sensory mechanism based on crystalline colloidal arrays. One such material, derivatized with a crown ether that binds to Pb²⁺, responds to Pb²⁺ in water with a visible color change.

Often, speed is one of the key advantages of sensors over more instrument-intensive analytical tools. With a senor that responds rapidly to its target analyte, it becomes possible to track real-time changes in the concentration of that substance. The figure at left is such an example. Here, neurons in the brain were stimulated for 10 seconds to cause dopamine release in a brain structure called the striatum. Simultaneously, both the stimulated dopamine release and its subsequent clearance from the extracellular space were monitored with a voltammetric microelectrode. The entire event lasted less than 20 seconds. Without a sensor capable of responding rapidly and real-time, such events cannot be observed. Experiments in the brain with these sensors are contributing to our understanding of brain disorders including Parkinson’s disease, schizophrenia and substance abuse.

All sensors operate based on molecular recognition. The very same molecular recognition can also be used to accomplish selective extractions. The Weber group is especially interested in microextractions that can be used for the selective extraction and preconcentration of bioactive species before their separation and determination by capillary electrophoresis. A derivative of the barbiturate receptor of Hamilton (shown complexed to phenobarbital) is the key factor in a polymer-based extraction. Thin polymer films can preconcentrate barbiturates in excess of 50-fold from microliter-sized samples allowing for analysis by capillary electrophoresis (CE). The figure shows CE separations of extracts of samples containing 10 barbiturates, 8 of which (*) bind strongly to the receptor, and two of which (arrows) do not. A comparison to extracts without the receptor (CE on the bottom) reveals the extraction selectivity: molecules that bind to the receptor have higher peaks.

ELECTROCHEMISTRY

As you have seen from the foregoing, several groups are working on electrochemistry. The Amemiya group employs scanning electrochemical microscopy and ion-transfer voltammetry, the Michael group uses microelectrodes and fast scan voltammetry, the Waldeck group creates self-assembled monolayers on gold electrodes and measures their properties, the Weber group has had a hand in developing and now uses routinely electrochemical detection as well as turning to rotating disc voltammetry to work out mechanistic questions.