News
Identifying multiple compounds from single neurons One of the challenges in characterizing neurons has been to simultaneously analyze multiple biologically active compounds. Analytical techniques have had to sacrifice either qualitative or quantitative information. Jonathan Sweedler and coworkers at the University of Illinois, UrbanaChampaign, combine CE with a wavelength-resolved laser-induced native fluorescence detection system to directly analyze aromatic monoamines and other compounds (more than 30 total) from single neurons of the mollusks .A/>/vsia californica and Pleurobranchaea californica. The analytes are identified by correlating CE migration times and the complete fluorescence emission spectra with those of standards. The correlation prevents the misi dentification of analytes with similar migration times, such as 5-hydroxytryptamine (serotonin) and tryptamine. They successfully identified, as interferences, compounds with fluorescence emission spectra that were shifted by as little as 5-10 nm. The detection limits for the cellular components were in the attomole to femtomole range. The analyses of standard mixtures and multiple injections of identified neurons were highly reproducible. The researchers use the technique to demonstrate how histochemically similar cells may actually appear very different in a single-cell assay, either in terms of concentrations or chemical components. They also compare homologous neurons of different species. (Neuron 1998,20,173-81)
Comparison of serotonergic metacerebral cells from (A) P. .clifornica and (B) A. californica and (C) a dorsal white eell from P. californica. (Adapted wiih permission. Copyright t198 Cell Press.) 172 A
Glucose detection in the FAST lane
(MBP) oiE. coli, based on changes in conformation that occur upon ligand binding. Because the glucose/galactose binding protein (GBP) is structurally related to MBP, an analogous approach was used to design FAST sites into GBP proteins. Engineered GBP could be the basis for a new class of fluorescent glucose sensors that may eliminate some of the problems associated with conventional glucose sensors, which rely on electrochemical detection of glucose oxidase. Electrode fouling, variation in oxygen levels, and the presence of inhibitors in the blood are just a few examples of the problems this approach could overcome. (J. Am. Chem. Socc.198,120, 7-11)
Biosensors have traditionally relied on naturally occurring enzymes or antibodies to provide specificity for a particular analyte. The problem is that natural proteins do not lend themselves to simple signal-transduction mechanisms, making it a challenge to develop instrumentation that is specific to the properties of the desired protein. Homme W. Hellinga and Jonathan S. Marvin of Duke University Medical Center have approached the problem from a new angle. Rather than trying to alter existing detection technologies they have genetically engineered proteins in such a way that the signal transduction functions directly integrated into them Fluorophores are sitespecifically introduced into a protein through various synthesis schemes. Changing the fluorescent reporter groups that respond to ligand binding changes the optical properties, which allows the authors to investigate various detection strategies. They have previously engineered integrated fluorescent allosteric signal transducer (FAST) functions Closed forms of MBP and GBP. Numbered spheres in the maltose-binding protein indicate attachment sites of fluorophores.
Native chromophores get excited Many biological fluorophores, including amino acids, neurotransmitters (melatonin and serotonin), and redox cofactors, such as flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NADH), have diverse emission spectra, making analysis of mixtures by single-photon (UVexcited) detection difficult. Alternatively, several laser lines could be used to excite these species; however, increased background and substantial overlap of emission and excitation spectra put constraints on detection capabilities when multiple wavelengths are involved. Jason B. Shear and co-workers at the University of Texas, Austin, have overcome these spectroscopic limitations and have developed a new approach for analyzing
Analytical Chemistry News & &eatures, March 1, 1998
complex biological mixtures. The method involves fractionation of native fluorophores by CE, followed by excitation of them by a minimum of two near-IR photons. All species are excited by a single, long-wavelength source, which eliminates problems with spectral overlap. The ability of CE coupled to multiphoton-excited (MPE) fluorescence detection to fractionate and detect individual components in a complex biological sample is demonstrated with a diluted baker's yeast homogenate. Lower detection limits of 110 nM for NADH and 38 nM for FAD are reported, compared with 1.4 uM for serotonin and 550 nM for melatonin. Photostability is found to be the primary factor limiting detectability. FAD and NADH are thought to be more photostable than melatonin and serotonin allowing them to be detected at lower concentrations.
