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Edward S. Yeung
The central theme of Dr. Yeung's program is the identification, development, evaluation, and application of new measurement concepts through an in-depth understanding of the associated fundamental chemical and physical principles. He recognizes that chemical analysis does not stop at just providing measurements, but must be directly involved in the development of science as a whole. To this end, his research is stimulated by selected, urgent problems in biology, medicine and materials, for which the proper analytical methodology is the key to their solutions.
Single Molecule Detection and Characterization
Dr. Yeung reached the ultimate level in microscale separations -- the isolation, manipulation and electrophoretic separation of a single molecule (227,319,336,365). This is possible by preparing an ultradilute solution (10-17 M) of the molecules and filling a narrow capillary tube together with the appropriate reaction substrates. When allowed to incubate, these molecules catalyze the conversion of the non-fluorescent substrates to generate thousands of fluorescent product molecules in the discrete zones where the molecules reside. A ground-breaking observation is that for the particular enzyme studied, each molecule can exhibit a different catalytic activity as a result of the many distinct tertiary structures created during folding and refolding (269,370,375,396,410). This promises to impact molecular modeling, drug design, and catalytic studies. This work was cited in C&E News, March 20, 1995, p. 38, and October 20, 1997, p. 37.
By using a novel laser-imaging system, Dr. Yeung can now follow the diffusional motion of individual fluorescent species in solution. The diffusion coefficient as well as the photochemical decomposition rate can be measured directly. This is the first demonstration of the determination of individual single-step rate constants in solution (262,286). He found that motion in a confined space can be very different compared to bulk diffusion. At chromatographic surfaces, proteins are simply trapped in the adjacent liquid layer rather than totally immobilized on the surface (276). These results represent the final frontier for sensitive detection and reveal unprecedented details about elementary reactions in solution and at surfaces. This was highlighted in C&E News, March 18, 1996, p. 33-34, and February 24, 1997, p. 10. The results also provide unprecedented insight into bulk separation processes (322,340,344,348,350,354,364,373,374,379,381,383,386,388,394, 398,399,420). Brand new separation modes were demonstrated based on these new insights (255,354,357,361).
In electrophoresis, the migration velocity is used for sizing DNA and proteins or for distinguishing molecules based on charge and hydrodynamic radius. Dr. Yeung demonstrated a high-throughput imaging approach that allows determination of the individual electrophoretic mobilities of many molecules at a time (up to 100,000 per second). This opens up the possibility of screening DNA or proteins within single biological cells for disease markers without performing polymerase chain reaction (304,308,309,317,341,343,349,352,362,389,391,401,416). Even spectra can be recorded for each molecule or nanoparticle (315,387,400,408,409,414,415,416).
Single Cell Analysis
The development of analytical techniques suitable for extremely small samples has been a major emphasis of Dr. Yeung's research (225,246,274,283,369,376). He was first to reach a major milestone -- theanalysis of the chemical contents of the fluid inside a single red blood cell (180). By applying the indirect fluorescence scheme he developed (113,114,122,123,129,133,139,141,144, 146,147,148,185,199,233), any ionic species became amenable to detection down to the 50 attomole range. Na and K ions can then be separated by capillary electrophoresis and detected by charge displacement with a cationic fluorophore upon excitation by a laser beam (219,224). Many questions of fundamental interest to biology can be studied with this technique, such as lactate/pyruvate correlations with aging (207), ATP/ADP as indicators of metabolism, ion transport across cell membranes, the variability of ionic concentrations among individual cells, and the role of small peptides in cell regulation. It may even be possible one day to identify chemical markers in diseased or cancer cells, long before pathological changes can be observed, and to perform pharmacokinetic ordrug efficacy studies on a single cell basis (221,226).
Dr. Yeung used another brand new strategy -- the detection of native fluorescence (175,196,222, 229,249,363,378) -- to perform the first separation and simultaneous measurement of various proteins in individual erythrocytes (189,223,237,251). Because no special pretreatment is involved and because laser excitation provides excellent signal-to-noise ratios, reliable quantitation ispossible. The results support the hypothesis that aging of the cells is responsible for gradual degradation of the proteins and the increased susceptibility to oxidative damage. This body of work was cited in C&E News, April 20, 1992, p. 29. Most recently, a miniaturized version of 2D-gel electrophoresis was developed for ultrasensitive protein mapping (358,372,384,403).
Dr. Yeung also used a high-power pulsed laser to irradiate each cell and literally vaporize it (202,211). The plasma produces atomic emission that allows the simultaneous determination of Na and K in single cells. This has been extended to the study of plant cells (368). In two most impressive developments (212,217,228), Dr. Yeung was able to quantify individual enzymes within single erythrocytes by a novel fluorescence enzyme assay and a unique particle-counting immunoassay. The detection limit of each is 800 molecules. The lactate dehydrogenase isoenzyme forms are individually quantified, opening the door for cancer diagnosis based on the ratios of these enzyme activities (243). This effort was cited in C&E News, March 28, 1994, p.42.
Dr. Yeung is the first to use capillary electrophoresis (CE) for following a dynamic event in a single cell (253,254,261,291). By continuously injecting over a long time, the changing chemical composition is encoded along the length of the capillary tube. The small inner diameter limits diffusion so that the temporal dependence can be read out later on in the form of an electropherogram (254,280). Individual release events of attomolar quantities from sub-femtoliter vesicles can be recorded. This represents the smallest real samples ever studied by CE. He further developed novel imaging methods for monitoring cellular processes (234,256,264,271,273,281, 286,290,295,328,366,367,382,385,393). Potential applications include the development and evaluation of pharmaceuticals and of industrial catalysts (327,332,334,392). This development was cited in C&E News, March 18, 1996, p. 5 and p.33. A brand new development is the real-time imaging of ATP in cells (303,359,360,371,395,402,411). This elusive but important molecule can be followed for the first time. This was cited in C&E News, January 29, 2001, p. 32-33. Novel sub-diffraction microscopies have also been developed (404,407,412,419).
High Speed DNA Sequencing
Separation by CE has shown good promise for substantially speeding up DNA sequencing. Dr. Yeung discovered several brand new separation media for DNA separation that is much faster and easier to make reproducibly (230,236,252,257,263,265,266,272,275,297,299,302,316,321). He also devised a unique separation scheme for larger DNAs in CE without a sieving medium (255). To gain another two tothree orders of magnitude in speed, some sort of multiplexing (parallel processing) scheme is clearly in order. Dr.Yeung developed an axial-beam excitation scheme for capillary electrophoresis (178). He used line focusing excitation geometry to simultaneously monitor 100 capillary tubes undergoing electrophoresis (191,215,231,284,285,292). Truly simultaneous multiplexing of capillary electrophoresis isthus achieved because the CCD (232) camera looks atall capillaries at all times, with data rates (240,248,301) fast enough for sequencing at > 1 base per s per lane for up to 1000 lanes, or the entire human genome in 35 days. This development was cited in C&E News, March 29, 1993, p.28, and in C&E News, July24, 1995, p.37.
While multiple capillary arrays have been demonstrated by Dr. Yeung and others to greatly facilitate DNA sequencing, for a long time people have been concerned about how to supply the samples fast enough to take advantage of multiple arrays. Dr. Yeung now has a fully integrated sample-preparation-cleanup-sequencing system in operation (259,279,298). Since everything is done on-line in connected capillary tubes, it should be possible to make this highly multiplexed as well with minimal amounts of sample (277,310,312,318,324,346,347,351,355). He is able to start with a drop of blood and derive a DNA fingerprint (genotype) automatically in a total of two hours (270,288). He was even able to produce DNA sequence from only a single bacterial colony (296) and detect mutations without knowing the detailed sequence (306,326). Even protein crystallization can benefit from this multiple capillary approach (377).
Although multiple capillary instrumentation is making a major impact on DNA sequencing and genotyping, Dr. Yeung successfully developed concepts for small molecule analysis with high throughput based on fluorescence (293,323,330,331,356) or absorption (294,329). This advance comes just in time to take on the analytical challenges of combinatorial synthesis. Since absorption at 214 nm is essentially a universal detector for CE, rugged and inexpensive instrumentation is therefore on the horizon for genotyping (300,320,326), peptide mapping (307,333,335,338,339), drug screening (311,337,345) and combinatorial analysis (313,342).
Laser Vaporization and Desorption
Laser-initiated microscopic events, including vaporization and desorption from surfaces and vapor deposition to surfaces, share the common problem of fluctuations in laser and surface properties from one pulse to the next. Dr. Yeung developed two new "total material" detectors to independently monitor these processes on a single shot basis based on refractive index gradients (117) and the acoustic wave (127) generated by the released material. This allowed the first studies of extremely dense laser plasmas by enhanced ionization (142,150) or reflection (155), where Dr.Yeung observed world-record spectral linewidths for atoms, up to 10nm or the equivalent of 1000 atmospheres of pressure. One can thus study, for the first time, the contributions of various reaction channels to the overall deposition process (166). Impressive results and new insights were obtained for thedeposition of carbon films (192,214) and for the study of matrix-assisted laser desorption (184,210,216,241,242,268,282,287). This body of work has been cited in C&E News, October 9, 1989, p.21-23. This also formed the basis of a new way of introducing micro-scale samples into the mass spectrometer by direct laser vaporization/ionization of the liquid (260). Even single mammalian cells can be studied in this way (278). Dr. Yeung also developed new additives for LDI that allow hydrophobic plant tissues to be imaged directly (368,390,397,405,406,417).
Laser Measurements near the Shot-Noise Limit
While lasers have been found useful for sensitive measurements of trace materials by providing larger analytical signals, such as in laser-excited fluorescence, they have not been shown tobe beneficial when relative intensities are monitored, such as in absorption, because intensity noise inherent to laser sources remains to be a limiting factor. Dr. Yeung developed a unique double-beam laser absorption detection scheme for capillary electrophoresis, producing a 25-fold improvement of detection limit over commercial systems (193,239). Dr. Yeung has been able to create a new universal detector for capillary electrophoresis based on indirect absorption (206). Even the liquid chromatographic schemes developed by Dr. Yeung (36, cited in C&E News, August 18, 1980, p. 24, and 54, cited in C&E News, October 10, 1983, p. 24) can be further improved by using this scheme, including absorption (203,213), thermal lens (204), and circular dichroism (209). In the future, absorption probes for laser-generated plumes (109,116,163) and for plasmas can be improved along similar lines. This accomplishment was highlighted in C&E News, April 12, 1993, p. 38.