2020-Now:    Full Professor, National Tsing Hua University, Taiwan.

2016-2020:    Associate Professor, National Tsing Hua University, Taiwan.

2011-2016:      Assistant Professor, National Tsing Hua University, Taiwan.

2007-2011:     Postdoc Research Fellow, Swiss Federal Institute of Technology (EPFL), Lausanne, Switzerland.

2001-2003:    Research Assistant, National University of Singapore.

New type of chemical probes

Chemical probes are important tools in basic biology research and medical diagnosis because they allow for sensitive, simple and specific detection of target molecules in complex environments, such as cell lysates, living cells, and in vivo. Currently, most of the chemical probes are reaction-based and designed for monitoring enzyme activities and reactive small molecules using fluorescence detection technique. Based on the fluorescence turn-on mechanism, they can be divided into two classes, dye-based fluorescent probe and caged-luciferin.

Recently, our lab has developed a new type of chemical probe based on the controlled binding of streptavidin and biotin. The rationale behind this new concept is based on the fact that biotin has extremely high binding affinity (Kd = 10−14 M) with streptavidin, while chemical modification at N’-1 urea nitrogen of biotin to form caged-biotin can dramatically reduce its streptavidin binding affinity (Kd ≈ 10−5 M). In the absence of the target analyte, the caged-biotin probe on the cell surface would not be able to bind with fluorophore conjugated streptavidin due to the low binding affinity of caged-biotin with streptavidin. The fluorophore conjugatedstreptavidin can then be washed away to eliminate any background fluorescence. In the presence of the target analyte to trigger biotin uncaging, fluorophore conjugated streptavidin would bind to the biotin probe. As there are multiple fluorophore units on one streptavidin molecule, significant signal amplification can be achieved. Furthermore, streptavidin conjugated with different bright fluorophores, such as Cy5, Cy3 or Alexa488 can be added on demand to generate the desired fluorescent signals. Thus, the detection is no longer restricted by the type of fluorescent dyes, such as in the case of fluorescent probes.

Near-infrared fluorescent switchable dyes

Our lab is also interested in developing new type of near-infrared (IR) fluorescent switchable dyes. Fluorescent dyes are a rapidly expanding area of research in chemical and biological sciences with multiple applications as biomolecule labels, enzyme substrates, environmental indicators and cellular stains. Fluorescent dyes that are excited and emit in the near-IR region are especially biocompatible and advantageous as they cause minimum damage to biological samples, have deep tissue penetration, and come under minimal interference from background auto-fluorescence by biomolecules in the living systems. Currently, most near-IR dyes are based on the cyanine and rhodamine scaffolds. Conventional strategies to obtain cyanine and rhodamine derivatives with appreciable bathochromic shift covering near-IR spectra include extending the p-conjugation and introducing heteroatoms and rigid bridges.

Recently, we have introduced a novel near-IR fluorescence switchable merocyanine dye that can be coupled with different protein ligands to interact with non-enzymatic proteins for the rapid fluorescence turn-on labeling and imaging in living cells and in vivo. In contrast to the typical p-conjugation extension approach, our dye achieves bathochromic shift by the formation of an unusual S-cis diene conformer. Although bathochromic shifts due to S-cis conformation was first reported by Woodward several decades ago and are well documented in many literatures, the application of this conformation in fluorescent dyes has not been demonstrated. This is the first time that a stable S-cis conformation has been successfully identified in a near-IR dye. In addition to the novel bathochromic shift mechanism, our new dye also exhibits fluorescence-switchable properties in response to polarity and viscosity. When different protein specific ligands were conjugated to the dye, the probes show a dramatic increase in fluorescence (up to 300-fold) in the presence of target proteins (SNAP-tag and hCAII proteins).