Browsing by Author "Chang, Ko-Shing"

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Novel Steroid-Sensing Model and Characterization of Protein Interactions Based on Fluorescence Anisotropy Decay
(2010-04-01)
Chang, Ko-Shing
;
Luo, Liyang
;
Chang, Chih-Wei
;
Huang, Yen-Chieh
;
Cheng, Chih-Yu
Intramolecular binding of a ligand with an alkyl link, (-CH(2))(3), covalently bound to a residue near the active site of the protein forms a novel steroid-sensing model. A genetically engineered Delta(5)-3-ketosteroid isomerase (KSI) was designed to conjugate uniquely with this ligand at its Cys-86 through the formation of a disulfide bond. The steady-state protein-ligand binding, mediated by hydrophobic interactions, was confirmed with fluorescence spectra, and the fluorophore-labeled peptide sequence was identified with tandem mass spectra. A comparison of steady-state fluorescence spectra of various fluorophore-labeled KS1 mutants reveals that the emission characteristics vary with environmental factors. An evaluation of the decay of the fluorescence anisotropy of the fluorophore indicates the existence of an intramolecular protein-ligand binding interaction. The measurement of time-resolved fluorescence anisotropy of various protein-ligand complexes yielded values of anisotropy decay representing the degrees of freedom of the fluorophore related to its location, inside or Outside the steroid-binding domain. When 19-norandrostenedione (19-NA) was added to this protein-ligand system, competitive binding between the ligand and the steroid was observed; this finding confirms the feasibility of the design of steroid detection with engineered KSI. On integration of this protein-ligand system with a silicon-based nanodevice (a p-type field-effect transistor with an Ultrathin body), it noncharged steroid, 19-NA, became detectable at it micromolar level (Biosens. Bioelectron. 2008, 23, 1883).
SENSING ELEMENT, MANUFACTURING METHOD THEREOF, AND BIOLOGICAL DETECTION SYSTEM EMPLOYING SUCH SENSING ELEMENT
(2010-03-25)
Sheu, Jeng-Tzong
;
Chen, Chen-Chia
;
Li, Yaw-Kuen
;
Chang, Ko-Shing
A sensing element includes a field-effect transistor (FET) with an ultra-thin channel, a reference electrode, a first and a second passivation layer, and a microchannel. The first and the second passivation layer enclose a first and a second portion of the FET, respectively. The microchannel is bonded to the first and the second passivation layer, such that the microchannel is extended across the channel of the ultra-thin channel FET. The ultra-thin channel has a chemically or physically modified surface. When an analyte to be tested passes through the microchannel and is in contact with the modified surface of the ultra-thin channel, it results in changes in the conductance of the ultra-thin channel FET. Trace detection may be conducted on the analyte by observing changes in the conductance. A method for manufacturing the sensing element and a biological detection system employing the sensing element are also provided.