Research

Biosensors

Analysis of the New Delhi Metallo-β-Lactamase (NDM)-Coding Gene via Biomolecular Logic Gate

利用核酸辨認(sequence-specific)機制發展傳染性病毒(Influenza Virus)及細菌抗藥性(NDM-1)的快速篩檢方法(Rapid Diagnostics)

The rapidly increasing collections of multi-drug resistant pathogens with enhanced capacity toward extended spectra of antibiotics have been posing a serious threat to the public health on a global scale^1-3. Recently, a potent enzyme New Delhi metallo-β-lactamase (NDM), which is capable of hydrolyzing multiple β-lactams including the antibiotics of the last resort, cabapenem^{4, 5}, was identified⁶ and has been reported to exist in various Enterobacteriaceae strains worldwide. Moreover, NDM is encoded in highly transferable genetic fragments and regularly transmitted along with variable resistance determinants of other antibiotics coded by the same plasmid^{7, 8}. All these features underscore the significance of monitoring the spread of encoding gene over bacteria, which facilitates precise medical prescriptions and prevents its further dissemination. Nevertheless, to date, there are few methods^9-11 developed to fast screen the existence of NDM, and none of the aforementioned assays were capable of accurately indicating its potential catalytic activity against antibiotics.

Motivated by need, in this communication we describe a newly strategic scheme, as shown in Scheme 1, for identification of multiple genetic segments locating distantly in a long DNA target. Distinct from the conventional sandwich configuration (designated as Terminus) in which nucleic acids are often captured at the terminus of the thread, the capture probe (M) is herein designed to hybridize with one of the targeted sequences residing at the midsection (I) of the analyte (MT). The other two targeted segments are (II & III) harboured at two sides of the captured position. Upon the two hybridization events of II & III with the FITC- and biotin-tagged reporter probes (F & B) respectively, the tagged molecules are recognized by enzyme complexes, horseradish peroxidase conjugated with anti-FITC antibody (α-FITC-HRP) and glucose oxidase linked with avidin (av-GOx). As a consequence, an enzymatic cascade reaction occurs upon addition of the substrate solution containing D-(+)-glucose (Glu) and 3,3’,5,5’-Tetramethylbenzidine (TMB(red)). Specifically, the bound GOx catalyzes the oxidation of glucose to generate H₂O₂, which can subsequently be reduced by HRP to produce oxidized TMB (TMB(ox)). This scheme configures an equivalent circuit composing of a parallel identity and AND gate, in connection to a series of AND gate (Fig. S1).

Sub-typing of Influenza Virus based on a Binary-Sequence Unzipped Molecular Beacon

Along our developing route to exploit new strategy aiming fast and high-fidelity diagnostics of multiple DNA targets, we developed an intelligent strategy based on molecular beacons (MBs), in cooperation with an assisting arming strand, to logically process dual inputs of hemagglutinin (HA) and neuraminidase (NA) in a AND gate format. Notably, the prerequisite for opening the designed MB in our study was distinct from conventional configurations reported in “1 input- 1 output fashion”, enabling this strategy suitable for high-fidelity binary-input determination. Systematic insights on the thermodynamic and kinetic parameters were also evaluated to facilitate appropriate designs of MB and the arming strand.

Bioelectrochemistry

氧化及還原態細胞色素(Cytochrome c)和粒腺體心脂質(Cardiolipin)交互作用之探討

Cytochrome c (cyt c), a protein inherent with a redox-centered heme in ferri/ferro states, is best known to carry electron from complex III-to-complex IV in respiratory process. A particular interest is raised recently, revealing that cytochrome c is able to function as a peroxidase upon binding to the mitochondrial glycerophospholipid cardiolipin (CL). The peroxidase activity promotes CL oxidation which is referred as an early step towards apoptosis. However the exact mechanism leading to apoptosis remains controversial. To this end, we uniquely perform electrochemical techniques to interrogate the interaction between cyt c and CL in connection to redox switch of cyt c.

Electrocatalysis

Synthesis and film formation of electroactive IrOx×nH₂O colloid/film towards effective catalysis for diverse substances

合成及分析具高催化活性奈米氧化銥膠體(IrOx colloids)於電解水及生化感測的應用

Converting solar energy to a usable form that can be stored and transported has attracted tremendous attention/interest in recent decade. Of various approach, developing light-driven water splitting system was deemed as one of efficient means as water is the most preferred source to offer protons and electrons. However, water-oxidation reaction requires moderate thermodynamic energy (Gibbs free energy of 237.17 kJ/mol), indicating a sluggish electron transfer kinetics even at high overpotential. This necessitates an efficient catalyst, in a consequence, to inspire an underway search worldwide to function in high turnover frequency (TOF) at low overpotential (h).

We report facile methods which are capable of efficiently forming electroactive IrOx×nH₂O film on a variety of electrodes towards effective catalysis of diverse substances (e.g., oxidation of water, reduction of H₂O₂ and O₂). By performing dual-potential pulsed amperometry (DPPA) to intermittently eliminate O₂ gas evolution through the electro-flocculation, the IrOx×nH₂O colloidal nanostructured electrode exhibiting improved stability and enhanced kinetics to OER was exploited.

Our Collaborators

Dr. Yaw-Kuen Li (李耀坤) Dept. Applied Chemistry, National Chiao Tung University

Dr. Dmitry Kolpashchikov Dept. Chemistry, University of Central Florida

Dr. Horng Yunn Dou (杜鴻運) Institute of Infectious Diseases and Vaccinology, National Health Research Institutes

Dr. Yu-Chieh Liao (廖玉潔) Institute of Population Health Sciences, National Health Research Institutes

Dr. Yuan-Hao Hsu (許員豪) Dept. Chemistry, Tunghai University

Prior Collaborators

Dr. Yukishige Kondo (近藤行成) Dept. Industrial Chemistry, Tokyo University of Science

Dr. Ja-an Annie Ho (何佳安) Dept. Biological Sci. and Tech., National Taiwan University

Dr. Jung-Jung Mu (慕蓉蓉)Center for Research, Diagnostics and Vaccine Development, Centers for Disease Control, Taiwan

Dr. You-Yin Chen(陳右穎)Dept. Biomedical Eng., National Yang-Ming University

Dr. Chi-How Peng (彭之皓) Dept. Chemistry, National Tsing Hua University