Research

Conducting DNA nanowire/nanodevice development

DNA is one of the most promising one-dimensional nanomaterials because of its adjustable length and self-assembly properties. It may be useful as a one-dimensional nanowire or nanodevice in electronic applications; however, it is of great interest to reveal the electrical conduction mechanism of DNA. The conductivity of DNA increases dramatically when it is doped with Ni ions to form Ni-DNA (M-DNA). Our studies demonstrate that Ni-DNA is a conducting nanowire. The the charges in Ni2+-doped DNA are transported through the Ni2+-mediated p-p stacking corridor.

Native bio-nanoparticle drug delivery system development

For hydrophobic drug delivery, it is important to consider the physicochemical properties of macromolecules, cell uptake, and the usage of the delivery system. A good hydrophobic drug delivery system (DDS) with native-like physicochemical properties and good cell permeability is highly desired. Modified natural transporter/protein would be an excellent candidate for DDS.

Low-density lipoprotein (LDL) is a native nanoparticle and its size is approximately 18–25 nm. Plasma LDL can be cleared from circulation through LDL receptor (LDLR)-mediated endocytosis. This allows for the transfer of LDL into endosomes, where the pH drops, causing LDL to dissociate from the receptor. The receptor is then recycled to the surface of the cell, while LDL is transferred into the lysosomes for degradation. This pathway could be useful for drug delivery, and may then be directed toward tumors expressing LDLR. It has been demonstrated that LDLR is overexpressed in various human cancer cell lines. A LDL-based hydrophobic/amphiphilic DDS can be internalized and degraded by regular cell metabolism, and these properties can be used in targeted therapies for cancer. In our previous study indicated that the reconstituted apoB lipoparticle (rABL) is an ideal carrier for transporting hydrophobic and amphiphilic compounds.

Ni-DNA nanowire device

DNA is an one-dimensional nanomaterials with the properties of self-assembly and length controllable by bio-synthesis. The electrical property of Ni-DNA has been characterized via AFM, demonstrating that Ni-DNA acts as a conducting nanowire. It has also been demonstrated that Ni-DNA acts like a conducting wire with an insulated shell to prevent intermolecular electron leakage. Furthermore, the device with the Ni2+-chelated DNA (Ni-DNA) connecting two gold electrodes, exhibited a negative differential resistance (NDR) behavior, when the measurement of current response in a cyclic-voltage scan (I-V) was performed under an ambient and water-free condition. Each Ni ion in Ni-DNA exhibits low (Ni2+) or high (Ni3+) oxidation state and can be switched sequentially by applying bias voltage with different polarities and writing times. The ratio of low and high oxidation states of Ni ions in Ni-DNA represents a programmable multi-state memory system with an added capacitive component, in which multi-state information can be written, read, and erased. This study also indicates the biomolecules-based self-organized nanostructure can be used as a template for nanodevices fabrication.

Ultra-sensitive biosensor platform development

1. Rapid and highly sensitive detection of Enterovirus 71 by using nanogoldenhanced electrochemical impedance spectroscopy

Enterovirus 71 (EV71) infection is an emerging infectious disease causing neurological complications and/or death within two to three days after the development of fever and rash. A low viral titre in clinical specimens makes the detection of EV71 difficult. Conventional approaches for detecting EV71 are time consuming, poorly sensitive, or complicated, and cannot be used effectively for clinical diagnosis. Furthermore, EV71 and Coxsackie virus A16 (CA16) may cross react in conventional assays. Therefore, a rapid, highly sensitive, specific, and user-friendly test is needed. We developed an EV71-specific nanogold-modified working electrode for electrochemical impedance spectroscopy in the detection of EV71. Our results show that EV71 can be distinguished from CA16, Herpes simplex virus, and lysozyme, with the modified nanogold electrode being able to detect EV71 in concentrations as low as 1 copy number/50 ml reaction volume, and the duration between sample preparation and detection being 11 min. This detection platform may have the potential for use in point-of-care diagnostics.

2. Human heptoglobin typing and concentration determination

Haptoglobin (Hp) is an acute phase protein that binds free hemoglobin (Hb), preventing Hb-induced oxidative damage in the vascular system. There are three phenotypes in Human Hp, which heterogeneous polymorphic structures and varying concentrations in plasma have been attributed to the cause of diseases, and outcome of clinical treatments. Different phenotypes of Hp may compose of the same subunits but different copy numbers, rendering their determination difficult by a single procedure. In this study, we have developed a simple, fast, reliable, and sensitive method, using label-free nanogold modified bioprobes coupled with self-development electrochemical impedance spectroscopy (EIS). By this method, probe surface charge transfer resistance is detected. The relative charge transfer resistance ratios for Hp 1-1, Hp 2-1, and Hp 2-2 were characterized. We were able to determine protein size difference within 3 nm, and the linear region of the calibration curve for Hp levels in the range of 90 pg/ml and 90 mg/ml (~1fM to 1pM). We surmise that similar approaches can be used to investigate protein polymorphism and altered protein-protein interaction associated with diseases.

• Ultra high sensitive biosensing platform have been developed in my lab.
• This platform can be used to detect infectious, emerging disease on site.
• This platform can be used to detect and quantify the proteins and chemicals.
• This platform can be used for detecting intermolecular interactions.

http://nanotechweb.org/cws/article/lab/46448

Protein folding and mechanism study

Protein is a macromolecule which posses self-organization and self-assembly properties. Protein folds spontaneously in the heterogeneous environment. However, some of them may aggregate with each other during the folding process. Therefore, both folded and aggregated protein molecules can be observed under certain folding condition. These two states, (unfolded/folded), coexist phenomenon indicates that the reaction of protein folding is a “first-order like phase transition” process. By combing experimental approaches and molecular simulation analysis, we can conclude that the protein aggregation process is a competitive process between “spontaneous folding” and “diffusion limited aggregation” reactions. Meanwhile, the protein self-stabilization time can be revealed. Moreover, by manipulating the folding process, room-temperature protein base molecular magnet can be synthesized. The protein selfassembly nano-pattern can be performed on silicon or metal substrate.

Single molecular studies

Tight-Turn Paperclip DNA Triplex in Solution

DNA triplex modulates gene expression by forming stable conformation in  physiological condition. However, it is not feasible to observe this unique molecular  structure of large molecule with 54 oligodeoxynucleotides directly by conventional nuclear magnetic approach. In this study, we observed directly single molecular images of paperclip DNA triplexes formation in a buffer solution of pH 6.0 by atomic force microscopy (AFM). Meanwhile, a diffuse “tail” of unwound DNAwas observed in pH8.0 solution. This designable  approach in visualizing the overall structures and shapes of oligo-DNAs at the single molecular level, by AFM, is applicable to other biopolymers as well.

Nanodiamonds

Nanodiamond ND is surrounded by layers of graphite on its surface. This unique structure feature creates unusual fluorescence spectra, which can be used as an indicator to monitor its surface modification. Meanwhile, the impurity, nitroso CuNvO inside the ND can be photolyzed by two-photon absorption, releasing NO to facilitate the formation of a sp3 diamond structure in the core of ND and transforming it into a sp2 graphite structure. Such a conformational transition enlarges the size of ND from 8 to 90 nm, resulting in a popcornlike structure. This transition reaction may be useful as nanoknives in biomedical application. Applied Physics Letters. 93,033905

Bio-photonics and mechanics

Triplex DNA formation/force analysis

We have detected unambiguously the dynamics of 17-mer DNA triplex dissociation mechanism in molecular level. Technique of fluorescence resonance energy transfer (FRET) has been used as an indicator for inter-molecular interaction in nanometer range, and atomic force microscopy (AFM) is employed to address the single molecule with sub-angstrom precision. By combining these two techniques, the triplex rupturing event is ascertained to involve one or a few triplex DNA molecule only. Meanwhile, the maximum DNA triplex rupture force is found between the pH of 4.12 and 5.00. This molecular mechanism is consistent with the macroscopic observation of NMR studies. These results indicated that the FRET-combining AFM detection system can be used for revealing molecular interaction in nano-meter scale.

Functional bio-nanomaterials development

Targeting nanoblast for cancer therapy by functionalized Nanodiamonds

It is highly desire to develop a therapeutic, observable nanoparticle complex for specifically targeting in cancer therapy. Growth hormone (GH) and its antagonists have been explored as cancer cell targeting molecules for both imaging and therapeutic applications. In this study, a low toxic, biocompatible, therapeutic, and observable GH-nanoparticle complex for specifically targeting growth hormone receptor (GHR) in cancer cells, has been synthesized by conjugating GH with green fluorescence protein and carboxylated nanodiamond. Moreover, we have shown that this complex can be triggered by laser irradiation to create a “nanoblast” and induce cell death in the non-small lung cancer cell line A549 via the apoptotic pathway. This laser-mediated cancer-targeting platform can be wildly used in cancer therapy.

Mechanism for ND nanoblast

Nanodiamond (ND) is surrounded by layers of graphite on its surface. This unique structure feature creates unusual fluorescence spectra, which can be used as an indicator to monitor its surface modification. Meanwhile, the impurity, nitroso (C-N=O) inside the ND can be photolyzed by two-photon absorption, releasing NO to facilitate the formation of a sp³ diamond structure in the core of ND and transforming it into a sp² graphite structure. Such a conformational transition enlarges the size of ND from 8 to 90 nm, resulting in a popcornlike structure. This transition reaction may be useful as nanoknives in biomedical application.

Biomaterials synthesis and characterization

First room temperature ferromagnetic protein in the world !!

The paper reports the methods of preparing molecular magnets and patterning of the molecules on a semiconductor surface. A highly magnetically aligned metallothionein containing Mn and Cd (Mn,Cd-MT-2) is first synthesized, and the molecules are then placed into nanopores prepared on silicon (0 0 1) surfaces using electron beam lithography and reactive ion-etching techniques. We have observed the self-assemble growth of the MT molecules on the patterned Si surface such that the MT molecules have grown into rod or ring type three dimensional nanostructures, depending on the patterned nanostructures on the surface. We also provide scanning electron microscopy, atomic force microscopy, and magnetic force microscope studies of the molecular nanostructures. This engineered molecule shows molecular magnetization and is biocompatible with conventional semiconductors. These features make Mn,Cd-MT-2 a good candidate for biological applications and sensing sources of new nanodevices. Using molecular self-assembly and topographical patterning of the semiconductor substrate, we can close the gap between bio-molecules and nanoelectronics built into the semiconductor chip.

Self-assembled molecular magnets on patterned silicon substrates:
Bridging bio-molecules with nanoelectronics

開發鈀奈米薄膜電化學感測平台用於病毒檢測與抑制分子篩選

Virus sensing and anti-infection molecules screening biosensing platform development by palladium nano-thin-film electrochemical spectroscopy