Hui-Hsin Lu

Diagnostic Devices for Isothermal Nucleic Acid Amplification

Since the development of the polymerase chain reaction (PCR) technique, genomic information has been retrievable from lesser amounts of DNA than previously possible. PCR-based amplifications require high-precision instruments to perform temperature cycling reactions; further, they are cumbersome for routine clinical use. However, the use of isothermal approaches can eliminate many complications associated with thermocycling. The application of diagnostic devices for isothermal DNA amplification has recently been studied extensively. In this paper, we describe the basic concepts of several isothermal amplification approaches and review recent progress in diagnostic device development.

Tip-enhanced fluorescence with radially polarized illumination for monitoring loop-mediated isothermal amplification on Hepatitis C virus cDNA

Abstract. A tip nanobiosensor for monitoring DNA replication was presented. The effects of excitation power and polarization on tip-enhanced fluorescence (TEF) were assessed with the tip immersed in fluorescein isothiocyanate solution first. The photon count rose on average fivefold with radially polarized illumination at 50 mW. We then used polymerase-functionalized tips for monitoring loop-mediated isothermal amplification on Hepatitis C virus cDNA. The amplicon-SYBR® Green I complex was detected and compared to real-time loop-mediated isothermal amplification. The signals of the reaction using 4 and 0.004  ng/μl templates were detected 10 and 30 min earlier, respectively. The results showed the potential of TEF in developing a nanobiosensor for real-time DNA amplification.

Optical Characterization of a 1-D Nanostructure by Dark-Field Microscopy and Surface Plasmon Resonance to Determine Biomolecular Interactions

This paper presents a multifunctional imaging system that combines dark-field microscopy (DFM) with spectroscopy to image nanostructures and identify their optical properties from absorption spectra. The optical resolving power of this system is determined using a 1-D nanostructure with pitches of 120, 390, and 770 nm with four formats of optical disks. These pattern sizes are verified by atomic force microscopy (AFM) first. The results demonstrate that the resolving power of current system setup can down to 86 nm. The resultant DFM images appear to be slightly larger than the AFM images. A 50-nm-thick gold film was then deposited on top of these nanostructures, and their absorption spectra were obtained to elucidate its optical properties, enhanced by surface plasmon resonance. The immobilization of streptavidin on the surface of gold-coated nanostructure causes the absorption spectra to shift from 600 to 610 nm. A protein nanoarray with a dot size of 50 nm was also imaged by DFM, and can be implemented as a potential biochemical diagnostic system on an optical disk format. Specimens of adenocarcinoma cells and ovary cancer cells were also imaged using this DFM system, and the nuclei structure and some cellular organs can be recognized using a 100× objective oil lens.

Detection of tip-enhanced fluorescence from loop-mediated isothermal amplification of hepatitis B virus by two-photon microscopy

Tip-enhanced fluorescence of localized DNA replication by loop-mediated isothermal amplification (LAMP) is a potential way to observe real-time biological reaction confined in nanometer scale. We successfully coated Bst polymerase on the apex (∼100 nm) of an atomic force microscope (AFM) tip and performed localized LAMP reaction of hepatitis B virus (HBV). By using this tip-based reaction, the replicated HBV DNA can be directly imaged to be 400∼500 nm spots by using two-photon excitation fluorescence microscopy.

RECOGNITION OF VOLATILE ORGANIC COMPOUNDS UTILIZING A PORTABLE ELECTRONIC NOSE

In this study, we proposed a portable electronic nose (e-nose) system based on a microcontroller (MSP430-FG439) combined with a tin oxide (SnO2) gas sensor, which was heated by a cyclic heating method, to recognize the volatile organic compounds (VOCs). We had demonstrated that this e-nose system can classify and quantify VOCs, such as methanol and ethanol. The sensitivity of the e-nose system had good linearity in the concentration range of 10–40 ppm of these two VOCs, respectively. This portable e-nose system was implemented with a microcontroller acted as CPU, an LCD for displaying information of gases in real time, a wireless communication system, ZigBee, and a warning system.

Metallic tip enhanced fluorescence for DNA replication monitoring

We have successfully performed localized loop-mediated isothermal reactions of hepatitis B virus (HBV) and hepatitis C virus (HCV) on the apex (50~100 nm) of metallic tips coated with Bst polymerases. The SYBR green molecules binding to the new formed HBV DNA inside the optical near fields were excited by two-photon fluorescence microscopy, and directly imaged in far field. Another reporter primer is used for HCV replication detection. Preliminary results are presented in this manuscript.

Nanodots array rapidly fabricated by Dip-Pen Nanolithography with temperature and humidity control

This study demonstrates the advantage of Dip-Pen Nanolithography (DPN) as a research and design tool for metal nano-structure fabrications. We design two different gold nano-structures, which are fabricated by DPN etching method with temperature and humidity control. The plasmon resonance frequencies of both structures are measured with dark field scattering spectroscopy. Our results show that with temperature and humidity control, DPN is highly potential in developing photonic circuit, solar cell and biomedical devices due to the rapid fabrication and cost effectiveness.

Fabrication of silicon dioxide nano array for bio-mimicking of molecular interactions

We employed atomic force microscopy (AFM) with bias control to fabricate oxided nanopatterns on silicon surface with feature size down to 50nm. The relationship of silicon dioxide nanopatterns against humidity was studied and then the optimal parameter was used to make oxide nanoarry for interaction of biotin and streptavidin. The scanning function of AFM was utilized to verify the different height of biomolecules. According to our experimental results, using nano biochip of silicon dioxide can decrease the monitoring scale to nanometer and can be the nano-platform for monitoring the behavior of biomolecular interaction. We anticipate mimicking the correlation of single molecular behavior and an array of biomolecular behavior to understand the coincidence of them.

Development and measurement of two-photon-base fluorescence correlation spectroscopy

We presented the fluorescence correlation spectroscopy (FCS) based on two-photon microscopy system to access physical parameters that give rise to fluctuations in fluorescence signal. As given excitation volume, the diffusion coefficient of molecules due to Brownian Movement into or out of excitation volume is determined. Further, depended on autocorrelation analysis based on diffusion coefficients of molecules, the fraction of bound species from measured samples containing the dye labeled anti-rabbit IgG and rabbit IgG can be separated. The mean standard deviation of diffusion coefficient measurement in our two-photon based system is about 0.064. The changes of diffusion coefficient with 15 nM concentration solution of fluorescein are 246.173 um^2/s to 35.1626 um^2/s with different concentration solution of glycerol ranging from 2.5% to 40%. Moreover, the maximal binding fraction of kinetics mode and end mode between Anti-Rabbit IgG and Rabbit IgG is 60% and 73%, respectively.