Chia-Hsien Hsu

AlGaN/GaN high electron mobility transistors for protein–peptide binding affinity study

Abstract Antibody-immobilized AlGaN/GaN high electron mobility transistors (HEMTs) were used to detect a short peptide consisting of 20 amino acids. One-binding-site model and two-binding-site model were used for the analysis of the electrical signals, revealing the number of binding sites on an antibody and the dissociation constants between the antibody and the short peptide. In the binding-site models, the surface coverage ratio of the short peptide on the sensor surface is relevant to the electrical signals resulted from the peptide–antibody binding on the HEMTs. Two binding sites on an antibody were observed and two dissociation constants, 4.404×10−11 M and 1.596×10−9 M, were extracted from the binding-site model through the analysis of the surface coverage ratio of the short peptide on the sensor surface. We have also shown that the conventional method to extract the dissociation constant from the linear regression of curve-fitting with Langmuir isotherm equation may lead to an incorrect information if the receptor has more than one binding site for the ligand. The limit of detection (LOD) of the sensor observed in the experimental result (∼10pM of the short peptide) is very close to the LOD (around 2.7–3.4pM) predicted from the value of the smallest dissociation constants. The sensitivity of the sensor is not only dependent on the transistors, but also highly relies on the affinity of the ligand-receptor pair. The results demonstrate that the AlGaN/GaN HEMTs cannot only be used for biosensors, but also for the biological affinity study.

Human immunodeficiency virus drug development assisted with AlGaN/GaN high electron mobility transistors and binding-site models

Human immunodeficiency virus (HIV) Reverse Transcriptase (RT)-immobilized AlGaN/GaN high electron mobility transistors (HEMTs) and binding-site models were used to find out the dissociation constants of the HIV RT-inhibitor complex and the number of the binding sites on RT for the inhibitor, Efavirenz. One binding site on the RT for the inhibitor is predicted and the dissociation constant extracted from the binding-site model is 0.212 nM. The AlGaN/GaN HEMTs and the binding-site-models are demonstrated to be good tools to assist drug developments by elucidating the dissociation constants and the number of binding sites, which can largely reduce the cost and time for drug developments.

Microwells support high-resolution time-lapse imaging and development of preimplanted mouse embryos

A vital aspect affecting the success rate of in vitro fertilization is the culture environment of the embryo. However, what is not yet comprehensively understood is the affect the biochemical, physical, and genetic requirements have over the dynamic development of human or mouse preimplantation embryos. The conventional microdrop technique often cultures embryos in groups, which limits the investigation of the microenvironment of embryos. We report an open microwell platform, which enables micropipette manipulation and culture of embryos in defined sub-microliter volumes without valves. The fluidic environment of each microwell is secluded from others by layering oil on top, allowing for non-invasive, high-resolution time-lapse microscopy, and data collection from each individual embryo without confounding factors. We have successfully cultured mouse embryos from the two-cell stage to completely hatched blastocysts inside microwells with an 89% success rate (n = 64), which is comparable to the success rate of the contemporary practice. Development timings of mouse embryos that developed into blastocysts are statistically different to those of embryos that failed to form blastocysts (p-value < 10(-10), two-tailed Students t-test) and are robust indicators of the competence of the embryo to form a blastocyst in vitro with 94% sensitivity and 100% specificity. Embryos at the cleavage- or blastocyst-stage following the normal development timings were selected and transferred to the uteri of surrogate female mice. Fifteen of twenty-two (68%) blastocysts and four of ten (40%) embryos successfully developed into normal baby mice following embryo transfer. This microwell platform, which supports the development of preimplanted embryos and is low-cost, easy to fabricate and operate, we believe, opens opportunities for a wide range of applications in reproductive medicine and cell biology.

Viscosity-dependent drain current noise of AlGaN/GaN high electron mobility transistor in polar liquids

The drain current fluctuation of ungated AlGaN/GaN high electron mobility transistors (HEMTs) measured in different fluids at a drain-source voltage of 0.5 V was investigated. The HEMTs with metal on the gate region showed good current stability in deionized water, while a large fluctuation in drain current was observed for HEMTs without gate metal. The fluctuation in drain current for the HEMTs without gate metal was observed and calculated as standard deviation from a real-time measurement in air, deionized water, ethanol, dimethyl sulfoxide, ethylene glycol, 1,2-butanediol, and glycerol. At room temperature, the fluctuation in drain current for the HEMTs without gate metal was found to be relevant to the dipole moment and the viscosity of the liquids. A liquid with a larger viscosity showed a smaller fluctuation in drain current. The viscosity-dependent fluctuation of the drain current was ascribed to the Brownian motions of the liquid molecules, which induced a variation in the surface dipole of the gate region. This study uncovers the causes of the fluctuation in drain current of HEMTs in fluids. The results show that the AlGaN/GaN HEMTs may be used as sensors to measure the viscosity of liquids within a certain range of viscosity.

Thermal stability and etching characteristics of electron beam deposited SiO and SiO2

We have studied the thermal stability and etching characteristics of electron beam deposited SiO and SiO2. Scanning electron microscopy, Auger, atomic form microscopy, and ellipsometry were used to analyze the surface morphology, roughness, and film composition as a function of annealing temperatures. Both SiO and SiO2 showed excellent thermal stability up to 400 °C anneal and refractive index, surface morphology and pattern edge definition of both films barely changed. For higher temperature anneal, based on Auger analysis results, the ratio of Si/O of SiO2 film stayed constant after 700 °C. However, the Si/O ratio of SiO film increased from 0.54 to 0.62 due to oxygen outdiffusion. Dry etch characteristics of SiO and SiO2 were investigated using SF6 and NF3 discharges in a Plasma Therm inductively coupled plasma system. Wet etches were performed using buffered HF and HF/H2O solutions. Dry etch rates of SiO2 were comparable with that of conventional plasma enhanced chemical vapor deposition SiO2. SiO2 etc...

Cell sorting in microfluidic systems using dielectrophoresis

Due to the rising requirement of accuracy and convenience in micro-size bio-chips, dielectrophoresis (DEP) has gained its popularity in research in the last decade for it could be utilized in various applications, such as trapping, sorting, and manipulation of micro- or nano- particles. Sorting cells using DEP is especially what researchers are fond of. Here, we proposed a design of bio-chip, which is able to encapsulate particles in droplets and sort them in one step.

Fertilization of Mouse Gametes in Vitro Using a Digital Microfluidic System

We demonstrated in vitro fertilization (IVF) using a digital microfluidic (DMF) system, so-called electrowetting on dielectric (EWOD). The DMF device was proved to be biocompatible and the DMF manipulation of a droplet was harmless to the embryos. This DMF platform was then used for the fertilization of mouse gametes in vitro and for embryo dynamic culture based on a dispersed droplet form. Development of the embryos was instantaneously recorded by a time-lapse microscope in an incubator. Our results indicated that increasing the number of sperms for IVF would raise the rate of fertilization. However, the excess of sperms in the 10 μL culture medium would more easily make the embryo dead during cell culture. Dynamic culture powered with EWOD can manipulate a single droplet containing mouse embryos and culture to the eight-cell stage. The fertilization rate of IVF demonstrated by DMF system was 34.8%, and about 25% inseminated embryos dynamically cultured on a DMF chip developed into an eight-cell stage. The results indicate that the DMF system has the potential for application in assisted reproductive technology.

A microfluidic device for single cell isolation from rare samples

Increasing evidence has shown that heterogeneity in physiology and pathology have important implications for the treatment of human diseases. Single cell isolation and characterization studies are crucial for unlocking the complex regulation pathways underlying various diseases like cancer. However, to date single cell isolation still faces many challenges, especially in processing rare samples. Here, we present a novel microfluidic array chip for single cell isolation for rare samples. Our design exempts the use of long tubing, thus decreases cell loss from dead volume and allows for more accurate control of flow rate using syringe pumps during the cell capture process. Our data showed that the device significantly improved single cell capture efficiency (~ 60 %) from small numbers of (50-500) cells in a small volume (5-10 μL). The device could be used for isolating single cells from rare samples of clinical specimens, thus potentially benefit disease diagnosis and therapy.

Effects of culture medium and volume on preimplantation development of mouse embryos

Assisted reproductive technologies (ART) is gaining its importance worldwide, and a crucial aspect of ART is to control and mimic the in vitro environment. The first step of ART is in vitro fertilization, and we demonstrate the fertilization rate of ~60% (N = 37) when the oocytes and the sperms were cultured in human tubal fluid medium (HTF) first. In this study, we used microwells of different volume, ranging from 16 to 9,813 nL, as the platform to culture pre-implant mouse embryos. Microwells made of polydimethylsiloxane (PDMS) are low-cost, easy to operate, and biocompatible. Besides, the fluidic environment of each microwell could be secluded from each other by layering oil on top, allowing for data collection from each individual embryo without confounding factors. We successfully cultured mouse embryos from the two-cell stage to blastocyst stage inside different volume of microwells with a ~80% successful rate (N = 588), which is comparable to the successful rate of the conventional microdrop methods. Among the five different volumes of microwells evaluated, the blastocyst rater were higher when embryos were cultured in 3,533 nL and 393 nL microwells.To our surprise, microwells, as small as 200 μm in diameter and 16 nL in volume could still support the embryo to develop to blastocyst with a ~75% successful rate (N = 78).

In vitro dynamic fertilization by using EWOD device

This research demonstrates the in vitro fertilization (IVF) technique by using the digital microfluidic (DMF) system. The DMF device has been proved with biocompatibility and used in this research for mouse gametes in vitro fertilization and embryos culture based on the dispersed droplet form. Dynamically moving, cell culture and observation can be achieved by the time-lapse microscopy in an incubator. The fertilization rate of IVF on DMF is 34.8%, and about 25% inseminated embryos dynamically cultured on DMF chip could develop into 8-cell stage. The result indicated that the DMF system has the potential used for assisted reproductive technology.