Tza-Huei Wang

Electrokinetics in micro devices for biotechnology applications

Electrokinetics is the study of the motion of bulk fluids or selected particles embedded in fluids when they are subjected to electric fields. With the recent developments in microfabrication, electrokinetics provides effective manipulation techniques in the micro and nano domains, which matches the length scale of various biological objects. The ability to manipulate objects down to molecular levels opens new avenues to exploit biological science and technology. Understanding of the fundamental characteristics and limitations of the forces becomes a crucial issue for successful applications of these force fields. In this paper, we review and examine the range of influence for electrokinetically manipulated biological objects in microdevices, which can lead to interesting applications in biotechnology.

Advances in microfluidic PCR for point-of-care infectious disease diagnostics

Global burdens from existing or emerging infectious diseases emphasize the need for point-of-care (POC) diagnostics to enhance timely recognition and intervention. Molecular approaches based on PCR methods have made significant inroads by improving detection time and accuracy but are still largely hampered by resource-intensive processing in centralized laboratories, thereby precluding their routine bedside- or field-use. Microfluidic technologies have enabled miniaturization of PCR processes onto a chip device with potential benefits including speed, cost, portability, throughput, and automation. In this review, we provide an overview of recent advances in microfluidic PCR technologies and discuss practical issues and perspectives related to implementing them into infectious disease diagnostics.

Quantitative Comparison of Intracellular Unpacking Kinetics of Polyplexes by a Model Constructed From Quantum Dot-FRET

A major challenge for non-viral gene delivery is gaining a mechanistic understanding of the rate-limiting steps. A critical barrier in polyplex-mediated gene delivery is the timely unpacking of polyplexes within the target cell to liberate DNA for efficient gene transfer. In this study, the component plasmid DNA and polymeric gene carrier were individually labeled with quantum dots (QDs) and Cy5 dyes, respectively, as a donor and acceptor pair for fluorescence resonance energy transfer (FRET). The high signal-to-noise ratio in QD-mediated FRET enabled sensitive detection of discrete changes in polyplex stability. The intracellular uptake and dissociation of polyplexes through QD-FRET was captured over time by confocal microscopy. From quantitative image-based analysis, distributions of released plasmid within the endo/lysosomal, cytosolic, and nuclear compartments formed the basis for constructing a three-compartment first-order kinetics model. Polyplex unpacking kinetics for chitosan, polyethylenimine, and polyphosphoramidate were compared and found to correlate well with transfection efficiencies. Thus, QD-FRET-enabled detection of polyplex stability combined with image-based quantification is a valuable method for studying mechanisms involved in polyplex unpacking and trafficking within live cells. We anticipate that this method will also aid the design of more efficient gene carriers.

Continuous dielectrophoretic bacterial separation and concentration from physiological media of high conductivity

Biological sample processing involves purifying target analytes from various sample matrices and concentrating them to a small volume from a large volume of crude sample. This complex process is the major obstacle for developing a microfluidic diagnostic platform. In this study, we present a microfluidic device that can continuously separate and concentrate pathogenic bacterial cells from complex sample matrices such as cerebrospinal fluid and whole blood. Having overcome critical limitations of dielectrophoretic (DEP) operation in physiological media of high conductivity, we utilized target specific DEP techniques to incorporate cell separation, medium exchange, and target concentration into an integrated platform. The proposed microfluidic device can uptake mL volumes of crude biological sample and selectively concentrate target cells into a submicrolitre volume, providing ~10(4) fold of concentration. We designed the device based on the electrokinetic theory and electric field simulation, and tested the device performance with different sample types. The separation efficiency of the device was as high as 97.0% for a bead mixture in TAE buffer and 94.3% and 87.2% for E. coli in human cerebrospinal fluid and blood, respectively. A capture efficiency of 100% was achieved in the concentration chamber. With a relatively simple configuration, the proposed device provides a robust method of continuous sample processing, which can be readily integrated into a fully automated microfluidic diagnostic platform for pathogen detection and quantification.

A surface topography assisted droplet manipulation platform for biomarker detection and pathogen identification

This paper reports a droplet microfluidic, sample-to-answer platform for the detection of disease biomarkers and infectious pathogens using crude biosamples. The platform exploited the dual functionality of silica superparamagnetic particles (SSP) for solid phase extraction of DNA and magnetic actuation. This enabled the integration of sample preparation and genetic analysis within discrete droplets, including the steps of cell lysis, DNA binding, washing, elution, amplification and detection. The microfluidic device was self contained, with all reagents stored in droplets, thereby eliminating the need for fluidic coupling to external reagent reservoirs. The device incorporated unique surface topographic features to assist droplet manipulation. Pairs of micro-elevations were created to form slits that facilitated efficient splitting of SSP from droplets. In addition, a compact sample handling stage, which integrated the magnet manipulator, the droplet microfluidic device and a Peltier thermal cycler, was built for convenient droplet manipulation and real-time detection. The feasibility of the platform was demonstrated by analysing ovarian cancer biomarker Rsf-1 and detecting Escherichia coli with real time polymerase chain reaction and real time helicase dependent amplification.

Quantum Dot Enabled Molecular Sensing and Diagnostics

Since its emergence, semiconductor nanoparticles known as quantum dots (QDs) have drawn considerable attention and have quickly extended their applicability to numerous fields within the life sciences. This is largely due to their unique optical properties such as high brightness and narrow emission band as well as other advantages over traditional organic fluorophores. New molecular sensing strategies based on QDs have been developed in pursuit of high sensitivity, high throughput, and multiplexing capabilities. For traditional biological applications, QDs have already begun to replace traditional organic fluorophores to serve as simple fluorescent reporters in immunoassays, microarrays, fluorescent imaging applications, and other assay platforms. In addition, smarter, more advanced QD probes such as quantum dot fluorescence resonance energy transfer (QD-FRET) sensors, quenching sensors, and barcoding systems are paving the way for highly-sensitive genetic and epigenetic detection of diseases, multiplexed identification of infectious pathogens, and tracking of intracellular drug and gene delivery. When combined with microfluidics and confocal fluorescence spectroscopy, the detection limit is further enhanced to single molecule level. Recently, investigations have revealed that QDs participate in series of new phenomena and exhibit interesting non-photoluminescent properties. Some of these new findings are now being incorporated into novel assays for gene copy number variation (CNV) studies and DNA methylation analysis with improved quantification resolution. Herein, we provide a comprehensive review on the latest developments of QD based molecular diagnostic platforms in which QD plays a versatile and essential role.

Cancer Cell Membrane-Coated Upconversion Nanoprobes for Highly Specific Tumor Imaging.

Cancer cell membrane-coated upconversion nanoprobes (CC-UCNPs) with immune escape and homologous targeting capabilities are used for highly specific tumor imaging. The combination of UCNPs with biomimetic cancer cell membranes embodies a novel materials design strategy and presents a compelling class of advanced materials.

Homogeneous point mutation detection by quantum dot-mediated two-color fluorescence coincidence analysis

This report describes a new genotyping method capable of detecting low-abundant point mutations in a homogeneous, separation-free format. The method is based on integration of oligonucleotide ligation with a semiconductor quantum dot (QD)-mediated two-color fluorescence coincidence detection scheme. Surface-functionalized QDs are used to capture fluorophore-labeled ligation products, forming QD-oligonucleotide nanoassemblies. The presence of such nanoassemblies and thereby the genotype of the sample is determined by detecting the simultaneous emissions of QDs and fluorophores that occurs whenever a single nanoassembly flows through the femtoliter measurement volume of a confocal fluorescence detection system. The ability of this method to detect single events enables analysis of target signals with a multiple-parameter (intensities and count rates of the digitized target signals) approach to enhance assay sensitivity and specificity. We demonstrate that this new method is capable of detecting zeptomoles of targets and achieve an allele discrimination selectivity factor >105.

Novel Methylation Biomarker Panel for the Early Detection of Pancreatic Cancer

Purpose: Pancreatic cancer is the fourth leading cause of cancer deaths and there currently is no reliable modality for the early detection of this disease. Here, we identify cancer-specific promoter DNA methylation of BNC1 and ADAMTS1 as a promising biomarker detection strategy meriting investigation in pancreatic cancer. Experimental Design: We used a genome-wide pharmacologic transcriptome approach to identify novel cancer-specific DNA methylation alterations in pancreatic cancer cell lines. Of eight promising genes, we focused our studies on BNC1 and ADAMTS1 for further downstream analysis, including methylation and expression. We used a nanoparticle-enabled methylation on beads (MOB) technology to detect early-stage pancreatic cancers by analyzing DNA methylation in patient serum. Results: We identified two novel genes, BNC1 (92%) and ADAMTS1 (68%), that showed a high frequency of methylation in pancreatic cancers (n = 143), up to 100% in PanIN-3 and 97% in stage I invasive cancers. Using the nanoparticle-enabled MOB technology, these alterations could be detected in serum samples (n = 42) from patients with pancreatic cancer, with a sensitivity for BNC1 of 79% [95% confidence interval (CI), 66%–91%] and for ADAMTS1 of 48% (95% CI, 33%–63%), whereas specificity was 89% for BNC1 (95% CI, 76%–100%) and 92% for ADAMTS1 (95% CI, 82%–100%). Overall sensitivity using both markers is 81% (95% CI, 69%–93%) and specificity is 85% (95% CI, 71%–99%). Conclusions: Promoter DNA methylation of BNC1 and ADAMTS1 is a potential biomarker to detect early-stage pancreatic cancers. Assaying the promoter methylation status of these genes in circulating DNA from serum is a promising strategy for early detection of pancreatic cancer and has the potential to improve mortality from this disease. Clin Cancer Res; 19(23); 6544–55. ©2013 AACR.

An open-access microfluidic model for lung-specific functional studies at an air-liquid interface

In an effort to improve the physiologic relevance of existing in vitro models for alveolar cells, we present a microfluidic platform which provides an air-interface in a dynamic system combining microfluidic and suspended membrane culture systems. Such a system provides the ability to manipulate multiple parameters on a single platform along with ease in cell seeding and manipulation. The current study presents a comparison of the efficacy of the hybrid system with conventional platforms using assays analyzing the maintenance of function and integrity of A549 alveolar epithelial cell monolayer cultures. The hybrid system incorporates bio-mimetic nourishment on the basal side of the epithelial cells along with an open system on the apical side of the cells exposed to air allowing for easy access for assays.