Showing papers by "Gwo-Bin Lee published in 2015"

Screening of aptamers specific to colorectal cancer cells and stem cells by utilizing On-chip Cell-SELEX

Abstract: Colorectal cancer (CRC) is the most frequently diagnosed cancer around the world, causing about 700,000 deaths every year. It is clear now that a small fraction of CRC, named colorectal cancer stem cells (CSCs) exhibiting self-renewal and extensive proliferative activities, are hard to be eradicated. Unfortunately, highly specific biomarkers for colorectal CSC (CR-CSCs) are lacking that prohibits the development of effective therapeutic strategies. This study designed and manufactured a novel microfluidic system capable of performing a fully automated cell-based, systematic evolution of ligands by exponential enrichment (SELEX) process. Eight CR-CSC/CRC-specific aptamers were successfully selected using the microfluidic chip. Three of the aptamers showed high affinities towards their respective target cells with a dissociation constant of 27.4, 28.5 and 12.3 nM, which are comparable to that of antibodies.

Selection of aptamers specific for glycated hemoglobin and total hemoglobin using on-chip SELEX

Abstract: Blood glycated hemoglobin (HbA1c) levels reflecting average glucose concentrations over the past three months are fundamental for the diagnosis, monitoring, and risk assessment of diabetes. It has been hypothesized that aptamers, which are single-stranded DNAs or RNAs that demonstrate high affinity to a large variety of molecules ranging from small drugs, metabolites, or proteins, could be used for the measurement of HbA1c. Aptamers are selected through an in vitro process called systematic evolution of ligands by exponential enrichment (SELEX), and they can be chemically synthesized with high reproducibility at relatively low costs. This study therefore aimed to select HbA1c- and hemoglobin (Hb)-specific single-stranded DNA aptamers using an on-chip SELEX protocol. A microfluidic SELEX chip was developed to continuously and automatically carry out multiple rounds of SELEX to screen specific aptamers for HbA1c and Hb. HbA1c and Hb were first coated onto magnetic beads. Following several rounds of selection and enrichment with a randomized 40-mer DNA library, specific oligonucleotides were selected. The binding specificity and affinity were assessed by competitive and binding assays. Using the developed microfluidic system, the incubation and partitioning times were greatly decreased, and the entire process was shortened dramatically. Both HbA1c- and Hb-specific aptamers selected by the microfluidic system showed high specificity and affinity (dissociation constant, Kd = 7.6 ± 3.0 nM and 7.3 ± 2.2 nM for HbA1c and Hb, respectively). With further refinements in the assay, these aptamers may replace the conventional antibodies for in vitro diagnostics applications in the near future.

Measurement of single leukemia cell's density and mass using optically induced electric field in a microfluidics chip

Abstract: We present a method capable of rapidly (∼20 s) determining the density and mass of a single leukemic cell using an optically induced electrokinetics (OEK) platform. Our team had reported recently on a technique that combines sedimentation theory, computer vision, and micro particle manipulation techniques on an OEK microfluidic platform to determine the mass and density of micron-scale entities in a fluidic medium; the mass and density of yeast cells were accurately determined in that prior work. In the work reported in this paper, we further refined the technique by performing significantly more experiments to determine a universal correction factor to Stokes' equation in expressing the drag force on a microparticle as it falls towards an infinite plane. Specifically, a theoretical model for micron-sized spheres settling towards an infinite plane in a microfluidic environment is presented, and which was validated experimentally using five different sizes of micro polystyrene beads. The same sedimentation process was applied to two kinds of leukemic cancer cells with similar sizes in an OEK platform, and their density and mass were determined accordingly. Our tests on mouse lymphocytic leukemia cells (L1210) and human leukemic cells (HL-60) have verified the practical viability of this method. Potentially, this new method provides a new way of measuring the volume, density, and mass of a single cell in an accurate, selective, and repeatable manner.

An integrated microfluidic system for screening of phage-displayed peptides specific to colon cancer cells and colon cancer stem cells.

Abstract: Affinity reagents recognizing biomarkers specifically are essential components of clinical diagnostics and target therapeutics. However, conventional methods for screening of these reagents often have drawbacks such as large reagent consumption, the labor-intensive or time-consuming procedures, and the involvement of bulky or expensive equipment. Alternatively, microfluidic platforms could potentially automate the screening process within a shorter period of time and reduce reagent and sample consumption dramatically. It has been demonstrated recently that a subpopulation of tumor cells known as cancer stem cells possess high drug resistance and proliferation potential and are regarded as the main cause of metastasis. Therefore, a peptide that recognizes cancer stem cells and differentiates them from other cancer cells will be extremely useful in early diagnosis and target therapy. This study utilized M13 phage display technology to identify peptides that bind, respectively, to colon cancer cells and colon cancer stem cells using an integrated microfluidic system. In addition to positive selection, a negative selection process was integrated on the chip to achieve the selection of peptides of high affinity and specificity. We successfully screened three peptides specific to colon cancer cells and colon cancer stem cells, namely, HOLC-1, HOLC-2, and COLC-1, respectively, and their specificity was measured by the capture rate between target, control, and other cell lines. The capture rates are 43.40 ± 7.23%, 45.16 ± 7.12%, and 49.79 ± 5.34% for colon cancer cells and colon cancer stem cells, respectively, showing a higher specificity on target cells than on control and other cell lines. The developed technique may be promising for early diagnosis of cancer cells and target therapeutics.

Cancer Cell-Specific Oligopeptides Selected by an Integrated Microfluidic System from a Phage Display Library for Ovarian Cancer Diagnosis

Abstract: Ovarian cancer is one of the leading causes of female mortality worldwide. Unfortunately, there are currently few high-specificity candidate oligopeptide targeting agents that can be used for early diagnosis of this cancer. It has been suggested that cancer-specific oligopeptides could be screened from a phage display library. However, conventional methods are tedious, labor-intensive, and time consuming. Therefore, a novel, integrated microfluidic system was developed to automate the entire screening process for ovarian cancer cell-specific oligopeptides. An oligopeptide screened with microfluidic chip-based technique was demonstrated to have high affinity to ovarian cancer cells and demonstrated relatively low binding to other cancer cells, indicating a high specificity. Furthermore, the developed method consumed relatively low volumes of samples and reagents; only 70 μL of reactant was used within the whole experimental process. Each panning process was also significantly shortened to only 7.5 hours. Therefore, the screened oligopeptide could be used to isolate ovarian cancer cells in a rapid manner, thus greatly expediting the diagnosis and its application as oligopeptide targeting agent for theranostics of this cancer.

Optically-controlled digital electrodeposition of thin-film metals for fabrication of nano-devices

Abstract: Precise micro-scale patterning of metal thin-films with semiconductor materials is a critical step in the fabrication of any micro-devices. Conventional metal patterning methods such as photolithography, laser-induced direct printing techniques, and micro-contact printing methods all have different disadvantages such as high capital equipment cost and low throughput efficiency. In this article, an optically-controlled digital electrodeposition (ODE) method for direct patterning of metal thin-films on a semiconductor substrate has been demonstrated. This method allows for dynamic patterning of custom micro-scale silver structures with high conductivity of 2 × 107 S/m in large scale within 10 seconds and could reach a smallest line width of 2.7 μm. The entire process is performed at room temperature and atmospheric pressure conditions, while requiring no photolithographic steps or metal nanoparticle inks. Utilizing this direct structural formation technique, a bottom-up protocol for rapidly assembling nanowire-based field-effect transistors has been demonstrated, which shows that this novel technique could potentially become an alternative, low-cost and flexible technology for fabricating integrated nano-devices.

Measurement of glycated hemoglobin levels using an integrated microfluidic system

Abstract: Diabetes mellitus (DM) is a common non-communicable disease worldwide. Since DM may lead to serious complications, its treatment and care relies on early diagnosis leading to strict glycemic control monitored by the periodic measurement of the patients’ blood glucose levels. However, glucose levels can easily fluctuate due to diet, exercise, insulin resistance, stress, and other factors. Alternatively, the percentage of glycated hemoglobin (HbA1c) in the blood reflects an average glucose concentration over the past 2–3 months with fewer variations due to non-glycemic factors. Therefore, the accurate measurement of the HbA1c in the blood provides a more effective route to diagnose and to monitor DM than a conventional blood glucose measurement. For the measurement of HbA1c, immunoassays have become a widely used method. Therefore, to demonstrate automatic HbA1c measurement, we have developed a microfluidic system to perform a chemiluminescence immunoassay on a single chip automatically except the treatment of samples was undertaken off-chip and required centrifugation. Compared to routine clinical protocols, which rely on bulky benchtop instruments, the developed system is more compact in size (3.8-cm wide and 6.8-cm long) and consumes less samples (volume = 1 μL) and reagents (volume = 150 μL). It is also less labor-intensive to perform an immunoassay using this chip. With further refinements in assay development, this device may be a promising replacement for conventional HbA1c measurement in the near future.

3-D Non-UV Digital Printing of Hydrogel Microstructures by Optically Controlled Digital Electropolymerization

Abstract: A technique using digital masks without ultraviolet light to rapidly print 3-D biopolymer structures with complex microarchitectures in a microfluidic chip has been demonstrated. In this approach, a customized system is used to project light images on a photoconductive substrate in order to create localized virtual electrodes when an alternating electric field is applied across the fluidic medium in an optically controlled digital electropolymerization chip. Upon these virtual electrodes, the localized electric fields are generated, which could activate the polymerization of acrylate-based molecules, such as poly(ethylene glycol) diacrylate (PEGDA), to form microstructures with the same shapes as the projected light images. We have demonstrated that the 3-D PEGDA microstructures with the customized shapes could be fabricated rapidly through a layer-by-layer process by applying a series of digital masks (projected light images). With our current projection and microscopy system, the fabrication of microhydrogel structures with a lateral resolution of 3 $\mu \text{m}$ and an adjustable thickness ranging from tens of nanometers to hundreds of micrometers has been demonstrated. In summary, this novel technique provides an efficient process for the rapid printing of the 3-D biopolymer-based microstructures, and could enable many future applications in a mechanoanalysis of cancer cells, tissue engineering, and drug screening. [2015-0110]

An integrated microfluidic platform for negative selection and enrichment of cancer cells

Abstract: Circulating tumor cells (CTCs), tumor cells that disseminate from primary tumors to the bloodstream, have recently emerged as promising indicators for cancer diagnosis and prognosis. However, the technical difficulties in isolating and detecting rare CTCs have limited the widespread applicability of this method to date. In this work, a new integrated microfluidic system integrating micromixers and micropumps capable of performing 'negative selection and enrichment' of CTCs was developed. By using anti-human CD45 antibodies-coated magnetic beads, leukocytes were effectively removed by applying an external magnetic force, leaving behind an enriched target cell population. The on-chip CTC recovery rate was experimentally found to be 70 ± 5% after a single round of negative selection and enrichment. Meanwhile, CD45 depletion efficiency was 83.99 ± 1.00% and could be improved to 99.84 ± 0.04% after three consecutive rounds of depletion. Notably, on-chip negative selection and enrichment was 58% faster and the repeated depletion could be processed automatically. These promising results suggested the developed microfluidic chip is potentiated for a standardized CTC isolation platform.

Continuous medium exchange and optically induced electroporation of cells in an integrated microfluidic system

Abstract: Optically induced electroporation (OIE) is a promising microfluidic-based approach for the electroporation of cell membranes. However, previously proposed microfluidic cell-electroporation devices required tedious sample pre-treatment steps, specifically, periodic media exchange. To enable the use of this OIE process in a practical protocol, we developed a new design for a microfluidic device that can perform continuous OIE; i.e., it is capable of automatically replacing the culture medium with electroporation buffers. Integrating medium exchanges on-chip with OIE minimises critical issues such as cell loss and damage, both of which are common in traditional, centrifuge-based approaches. Most importantly, our new system is suitable for handling small or rare cell populations. Two medium exchange modules, including a micropost array railing structure and a deterministic lateral displacement structure, were first adopted and optimised for medium exchange and then integrated with the OIE module. The efficacy of these integrated microfluidic systems was demonstrated by transfecting an enhanced green fluorescent protein (EGFP) plasmid into human embryonic kidney 293T cells, with an efficiency of 8.3%. This result is the highest efficiency reported for any existing OIE-based microfluidic system. In addition, successful co-transfections of three distinct plasmids (EGFP, DsRed and ECFP) into cells were successfully achieved. Hence, we demonstrated that this system is capable of automatically performing multiple gene transfections into mammalian cells. A microfluidic device offers a more streamlined approach for introducing foreign genes into mammalian cells. So-called ‘lab-on-a-chip’ systems could simplify and improve the efficiency of many biological techniques by employing microscale channels and valves to precisely control the movement of fluids, cells and reagents. Researchers led by Gwo-Bin Lee of National Tsing Hua University, Hsinchu, have devised such a system to manage the targeted genetic modification of cultured cells via a process known as electroporation. Lee and colleagues constructed microfluidic devices that employ polymeric structures to direct a continuously flowing stream of cells out of their culture medium and into a specialized module for light-induced electroporation. Their approach achieves remarkable efficiency in introducing individual genes into human cells, and the researchers were even able to use their device to deliver three different plasmids simultaneously.

Generation of murine induced pluripotent stem cells by using high-density distributed electrodes network.

Abstract: This study reports a robust method of gene transfection in a murine primary cell model by using a high-density electrodes network (HDEN). By demonstrating high cell viability after gene transfection and successful expression of transgenes including fluorescent proteins, the HDEN device shows great promise as a solution in which reprogramming efficiency using non-viral induction for generation of murine induced pluripotent stem cells (iPSCs) is optimized. High and steady transgene expression levels in host cells of iPSCs can be demonstrated using this method. Moreover, the HDEN device achieved successful gene transfection with a low voltage of less than 180 V while requiring relatively low cell numbers (less than 1.5 × 10(4) cells). The results are comparable to current conventional methods, demonstrating a reasonable fluorescent-plasmid transfection rate (42.4% in single transfection and 24.5% in triple transfection) and high cell viability of over 95%. The gene expression levels of each iPSC factor was measured to be over 10-fold higher than that reported in previous studies using a single mouse embryonic fibroblast cell. Our results demonstrate that the generation of iPSCs using HDEN transfection of plasmid DNA may be a feasible and safe alternative to using viral transfection methods in the near future.

High-density distributed electrodes network for generation of murine induced pluripotent stem cells

Abstract: High-density electrodes network (HDEN) demonstrates a flexible electroporation procedure for gene transfection in different cell types. In this study, the HDEN device shows its capacity as a great candidate in optimization of efficiency in non-viral induction of murine induced pluripotent stem cells (iPSCs) by demonstrating successful expression of different kinds of transgenes of fluorescent proteins. High and steady expression of iPSCs gene expression has been observed. A reasonable transfection rate and viability in different cell types have been demonstrated as well. It implies that a new alternative of iPSCs generation with a combination of plasmid DNA induction and HDEN may be feasible in the near future.

Test kit and method for rapidly detecting live bacterium and test kit and method for rapidly determining appropriate antibiotic

Abstract: A test kit for rapidly detecting a live bacterium is disclosed. The test kit for rapidly detecting a live bacterium includes ethidium monoazide, a magnetic bead, a first primer pair and a microfluidic chip. A test kit for rapidly determining an appropriate antibiotic is also disclosed. The test kit for rapidly determining an appropriate antibiotic includes ethidium monoazide, a magnetic bead, four primer pairs and a microfluidic chip.

An integrated microfluidic system with field-effect-transistor-based biosensors for automatic highly-sensitive C-reactive protein measurement

Abstract: Rapid and accurate diagnosis of C-reactive protein (CRP) is crucial for preventing cardiovascular diseases because it is a well-known biomarker for evaluating risks of cardiovascular diseases. Our previous work has shown that a microfluidic system equipped with a field-effect-transistor (FET)-based biosensor could detect CRP in 0.1X PBS and provided a limit of detection (LOD) of 26 pM CRP without gate bias. To improve the LOD, a new microfluidic device with a new methodology for measuring FET-based biosensors is presented in this study. Not only can the proposed system work in a solution with a physiological salt concentration but it also detects CRP with ultra-high sensitivity in an automatic fashion. This is the first time that a FET-based biosensor can effectively and automatically detect CRP in a physiological salt concentration without decreasing the sensitivity. The LOD of CRP using aptamer-immobilized AlGaN/GaN high-electron-mobility transistors (HEMTs) was experimentally found to be 1fM, demonstrating the superior performance of this new technique. It may be used as a point-of-care device for CRP detection in the near future.

Optically-induced cell fusion on microfluidic chip utilizing locally enhanced electric field

Abstract: This work reports a new approach called optically-induced cell fusion (OICF) which integrates cell-pairing microstructures and an optically-induced system to achieve cell fusion with high yields and efficiency. It is the first time in literature that cell-pairing SU8 microstructures were combined with optically-induced “virtual” electrodes to form the OICF system such that precise cell-pairing and high-yield cell fusion could be achieved. Experimental results showed that HeLa cells and MCF-7 cells could be successfully fused using this new approach. Furthermore, the new method allows one to selectively fuse cells by using addressable light patterns. It is therefore promising for further biomedical applications.

Multiple influenza virulent diagnosis by utilizing a single-aptamer based microfluidic system

Abstract: Aptamers that could bind with specific target have great potential as artificial antibodies for diagnostic applications. However, the DNA folding of conformation may change under different environment conditions, thus affecting their affinity and specificity towards target molecules. In this study, a novel microfluidic system was developed that used single influenza universal aptamer for multiple-type influenza virulent diagnosis under different environment conditions. A nanogold and single-aptamer based microfluidic system was demonstrated for successful detection of influenza viruses. The developed microsystem has a great potential for point-of-care applications.

Optimization of drug cocktail on an integrated microfluidic system

Abstract: Optimization of drug cocktails has been an important issue for a number of therapies of complicated diseases; however, it is an extremely task using traditional methods. Here we demonstrate a new microfluidic platform capable of automatically dispensing, mixing a small amount of drug combinations, and adding them to the cell cultures such that a precise and fine-tuned drug cocktails may be formed for the subsequent cell-level testing. Our proposed system could decrease manual bias and enhance the throughput of drug cocktail formulation on an automated and minituriazed microfluidic system.