Jong-Myeon Park

One-step pathogen specific DNA extraction from whole blood on a centrifugal microfluidic device

We could, for the first time, demonstrate a fully integrated pathogen specific DNA extraction from whole blood utilizing centrifugal microfluidics on a polymer based CD platform. By combining the TS-LIMBS (target separation and laser-irradiated magnetic bead system) and centrifugal microfluidics using the novel LIFM (the laser irradiated ferrowax microvalves), DNA extraction experiments from whole blood spiked with Hepatitis B virus (HBV) were conducted. The total process was finished within 12 minutes with only one manual step of loading 100 muL of whole blood. Real-time PCR results showed that the concentration of DNA prepared on a CD was as good as that of the samples prepared in conventional bench top method.

Efficient Isolation and Accurate In Situ Analysis of Circulating Tumor Cells Using Detachable Beads and a High-Pore-Density Filter**

Circulating tumor cells (CTCs) in the bloodstream of cancer patients may indicate the likelihood and severity of metastatic progression. Identification, enumeration, and characterization of CTCs may provide a minimally invasive method for assessing cancer status of patients and prescribing personalized anticancer therapy. However, examination of CTCs requires isolation of these cells from whole blood of patients, which is difficult owing to their low quantity (around one CTC per 10 non-cancerous hematopoietic cells in patient blood). Many techniques are used to isolate CTCs, including density gradient centrifugation and dielectrophoresis, but methods using either size-based exclusion or affinity-based enrichment are common. Size-based exclusion assumes that CTCs are larger than most hematopoietic cells and removes all cells smaller than a pre-determined size threshold. Affinity-based enrichment relies on the expression of surface proteins specific to cancer cells and absent in hematopoietic cells. These methods generally use antibody-conjugated magnetic beads and enrich for CTCs by magnetic separation, such as the CellSearch system. Owing to their heterogeneous nature, it may be practically impossible to isolate CTCs with high isolation efficiency using the aforementioned methods. Some CTCs are reported to be nearly identical in size or even smaller than leukocytes, making them difficult to discriminate by size. As for protein expression, epithelial markers, such as EpCAM (epithelial cell adhesion molecule), are downregulated during epithelialto-mesenchymal transition (EMT). Furthermore, the type and expression levels of surface proteins may vary greatly depending on cancer histological subtype. Considering these variations, finding the right antibody or combination of antibodies that consistently captures all CTCs may prove to be difficult. To maximize isolation efficiency, we devised a dual-mode isolation strategy that combines affinity-based enrichment and size-based exclusion. By using microbeads conjugated with CTC-specific antibodies, the size of CTCs can be augmented to enable better discrimination against leukocytes. Subsequent size filtration isolates bead-bound CTCs, allowing the recovery of even smaller-sized CTCs. However, all beadbased capture methods have the inherent limitation of prohibiting accurate image analysis, which is due to optical distortion created by the presence of beads attached to cells. The attached beads not only impede observation of cellular morphology but can actually alter fluorescence signal intensities (Figure 1), demonstrating the incompatibility of in situ quantitative analysis with bead-based capture methods (see the Supporting Information). Accurate quantification of protein expression can lead to better clinical management, particularly in regards to personalized therapy. The expression levels of predictive biomarkers, such as HER2 and EGFR, are commonly used to match patients with appropriate treatment strategies and predict the effectiveness of anticancer drugs. Therefore, it is important to accurately characterize CTCs, and removal of beads from the surface of CTCs prior to imaging is necessary. Thus, we have developed a new method to detach beads from beadbound cells: By inserting a photocleavable linker between the bead surface and conjugated antibodies, it is possible to remove the attached beads from cells by light irradiation without affecting cell viability. Herein, we demonstrate a novel approach for isolation and subsequent in situ protein-expression analysis of CTCs using detachable beads termed RIA (reversible bead attachment for cell isolation and analysis). Scheme 1 illustrates the entire RIA process. Detachable beads conjugated to CTC-specific antibodies bind to CTCs in whole blood of patients. After incubation, the entire sample is filtered through a high-pore-density membrane filter chip, which contains a maximal number of uniform-sized (pore diameter 8 mm) evenly spaced circular pores (distance between pores 5 mm). This step eliminates almost all hematopoietic cells, while CTCs remain on the filter surface. The [*] H. J. Lee, Dr. J. H. Oh, J. M. Oh, J. M. Park, Dr. J. G. Lee, Dr. M. S. Kim, Dr. Y. J. Kim, Dr. H. J. Kang, Dr. S. S. Lee, Dr. N. Huh In Vitro Diagnostics Lab, Bio Research Center, Samsung Biomedical Research Institute, Samsung Advanced Institute of Technology Gyeonggi-do 446-712 (Korea) E-mail: soosuk@samsung.com namhuh@samsung.com H. J. Lee, Prof. J. W. Choi Interdisciplinary Program of Integrated Biotechnology, Department of Chemical & Biomolecular Engineering Sogang University, Seoul 121-742, Korea E-mail: jwchoi@sogang.ac.kr

Simultaneous capture and in situ analysis of circulating tumor cells using multiple hybrid nanoparticles

Using hybrid nanoparticles (HNPs), we demonstrate simultaneous capture, in situ protein expression analysis, and cellular phenotype identification of circulating tumor cells (CTCs). Each HNP consists of three parts: (i) antibodies that bind specifically to a known biomarker for CTCs, (ii) a quantum dot that emits fluorescence signals, and (iii) biotinylated DNA that allows capture and release of CTC-HNP complex to an in-house developed capture & recovery chip (CRC). To evaluate our approach, cells representative of different breast cancer subtypes (MCF-7: luminal; SK-BR-3: HER2; and MDA-MB-231: basal-like) were captured onto CRC and expressions of EpCAM, HER2, and EGFR were detected concurrently. The average capture efficiency of CTCs was 87.5% with identification accuracy of 92.4%. Subsequently, by cleaving the DNA portion with restriction enzymes, captured cells were released at efficiencies of 86.1%. Further studies showed that these recovered cells are viable and can proliferate in vitro. Using HNPs, it is possible to count, analyze in situ protein expression, and culture CTCs, all from the same set of cells, enabling a wide range of molecular- and cellular-based studies using CTCs.

A Trachea‐Inspired Bifurcated Microfilter Capturing Viable Circulating Tumor Cells via Altered Biophysical Properties as Measured by Atomic Force Microscopy

Circulating tumor cells (CTCs), though exceedingly rare in the blood, are nonetheless becoming increasingly important in cancer diagnostics. Despite this keen interest and the growing number of potential clinical applications, there has been limited success in developing a CTC isolation platform that simultaneously optimizes recovery rates, purity, and cell compatibility. Herein, a novel tracheal carina-inspired bifurcated (TRAB) microfilter system is reported, which uses an optimal filter gap size satisfying both 100% theoretical recovery rate and purity, as determined by biomechanical analysis and fluid-structure interaction (FSI) simulations. Biomechanical properties are also used to clearly discriminate between cancer cells and leukocytes, whereby cancer cells are selectively bound to melamine microbeads, which increase the size and stiffness of these cells. Nanoindentation experiments are conducted to measure the stiffness of leukocytes as compared to the microbead-conjugated cancer cells, with these parameters then being used in FSI analyses to optimize the filter gap size. The simulation results show that given a flow rate of 100 μL min(-1), an 8 μm filter gap optimizes the recovery rate and purity. MCF-7 breast cancer cells with solid microbeads are spiked into 3 mL of whole blood and, by using this flow rate along with the optimized microfilter dimensions, the cell mixture passes through the TRAB filter, which achieves a recovery rate of 93% and purity of 59%. Regarding cell compatibility, it is verified that the isolation procedure does not adversely affect cell viability, thus also confirming that the re-collected cancer cells can be cultured for up to 8 days. This work demonstrates a CTC isolation technology platform that optimizes high recovery rates and cell purity while also providing a framework for functional cell studies, potentially enabling even more sensitive and specific cancer diagnostics.

Highly dense, optically inactive silica microbeads for the isolation and identification of circulating tumor cells.

Efficient isolation of circulating tumor cells (CTCs) from whole blood is a major challenge for the clinical application of CTCs. Here, we report an efficient method to isolate CTCs from whole blood using highly dense and transparent silica microbeads. The surfaces of silica microbeads were fully covered with an antibody to capture CTCs, and blocked by zwitterionic moieties to prevent the non-specific adsorption of blood cells. Owing to the high density of the silica microbeads, the complexation of CTCs with silica microbeads resulted in the efficient sedimentation of CTC-microbead complexes, which enabled their discrimination from other blood cells in density gradient media. Model CTCs (MCF-7, HCC827, and SHP-77) with various levels of epithelial cell adhesion molecule (EpCAM) were isolated efficiently, especially those with low EpCAM expression (SHP-77). Moreover, the transparency of silica microbeads enabled CTCs to be clearly identified without interference caused by microbeads. The improved sensitivity resulted in increased CTC recovery from patient samples compared with the FDA-approved CellSearch system (14/15 using our method; 5/15 using the CellSearch system). These results indicate that the isolation method described in this report constitutes a powerful tool for the isolation of CTCs from whole blood, which has important applications in clinical practice.

Triggering navigators for innovative system design: The case of lab-on-a-chip technology

Technology innovation is critical for organizational and economic development and thus has been widely discussed. In spite of the extensive research, however, the research community has made few efforts to guide the direction of innovation, considering the general evolutionary patterns of technical systems. Therefore, this research suggests a novel approach to predict prioritized directions of innovation as well as to create the most promising design of practical concept design. For the purpose, firstly, we analyze the main functions of a target system using function analysis techniques. Then we develop an evolution opportunity map based on the Laws of Technical Systems Evolution in TRIZ, from which we generate the most suitable concept for a next-generation system. Finally, we analyze technical challenges that might arise in realizing the concept using Su-field analysis, which can help to obtain a feasible solution for it. Samsung Advanced Institute for Technology in Korea adopted the suggested approach to the new technology generation process, and verified its practicability and utility. We expect this research to be useful for those in charge of innovation management or new product development processes.

Vertical Magnetic Separation of Circulating Tumor Cells for Somatic Genomic-Alteration Analysis in Lung Cancer Patients.

Efficient isolation and genetic analysis of circulating tumor cells (CTCs) from cancer patients’ blood is a critical step for clinical applications using CTCs. Here, we report a novel CTC-isolation method and subsequent genetic analysis. CTCs from the blood were complexed with magnetic beads coated with antibodies against the epithelial cell adhesion molecule (EpCAM) and separated vertically on a density-gradient medium in a modified well-plate. The recovery rate of model CTCs was reasonable and the cell purity was enhanced dramatically when compared to those parameters obtained using a conventional magnetic isolation method. CTCs were recovered from an increased number of patient samples using our magnetic system vs. the FDA-approved CellSearch system (100% vs. 33%, respectively). In 8 of 13 cases, targeted deep sequencing analysis of CTCs revealed private point mutations present in CTCs but not in matched tumor samples and white blood cells (WBCs), which was also validated by droplet digital PCR. Copy-number alterations in CTCs were also observed in the corresponding tumor tissues for some patients. In this report, we showed that CTCs isolated by the EpCAM-based method had complex and diverse genetic features that were similar to those of tumor samples in some, but not all, cases.

One-Step Pathogen Specific DNA Extraction from Whole Blood on a Centrifugal Microfluidic Device

We could, for the first time, demonstrate a fully integrated pathogen specific DNA extraction from whole blood utilizing centrifugal microfluidics on a polymer based CD platform. By combining the TS-LIMBS (target separation and laser-irradiated magnetic bead system) and centrifugal microfluidics using the novel LIFM (the laser irradiated ferrowax microvalves), DNA extraction experiments from whole blood spiked with Hepatitis B virus (HBV) were conducted. The total process was finished within 12 minutes with only one manual step of loading 100 muL of whole blood. Real-time PCR results showed that the concentration of DNA prepared on a CD was as good as that of the samples prepared in conventional bench top method.

Enhancement of stress migration tolerance by in situ high temperature annealing

Good contact filling and improved resistance against stress migration (SM) of thin Al films are presented in this paper. Aluminum films were deposited at room temperature without substrate bias and then annealed in situ. The annealing was done at a temperature range of 400 °C∼550 °C in a vacuum‐isolated modular sputtering system. Open failure rate was evaluated in metal lines after baking at high temperatures for a long period of time (150 °C∼350 °C, 100 hrs∼1500 hrs). Void observation, impurity analysis, and stress measurement were carried out by using SEM and TEM, SIMS, and stress measurement system, respectively. The resistance against SM and against electromigration (EM) were found to be improved by annealing. These results may be attributed to grain growth during in situ anealing, leading to denser packing of Al atoms and fewer number of misfits between the grain boundaries.In conclusion, thin Al films after in situ annealing showed better contact filling and reliability.