Seyong Kwon

Quantum dot-based immunoassay enhanced by high-density vertical ZnO nanowire array

In this paper, we report an efficient and high-performance immunoassay platform by combining high-density vertical ZnO nanowire array with photostable quantum dot (QD) labeling. The ZnO nanowire array provides a large surface area for the immobilization of biomolecules, which makes it an efficient substrate for the immunoreaction of biomolecules. When a sandwich immunoassay with QD label was conducted on various substrates, the ZnO nanowire substrate showed stronger fluorescence signal than ZnO thin film and bare glass substrates by 3.8 and 8.5 times, respectively. We found that the fluorescence resonance energy transfer (FRET) from QD to ZnO nanowire could be suppressed by extending their distance with multilayer biotin-streptavidin complex. In addition, we demonstrated the QD-based immunoassay of carcinoembryonic antigen (CEA) on a ZnO nanowire substrate, showing an excellent immunoassay performance with a very low detection limit (0.001 ng/mL) and a large detection range up to 100 ng/mL.

Quantitative proteomic profiling of breast cancers using a multiplexed microfluidic platform for immunohistochemistry and immunocytochemistry

This paper describes a multiplexed microfluidic immunohistochemistry (IHC)/immunocytochemistry (ICC) platform for quantitative proteomic profiling in breast cancer samples. Proteomic profiling via ICC was examined for four breast cancer cell lines (AU-565, HCC70, MCF-7, and SK-BR-3). The microfluidic device enabled 20 ICC assays on a biological specimen at the same time and a 16-fold decrease in time consumption, and could be used to quantitatively compare the expression level of each biomarker. The immunohistochemical staining from the microfluidic system showed an accurate localization of protein and comparable quality to that of the conventional IHC method. Although AU-565 and SK-BR-3 cell lines were classified by luminal subtype and adenocarcinomas and were derived from the same patient, weak p63 expression was seen only in SK-BR-3. The HCC70 cell line showed a triple-negative (estrogen receptor-negative/progesterone receptor-negative/human epidermal growth factor receptor 2-negative) phenotype and showed only cytokeratin 5 expression, a representative basal/myoepithelial cell marker. To demonstrate the applicability of the system to clinical samples for proteomic profiling, we were also able to apply this platform to human breast cancer tissue. This result indicates that the microfluidic IHC/ICC platform is useful for accurate histopathological diagnoses using numerous specific biomarkers simultaneously, facilitating the individualization of cancer therapy.

Cellular Hydrogel Biopaper for Patterned 3D Cell Culture and Modular Tissue Reconstruction

Microarchitectured freestanding cellular hydrogel biopaper as a novel 3D cell culture or tissue reconstruction module is reported. New harvesting, transfer, and assembly techniques are used to construct laminated tissue composites of the biopaper, such as hepatic hydrogel sheet modules with augmented liver function for stratified 3D hepatic tissue reconstruction.

Automated Measurement of Multiple Cancer Biomarkers Using Quantum-Dot-Based Microfluidic Immunohistochemistry

We report an automated multiple biomarker measurement method for tissue from cancer patients using quantum dot (QD)-based protein detection combined with reference-based protein quantification and autofluorescence (AF) removal. For multiplexed detection of biomarkers in tissue samples, visualization of QDs on cytokeratin was performed to create a multichannel microfluidic device on sites with dense populations of tumor cells. Three major breast cancer biomarkers (i.e., estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2) were labeled using QDs successively on cancer cells in tissue sections. For the automated measurement of biomarkers, a cytokeratin-based biomarker normalization method was used to measure the averaged expression of proteins. A novel AF-removal algorithm was developed, which normalizes the reference AF spectra reconstructed from unknown AF spectra based on random sampling. For accurate quantification of QDs, we automatically and accurately removed the AF signal from 344 spots of QD-labeled tissue samples using 240 reference AF spectra. Using analytical data with 10 tissue samples from breast cancer patients, the measured biomarker intensities were in good agreement with the results of conventional analyses.

Breast cancer diagnostics using microfluidic multiplexed immunohistochemistry.

A quantitative, reproducible, fast and inexpensive multiplexed immunohistochemistry (IHC) system might play a locomotive role in drug screening and personalized medicine. Currently, fully automated IHC machines and sequential multiplexed IHC methods based upon multiple color reagents have been developed, with the evolution of such methods having revealed novel biological findings over the conventional IHC method, which is time consuming and labor intensive. We describe a novel parallel multiplexed IHC method using a microfluidic multiplexed immunohistochemistry (MMIHC) device for quantitative pathological diagnosis of breast cancer. The key factors for success of parallel multiplexed IHC are the fabrication of a robust microfluidic device, the interface between the device and a tissue slide, and an accurate fluidic control for multiple IHC reagents. In order to apply conventional thin-section tissues into on-chip systems without any additional modification process, a tissue slide-compatible assembler was developed for optimal compatibility of conventional IHC methods. With this approach, a perfect fluid control for various solutions was demonstrated without any leakage, bubble formation or cross-contamination. The results presented in this chapter indicate that the microfluidic IHC protocol developed can provide the possibility of tailored cancer treatments as well as precise histopathological diagnosis using numerous specific biomarkers.

A quantum dot-based microfluidic multi-window platform for quantifying the biomarkers of breast cancer cells.

Conventional molecular profiling methods using immunochemical assays have limits in terms of multiplexity and the quantification of biomarkers in investigation of cancer cells. In this paper, we demonstrate a quantum dot (QD)-based microfluidic multiple biomarker quantification (QD-MMBQ) method that enables labeling of more than eight proteins immunochemically on cell blocks within 1 h, in a quantitative manner. An internal reference, β-actin, was used as a loading control to compensate for differences in not only the cell number but also in staining quality among specimens. Furthermore, the microfluidic blocking method exhibited less nonspecific binding of QDs than the conventional static blocking method.

A Microfluidic Immunostaining System Enables Quality Assured and Standardized Immunohistochemical Biomarker Analysis

Immunohistochemistry (IHC) plays an important role in biomarker-driven cancer therapy. Although there has been a high demand for standardized and quality assured IHC, it has rarely been achieved due to the complexity of IHC testing and the subjective validation-based process flow of IHC quality control. We present here a microfluidic immunostaining system for the standardization of IHC by creating a microfluidic linearly graded antibody (Ab)-staining device and a reference cell microarray. Unlike conventional efforts, our system deals primarily with the screening of biomarker staining conditions for quantitative quality assurance testing in IHC. We characterized the microfluidic matching of Ab staining intensity using three HER2 Abs produced by different manufacturers. The quality of HER2 Ab was also validated using tissues of breast cancer patients, demonstrating that our system is an efficient and powerful tool for the standardization and quality assurance of IHC.

Microfluidic on-chip immunohistochemistry directly from a paraffin-embedded section

We present here a novel microfluidic platform that can perform microfluidic on-chip immunohistochemistry (IHC) processes on a formalin-fixed paraffin-embedded section slide. Unlike previous microfluidic IHC studies, our microfluidic chip made of organic solvent-resistant polyurethane acrylate (PUA) is capable of conducting on-chip IHC processes consecutively. A narrow channel wall structure of the PUA chip shows effective sealing by pressure-based reversible assembly with a section slide. We performed both on-chip IHC and conventional IHC processes and compared the IHC results based on the immunostaining intensity. The result showed that the effects of the on-chip deparaffinization, antigen retrieval, and immunoreaction processes on the IHC result were equivalent to conventional methods while reducing the total process time to less than 1/2. The experiment with breast cancer tissue shows that human epidermal growth factor receptor 2 (HER2) classification can be performed by obtaining a clearly distinguishable immunostaining intensity according to the HER2 expression level. We expect our on-chip microfluidic platform to provide a facile technique suitable for miniaturized, automated, and precise diagnostic devices, including a point-of-care device.

Microfluidic immunocytochemical staining system for efficient immunoreaction

Immunocytochemistry (ICC) has been used for assessing antibodies that target specific antigens in a cell via specific epitopes. Although ICC has been regarded as a well established method, it requires many steps for immunoreactions and long time over 4 h. It means that more efficient immunocytochemical staining method should be developed. Moreover, many biological studies demand quantitative comparison for expression of various antibodies. Here, we have developed a novel microfluidic immunocytochemical staining method to improve the efficiency of color reaction and to enable quantitative comparison for various antibodies on a cell specimen. To realize fluid control with microvalves and uniform fluid velocity across the reaction channels of the device, two-step multilayer soft lithography technique was introduced. The system was automatically controlled for reagents and flow rate, solving the variability of ICC staining and the labor intensiveness of ICC. The microfluidic system made efficient mass transport for reagents and antibodies at near the sample and the phenomenon was proved by computational fluid dynamics (CFD) study. Results showed that reaction time was more significantly affected in immunoreaction than flow rate.

Abstract 3992: Proteomic profiling of breast tumors using a microfluidic quantitative immunohistochemistry platform

In breast cancers, one of the most troublesome characteristics for cure is the heterogeneity in point of genetic alterations, treatment-response variability, and histopathological features. This characteristic urgently requires personalized tailored therapy, getting out of a simple and widespread prescription. Therefore, the investigation of multiple biomarkers is inevitable for complete recovery. Immunohistochemistry (IHC) has been a major diagnostic method in various malignancies and many studies showing the relationship between immunohistochemical profiles and molecular classification support that IHC might play a significant role in subclassification of breast cancer patients for personalized medicine. However, conventional IHC method is not adequate for examination of multiple biomarkers since it requires consumption of many tissue slices and proportionally increases diagnostic cost which can give a big burden to patients. In addition, subjective decision for biomarker expression is another major problem. Therefore, an innovative multiplexed IHC platform satisfying not only investigation of multiple biomarkers with reducing consumption of tissue slice but also quantification for the biomarkers is the current demand for personalized medicine. Here, a novel microfluidic multiplexed and quantitative IHC platform was developed for proteomic profiling of breast tumors. The large-scale microfluidic device was designed to have 20 microchannels, and multilayer soft lithography and two-step lithography technologies were used for fabrication. Fluidic resistance was considered for equivalent flow rate of individual reaction channel. Ten biomarkers known as predictive and prognostic indicators in breast cancers, including SMM-HC, CK5, CK14, E-Cadherin, p53, ER, p63, PR, HER2, and Ki-67, were examined on four breast cancer cell lines (MCF-7, SK-BR-3, AU-565, and HCC70) to confirm the massive multiplexed IHC. Results showed that the platform successfully realized the expressions of each biomarker on a cellblock at the same time and proteomic profiling for 10 biomarkers could be quantitatively analyzed from the image analysis. The platform was also applied to a breast cancer tissue sample and the result was corresponded to the results of conventional IHC method. This microfluidic platform is expected to realize precise subtyping of cancer patients by the proteomic profiling even though the size of tumor samples acquired by operation has been getting smaller due to early detection and neoadjuvant therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3992.