Soo Suk Lee

DNAzyme Molecular Beacon Probes for Target-Induced Signal-Amplifying Colorimetric Detection of Nucleic Acids

A novel DNAzyme molecular beacon (DNAzymeMB) strategy was developed for target-induced signal-amplifying colorimetric detection of target nucleic acids. The DNAzymeMB, which exhibits peroxidase activity in its free hairpin structure, was engineered to form a catalytically inactive hybrid through hybridization with a blocker DNA. The presence of target DNA leads to dissociation of the DNAzymeMB from the inactive hybrid through hybridization with the blocker DNA. This process results in recovery of the catalytically active DNAzymeMB, which can catalyze a colorimetric reaction that signals the presence of the target DNA. In addition, a primer was rationally designed to anneal to the blocker DNA of the blocker/target DNA duplex and displace the bound target DNA during the extension reaction. The released target DNA triggers the next cycle involving hybridization with blocker DNA, DNAzymeMB dissociation, primer extension, and target displacement. This unique amplifying strategy leads to the generation of multiple numbers of active DNAzymeMB molecules from a single target molecule and gives a detection limit down to 1 pM, a value that is nearly 3 or 5 orders of magnitude lower than those of previously reported DNAzyme molecular beacon-based DNA detection methods.

Highly Efficient Assay of Circulating Tumor Cells by Selective Sedimentation with a Density Gradient Medium and Microfiltration from Whole Blood

Isolation of circulating tumor cells (CTCs) by size exclusion can yield poor purity and low recovery rates, due to large variations in size of CTCs, which may overlap with leukocytes and render size-based filtration methods unreliable. This report presents a very sensitive, selective, fast, and novel method for isolation and detection of CTCs. Our assay platform consists of three steps: (i) capturing CTCs with anti-EpCAM conjugated microbeads, (ii) removal of unwanted hematologic cells (e.g., leukocytes, erythrocytes, etc.) by selective sedimentation of CTCs within a density gradient medium, and (iii) simple microfiltration to collect these cells. To demonstrate the efficacy of this assay, MCF-7 breast cancer cells (average diameter, 24 μm) and DMS-79 small cell lung cancer cells (average diameter, 10 μm) were used to model CTCs. We investigated the relative sedimentation rates for various cells and/or particles, such as CTCs conjugated with different types of microbeads, leukocytes, and erythrocytes, in order to maximize differences in the physical properties. We observed that greater than 99% of leukocytes in whole blood were effectively removed at an optimal centrifugal force, due to differences in their sedimentation rates, yielding a much purer sample compared to other filter-based methods. We also investigated not only the effect of filtration conditions on recovery rates and sample purity but also the sensitivity of our assay platform. Our results showed a near perfect recovery rate (~99%) for MCF-7 cells and very high recovery rate (~89%) for DMS-79 cells, with minimal amounts of leukocytes present.

Surface acoustic wave immunosensor for real-time detection of hepatitis B surface antibodies in whole blood samples

We demonstrate an application of Love wave mode surface acoustic wave (SAW) immunosensor to detect hepatitis B surface antibody (HBsAb) in aqueous conditions. SiO(2) guiding layer was deposited on 36 degrees YX-LiTaO(3) piezoelectric single crystal substrate to protect the electrodes and to trap the acoustic energy near the surface, and hepatitis B surface antigen (HBsAg) was immobilized on the sensing area. The resonance frequency shift was monitored to detect specific binding of HBsAb to immobilized HBsAg. To eliminate the effects of other physical factors except for the mass change, the resonance frequency was compared to that of a reference SAW device coated with bovine serum albumin (BSA) to block binding of HBsAb. The guiding layer thickness with maximum mass sensitivity was found to be 5 microm, which was in agreement with the theoretical calculation, and the center resonance frequency was around 199 MHz. The sensor showed binding specificity to HBsAb and a linear relationship between the frequency shift and the antibody concentration with sensitivity of 0.74 Hz/(pg/microl) and detection limit below 10 pg/microl. In addition, our SAW immunosensor successfully detected HBsAb in whole blood samples without any pretreatment, opening up its applicability in fast label-free protein detection methods.

Sensitive and simultaneous detection of cardiac markers in human serum using surface acoustic wave immunosensor.

We present a rapid and sensitive surface acoustic wave (SAW) immunosensor that utilizes gold staining as a signal enhancement method. A sandwich immunoassay was performed on sensing area of the SAW sensor, which could specifically capture and detect cardiac markers (cardiac troponin I (cTnI), creatine kinase (CK)-MB, and myoglobin). The analytes in human serum were captured on gold nanoparticles (AuNPs) that were conjugated in advance with detection antibodies. Introduction of these complexes to the capture antibody-immobilized sensor surface resulted in a classic AuNP-based sandwich immunoassay format that has been used for signal amplification. In order to achieve further signal enhancement, a gold staining method was performed, which demonstrated that it is possible to obtain gold staining-mediated signal augmentation on a mass-sensitive device. The sensor response due to gold staining varied as a function of cardiac marker concentration. We also investigated effects of increasing operating frequency on sensor responses. Results showed that detection limit of the SAW sensor could be further improved by increasing the operating frequency.

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.

Continuous labeling of circulating tumor cells with microbeads using a vortex micromixer for highly selective isolation.

Circulating tumor cells (CTCs) are identified in transit within the blood stream of cancer patients and have been proven to be a main cause of metastatic disease. Current approaches for the size-based isolation of CTCs have encountered technical challenges as some of the CTCs have a size similar to that of leukocytes and therefore CTCs are often lost in the process. Here, we propose a novel strategy where most of the CTCs are coated by a large number of microbeads to amplify their size to enable complete discrimination from leukocytes. In addition, all of the microbead labeling processes are carried out in a continuous manner to prevent any loss of CTCs during the isolation process. Thus, a microfluidic mixer was employed to facilitate the efficient and selective labeling of CTCs from peripheral blood samples. By generating secondary vortex flows called Taylor-Gortler vortices perpendicular to the main flow direction in our microfluidic device, CTCs were continuously and successfully coated with anti-epithelial cell adhesion molecule-conjugated beads. After the continuous labeling, the enlarged CTCs were perfectly trapped in a micro-filter whereas all of the leukocytes escaped.

A DNA intercalation-based electrochemical method for detection of Chlamydia trachomatis utilizing peroxidase-catalyzed signal amplification

A sensitive electrochemical DNA detection method for the diagnosis of sexually transmitted disease (STD) caused by Chlamydia trachomatis was developed. The method utilizes a DNA-intercalating agent and a peroxidase promoted enzymatic precipitation reaction and involves the following steps. After hybridization of the target C. trachomatis gene with an immobilized DNA capture probe on a gold electrode surface, the biotin-tagged DNA intercalator (anthraquinone) was inserted into the resulting DNA duplex. Subsequently, the polymeric streptavidin/peroxidase complex was applied to the biotin-decorated electrode. Peroxidase catalyzed 4-chloronaphthol to produce insoluble product, which is precipitated on the electrode surface in the presence of hydrogen peroxide. Cyclic voltammograms with the gold electrode exhibited a peak current of ferrocenemethanol in electrolyte, which decreased in a proportional way to increasing concentration of target DNA owing to insulation of electrode surface by the growing insoluble precipitate. Using this strategy, we were able to detect picomolar concentrations of C. trachomatis gene in a sample taken from a real patient.

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.

A rapid and facile signal enhancement method for microcantilever-based immunoassays using the agglomeration of ferromagnetic nanoparticles

A rapid and facile signal enhancement method for detecting alpha-fetoprotein (AFP) was developed using the magnetic agglomeration of ferromagnetic nanoparticles and microcantilever sensors. The resonance frequency and deflection of the cantilevers were found to be more than 10-fold greater than that before physical agglomeration of the free nanoparticles around the magnetized nanoparticles.