Fluorescence Immunoassay of Prostate-Specific Antigen Using 3D Paddle Screw-Type Devices and Their Rotating System.

In this paper, we present a sensitive and highly reproducible fluorescence immunosensor for detecting PSA in human serum. A unique feature of this study is that it uses creatively designed paddle screw-type devices and their custom-made rotating system for PSA immunoassay. The paddle screw devices were designed to maximize the surface-to-volume ratio over which the immunoassay reaction could occur to improve detection sensitivity. This paddle screw-based immunoassay offers an accessible and efficient method with a short analysis time of less than 30 min. Active rotation of the paddle screw plays a crucial role in fast and accurate analysis of PSA. Additionally, a paddle screw-based immunoassay and subsequent fluorescence detection using a custom prototype fluorescence detection system were compared to a typical well plate-based immunoassay system. Results of PSA detection in human serum showed that the detection sensitivity through the paddle screw-based analysis improved about five times compared to that with a well plate-based analysis.

Isolation and Characterization of Exosomes from Cancer Cells Using Antibody-Functionalized Paddle Screw-Type Devices and Detection of Exosomal miRNA Using Piezoelectric Biosensor.

Exosomes are small extracellular vesicles produced by almost all cell types in the human body, and exosomal microRNAs (miRNAs) are small non-coding RNA molecules that are known to serve as important biomarkers for diseases such as cancer. Given that the upregulation of miR-106b is closely associated with several types of malignancies, the sensitive and accurate detection of miR-106b is important but difficult. In this study, a surface acoustic wave (SAW) biosensor was developed to detect miR-106b isolated from cancer cells based on immunoaffinity separation technique using our unique paddle screw device. Our novel SAW biosensor could detect a miR-106b concentration as low as 0.0034 pM in a linear range from 0.1 pM to 1.0 µM with a correlation coefficient of 0.997. Additionally, we were able to successfully detect miR-106b in total RNA extracted from the exosomes isolated from the MCF-7 cancer cell line, a model system for human breast cancer, with performance comparable to commercial RT-qPCR methods. Therefore, the exosome isolation by the paddle screw method and the miRNA detection using the SAW biosensor has the potential to be used in basic biological research and clinical diagnosis as an alternative to RT-qPCR.

Simultaneous Detection of Exosomal microRNAs Isolated from Cancer Cells Using Surface Acoustic Wave Sensor Array with High Sensitivity and Reproducibility.

We present a surface acoustic wave (SAW) sensor array for microRNA (miRNA) detection that utilizes photocatalytic silver staining on titanium dioxide (TiO2) nanoparticles as a signal enhancement technique for high sensitivity with an internal reference sensor for high reproducibility. A sandwich hybridization was performed on working sensors of the SAW sensor array that could simultaneously capture and detect three miRNAs (miRNA-21, miRNA-106b, and miRNA-155) known to be upregulated in cancer. Sensor responses due to signal amplification varied depending on the concentration of synthetic miRNAs. It was confirmed that normalization (a ratio of working sensor response to reference sensor response) screened out background interferences by manipulating data and minimized non-uniformity in the photocatalytic silver staining step by suppressing disturbances to both working sensor signal and reference sensor signal. Finally, we were able to successfully detect target miRNAs in cancer cell-derived exosomal miRNAs with performance comparable to the detection of synthetic miRNAs.

Sensitive detection of microRNA using QCM biosensors: sandwich hybridization and signal amplification by TiO2 nanoparticles.

MicroRNA-21 (miR-21) is known to act as an important biomarker for cancer, in that its up-regulation is closely related to several types of malignant tumor. Sensitive and accurate detection of miR-21 using a biosensor is highly challenging. In this study, sensitive and selective detection technology for miR-21 molecules using a quartz crystal microbalance (QCM) biosensor was developed. Sandwich hybridization between miR-21 and specially designed probes and a subsequent TiO2 photocatalytic silver enhancement reaction were the driving forces for sensitive detection with high selectivity for miR-21. Using this strategic approach under optimal conditions, the novel QCM biosensor can detect miR-21 with a LOD of 0.87 pM over the entire linear range from 0.1 pM to 10 µM, with a correlation coefficient of 0.988. In addition, the developed QCM biosensor was very effective in the quantification of miR-21 in serum samples, so the proposed miRNA detection method offers great potential for the diagnosis of early disease, such as cancer and vascular diseases, and could be an excellent alternative for biological research and clinical diagnosis.

QCM sensing of miR-21 by formation of microRNA-DNA hybrid duplexes and intercalation on surface-functionalized pyrene.

MicroRNAs (miRNAs) are small non-coding RNA molecules that serve as important biomarkers for a variety of diseases such as cancer and vascular disease. However, sensitive and accurate detection of miR-21 is very challenging in that up-regulation of miR-21 is highly associated with several types of malignant tumors. Here, quartz crystal microbalance (QCM) biosensors were developed for sensitive and specific detection of miR-21 through formation of miR-21-DNA hybrid duplexes and non-specific intercalation of surface-modified pyrene molecules. High selectivity for miR-21 over other miRNAs came from the specific hybridization between miR-21 and gold nanoparticle (AuNP)-conjugated complementary oligonucleotides of miR-21. High sensitivity was obtained through formation of intercalated complexes on the surface with subsequent gold staining signal amplification. Under optimum condition using this strategic approach, our novel QCM biosensors could detect miR-21 concentration as low as 3.6 pM in the entire linear range from 2.5 pM to 2.5 µM with a correlation coefficient of 0.989. In addition, these sensors did not work at all for other miRNAs based on their high selectivity. miR-21 in human brain total RNA and total RNA extracted from A549 cell line could also be successfully detected. Therefore, miRNA detection technology using QCM biosensors and their detection mechanisms have potential as alternatives in biological studies and clinical diagnosis.

Sensitivity and reproducibility improvements in a human plasma immunoassay with removal of clotting factors.

Interferences in human plasma immunoassay are severe challenge that affects the sensitivity and reproducibility of the assay. The clotting factor fibrinogen is a negatively charged protein and is one of the most common sources of interference in immunoassays, and its removal increases the sensitivity and reproducibility. Here, we present a highly sensitive and reproducible method for the detection of prostate specific antigen (PSA) in human plasma immunoassays. Protamine sulfate, a highly positively charged protein, was used to precipitate fibrinogen via ionic interaction to improve the sensitivity and reproducibility of human plasma immunoassay. In a sandwich ELISA for PSA using plasma and protamine-treated plasma samples, the limit of detection was improved from 413 pg/mL in plasma to 235 pg/mL in protamine-treated plasma samples, and the coefficient of variation known as a measure of reproducibility was significantly lowered by protamine treatment. The use of protamine sulfate in human plasma immunoassays for detection of PSA using quartz crystal microbalance (QCM) biosensors resulted in increased sensitivity and reproducibility by about 2-fold and 3-fold, respectively, relative to when not using protamine sulfate. Based on these results, protamine sulfate was the best choice to increase the sensitivity and reproducibility in immunoassays using plasma samples.

Highly sensitive piezoelectric immunosensors employing signal amplification with gold nanoparticles.

We present a quartz crystal microbalance (QCM) immunosensor for highly sensitive detection of prostate-specific antigen (PSA) in a human serum immunoassay. In particular, in this study, we employed signal amplification using and enlarging gold nanoparticles. Because QCM measures the change of resonance frequency according to the mass change occurring on the sensor surface, we could quantitatively analyze PSA based on a tremendous increase in mass by sandwich immunoassay using AuNP-conjugated anti-PSA-detecting antibody enhanced with subsequent gold staining. The limit of detection of the PSA immunoassay in human serum without gold staining enhancement was 687 pg ml-1 but was 48 pg ml-1 with the gold staining-mediated signal amplification. That is, amplifying the signal resulted in increased sensitivity and reproducibility of immunoassay in a human serum.

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.

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.

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.

SSA-MOA: a novel CTC isolation platform using selective size amplification (SSA) and a multi-obstacle architecture (MOA) filter.

Circulating tumor cells (CTCs) have gained increasing attention as physicians and scientists learn more about the role these extraordinarily rare cells play in metastatic cancer. In developing CTC technology, the critical criteria are high recovery rates and high purity. Current isolation methods suffer from an inherent trade-off between these two goals. Moreover, ensuring minimal cell stress and robust reproducibility is also important for the clinical application of CTCs. In this paper, we introduce a novel CTC isolation technology using selective size amplification (SSA) for target cells and a multi-obstacle architecture (MOA) filter to overcome this trade-off, improving both recovery rate and purity. We also demonstrate SSA-MOA's advantages in minimizing cell deformation during filter transit, resulting in more stable and robust CTC isolation. In this technique, polymer microbeads conjugated with anti-epithelial cell adhesion molecules (anti-EpCAM) were used to selectively size-amplify MCF-7 breast cancer cells, definitively differentiating from the white blood cells (WBCs) by avoiding the size overlap that compromises other size selection methods. 3 µm was determined to be the optimal microbead diameter, not only for size discrimination but also in maximizing CTC surface coverage. A multi-obstacle architecture filter was fabricated using silicon-on-glass (SOG) technology-a first such application of this fabrication technique-to create a precise microfilter structure with a high aspect ratio. The filter was designed to minimize cell deformation as simulation results predicted that cells captured via this MOA filter would experience 22% less moving force than with a single-obstacle architecture. This was verified by experiments, as we observed reliable cell capture and reduced cell deformation, with a 92% average recovery rate and 351 peripheral blood leukocytes (PBL) per millilitre (average). We expect the SSA-MOA platform to optimize CTC recovery rates, purity, and stability, increasing the sensitivity and reliability of such tests, thereby potentially expanding the utilization of CTC technologies in the clinic.

Electrochemical detection of DNA mutations on a PNA-modified electrode utilizing a single-stranded DNA specific endonuclease.

Utilizing a peptide nucleic acid (PNA)-modified electrode and a single-stranded DNA specific endonuclease, a novel electrochemical method to identify DNA mutations has been developed and represents a totally new strategy for the electrochemical diagnosis of human genetic mutations.

Serum starvation induces G1 arrest through suppression of Skp2-CDK2 and CDK4 in SK-OV-3 cells.

Recent studies have suggested that Skp2, an SCF-type ubiquitin ligase, positively regulates cell cycle through degradation of p27, which is an inhibitor of cyclin-dependent kinase 2 (CDK2), which drives cells from the G1 to S phase of cell cycles. In the present study, we examined key regulatory proteins involved in serum starvation-induced cell cycle arrest in human ovarian cancer cells, SK-OV-3. Cell cycle analysis showed that cells were arrested at the G1 phase after serum starvation. Western blot analysis showed that the protein levels of CDK4 and CDK2 were significantly decreased in SK-OV-3 cells. Consistently, Roscovitine, an inhibitor of CDK2, induced cell cycle arrest in normally proliferating cells and a chemical inhibitor of CDK4, 3-ATA [3-Amino-9-thio(10H)-acridone], was found to induce growth arrest. We also found that the protein level of Skp2 was dramatically decreased in response to serum starvation. Moreover, CDK2 protein, which allows cell cycle transit from the G1 to the S phase, was decreased when the Skp2 expression was inhibited by specific siRNA of Skp2, but CDK4 was not decreased. Therefore, these results suggest that serum starvation induces G1 arrest through suppression of Skp2-dependent CDK2 activity and Skp2-independent CDK4 activity in human SK-OV-3 ovarian cancer cells.

Ascorbate (vitamin C) induces cell death through the apoptosis-inducing factor in human breast cancer cells.

Although ascorbate (Vitamin C) has been shown to inhibit cell growth and induce cell death in variety of cancer cells, results reported in other studies are inconsistent with this conclusion. It was previously reported that ascorbate induces apoptosis in human breast cancer cells. However, the molecular mechanism for this is not clear. In this study, we demonstrate that ascorbate induces cell death through the apoptosis-inducing factor (AIF) in the human breast cancer cell lines, SK-BR3 and Hs578T, but not in a normal breast cell line, Hs578. Ascorbate treatment caused the nuclear translocation of AIF, which is retained in the mitochondria in healthy cells, but caspase cleavage is not induced. Moreover, MG132, an inhibitor of AIF release from mitochondria, blocked the induction of cell death. Furthermore, cells that had been treated with human AIF-specific siRNA resisted cell death induced by ascorbate, implying that the translocation of AIF from mitochondria to the nucleus is responsible for ascorbate-mediated cell death. Therefore, these results suggest that ascorbate activates a caspase-independent and AIF-mediated cell death pathway in human breast cancer cells, SK-BR3, and Hs578T.