Microfluidic chip for rapid and selective isolation of tumor-derived extracellular vesicles for early diagnosis and metastatic risk evaluation of breast cancer.

The epithelial-to-mesenchymal transition (EMT) index in cancer is a complementary approach for estimating metastatic risk. Considering the demand for evaluating metastatic risk based on liquid biopsies, tumor-derived extracellular vesicles (EVs) can be exploited to generate the EMT index. For the generation of EVs-based EMT index, it is essential to selectively isolate each epithelial cell and mesenchymal cell-derived EVs. This study proposes a novel microfluidic chip for selectively separating two types of EVs in an efficient and timely manner. The microfluidic chip is fully integrated with a micromixer for the creation of efficient collision between EVs and specific antibody-coated microbeads (7 and 15 µm in diameter) and a hydrodynamic particle separator for the stratification of EVs bound microbeads according to the sizes of microbeads. Using this chip, over 90% of EVs expressing the epithelial marker (epithelial cell adhesion molecule, EpCAM) and the mesenchymal marker (CD49f) can be selectively isolated within 6.7 min per 100 µL of sample volume. The clinical relevance of EMT is investigated using plasma samples from 20 breast cancer patients and 10 age-matched controls. The EMT index produced from the microfluidic chip is in a good agreement with the conventional tissue-based EMT index and is significantly high in patients with aggressive breast cancer subtypes, compared with healthy controls. In addition, the patients with high scores on the EMT index (≥5) shows recurrence within 5 years after adjuvant treatment. Predicting EMT-index-based metastatic risk using our microfluidic chip can be beneficial for cancer diagnosis and prognosis.

Detachable microfluidic device implemented with electrochemical aptasensor (DeMEA) for sequential analysis of cancerous exosomes.

The quantification of cancer-derived exosomes has a strong potential for minimally invasive diagnosis of cancer during its initial stage. As cancerous exosomes form a small fraction of all the exosomes present in blood, ultra-sensitive detection is a prerequisite for the development of exosome-based cancer diagnostics. Herein, a detachable microfluidic device implemented with an electrochemical aptasensor (DeMEA) is introduced for highly sensitive and in-situ quantification of cancerous exosomes. To fabricate the aptasensor, a nanocomposite was applied on the electrode surface followed by electroplating of gold nanostructures. Subsequently, an aptamer against an epithelial cell adhesion molecule is immobilized on the electrode surface to specifically detect cancer-specific exosomes. A microfluidic vortexer is then constructed and implemented in the sensing system to increase the collision between the exosomes and sensing surface using hydrodynamically generated transverse flow. The microfluidic vortexer was integrated with the aptasensor via a 3D printed magnetic housing. The detachable clamping of the two different devices provides an opportunity to subsequently harvest the exosomes for downstream analysis. The DeMEA has high sensitivity and specificity with an ultra-low limit of detection of 17 exosomes/µL over a wide dynamic range (1 × 102 to 1 × 109) exosomes/µL in a short period. As proof of the concept, the aptasensor can be separated from the 3D printed housing to harvest and analyze the exosomes by real-time polymerase chain reaction. Moreover, the DeMEA quantifies the exosomes from plasma samples of patients with breast cancer at different stages of the disease. The DeMEA provides a bright horizon for the application of microfluidic integrated biosensors for the early detection of cancerous biomarkers.

On-chip isolation and enrichment of circulating cell-free DNA using microfluidic device.

Circulating cell-free DNA (cfDNA), containing cancer-specific DNAs derived from tumor cells, plays an important role in real-time monitoring of disease progression. Due to the abnormal growth of cancer and the promotion of cancer cell apoptosis by chemotherapy, the higher cfDNA concentration than healthy individuals is closely correlated with the diagnosis and treatment of cancer. Also, the mutation detection in tumor cell-derived cfDNA can be used to predict tumor progression. Human blood contains many blood cells (red blood cells, white blood cells, and platelets), proteins, extracellular vesicles, and so on. These blood components act as the inhibitors when the cfDNA is analyzed using polymerase chain reaction. So, analysis of cfDNA using whole blood directly may affect the sensitivity of the analysis or result in false-negative. The conventional methods of cfDNA isolation, such as silica absorption and polymer-mediated enrichment, are labor-intensive and time-consuming processes that can also lead to the loss of cfDNA in cumbersome procedures. Here, we designed an integrated microfluidic chip capable of on-chip cfDNA extracting to reduce sample loss and processing time. Our proposed device minimizes the number of experimental steps from 5 to 1, the total processing time from 42 to 19 min, and the required volume of washing reagents from 2 to 0.4 ml for cfDNA enrichment compared to the conventional method.

Progress in Circulating Tumor Cell Research Using Microfluidic Devices.

Circulating tumor cells (CTCs) are a popular topic in cancer research because they can be obtained by liquid biopsy, a minimally invasive procedure with more sample accessibility than tissue biopsy, to monitor a patient's condition. Over the past decades, CTC research has covered a wide variety of topics such as enumeration, profiling, and correlation between CTC number and patient overall survival. It is important to isolate and enrich CTCs before performing CTC analysis because CTCs in the blood stream are very rare (0⁻10 CTCs/mL of blood). Among the various approaches to separating CTCs, here, we review the research trends in the isolation and analysis of CTCs using microfluidics. Microfluidics provides many attractive advantages for CTC studies such as continuous sample processing to reduce target cell loss and easy integration of various functions into a chip, making "do-everything-on-a-chip" possible. However, tumor cells obtained from different sites within a tumor exhibit heterogenetic features. Thus, heterogeneous CTC profiling should be conducted at a single-cell level after isolation to guide the optimal therapeutic path. We describe the studies on single-CTC analysis based on microfluidic devices. Additionally, as a critical concern in CTC studies, we explain the use of CTCs in cancer research, despite their rarity and heterogeneity, compared with other currently emerging circulating biomarkers, including exosomes and cell-free DNA (cfDNA). Finally, the commercialization of products for CTC separation and analysis is discussed.

A microfluidic electrochemical aptasensor for enrichment and detection of bisphenol A.

Bisphenol A (BPA) is an organic monomer used to make common consumer goods such as plastic containers, sports equipment, and cosmetics which are heavily produced worldwide. A growing interest has been drawn to general public as BPA is one of the major endocrine disrupting chemicals threating human health. To date, numerous BPA sensors have been attempted to be developed but important challenges still remained such as limited linearity range, easy to use, and long term response time. To address the present issues, a microfluidic channel should be integrated into an electrochemical aptasensor and it is called Geometrically Activated Surface Interaction (GASI) chip. The vigorous generation of the micro-vortex in the GASI fluidic chamber provides the high collision chances between BPA and anti-BPA aptamer (BPAPT) and consequently more BPA molecules can be captured on the aptasensor surface, which finally results in high sensitivity of the aptasensor. To construct the integrated aptasensor, a miniaturized gold electrode is fabricated using shadow mask and e-beam evaporation process. Afterward, BPAPT is immobilized on a nanostructured gold electrode via thiol chemistry, and other terminus of the aptamer is labeled with a ferrocene (Fc) redox probe. Then, the microfluidic channel is mounted over the miniaturized gold electrode to introduce and enrich BPA to the aptasensor. Upon the specific interaction between BPA and its aptamer, configuration of aptamer is changed so that Fc tag approaches to the electrode surface and direct oxidation signal of Fc and BPA are followed as analytical signals. The unique microfluidic integrated electrochemical aptasensor delivers a wide linear dynamic range over 5 × 10-12 to 1 × 10-9 M, with a limit of detection 2 × 10-13 M. This aptasensor provides a precise platform for simple, selective and more importantly rapid detection of BPA. Such kind of sensing platforms can serve as a fertile ground for designing miniaturized portable sensors.

Graphene-Iodine Nanocomposites: Highly Potent Bacterial Inhibitors that are Bio-compatible with Human Cells.

Graphene-composites, capable of inhibiting bacterial growth which is also bio-compatible with human cells have been highly sought after. Here we report for the first time the preparation of new graphene-iodine nano-composites via electrostatic interactions between positively charged graphene derivatives and triiodide anions. The resulting composites were characterized by X-ray photoemission spectroscopy, UV-spectroscopy, Raman spectroscopy and Scanning electron microscopy. The antibacterial potential of these graphene-iodine composites against Klebsiella pneumonia, Pseudomonas aeruginosa, Proteus mirobilis, Staphylococcus aureus, and E. coli was investigated. In addition, the cytotoxicity of the nanocomposite with human cells [human white blood cells (WBC), HeLa, MDA-MB-231, Fibroblast (primary human keratinocyte) and Keratinocyte (immortalized fibroblast)], was assessed. DGO (Double-oxidizes graphene oxide) was prepared by the additional oxidation of GO (graphene oxide). This generates more oxygen containing functional groups that can readily trap more H(+), thus generating a positively charged surface area under highly acidic conditions. This step allowed bonding with a greater number of anionic triiodides and generated the most potent antibacterial agent among graphene-iodine and as-made povidone-iodine (PVP-I) composites also exhibited nontoxic to human cells culture. Thus, these nano-composites can be used to inhibit the growth of various bacterial species. Importantly, they are also very low-cytotoxic to human cells culture.

Isolation and enrichment of circulating biomarkers for cancer screening, detection, and diagnostics.

Much research has been performed over the past several decades in an attempt to conquer cancer. Tissue biopsy is the conventional method for gathering biological materials to analyze cancer and has contributed greatly to the understanding of cancer. However, this method is limited because it is time-consuming (requires tissue sectioning, staining, and pathological analysis), costly, provides scarce starting materials for multiple tests, and is painful. A liquid biopsy, which analyzes cancer-derived materials from various body fluids using a minimally invasive procedure, is more practical for real-time monitoring of disease progression than tissue biopsy. Biomarkers analyzable through liquid biopsy include circulating tumor cells (CTCs), exosomes, circulating cell-free DNA (cfDNA), miRNA, and proteins. Research on CTCs has been actively conducted because CTCs provide information on the whole cell, unlike the other biomarkers mentioned above. However, owing to the rarity and heterogeneity of CTCs, CTC research faces many critical concerns. Although exosomes and cfDNA have some technical challenges, they are being highlighted as new target materials. That is because they also have genetic information on cancers. Even though the number of exosomes and cfDNA from early stage cancer patients are similar to healthy individuals, they are present in high concentrations after metastasis. In this article, we review several technologies for material analyses of cancer, discuss the critical concerns based on hands-on experience, and describe future directions for cancer screening, detection, and diagnostics.