Advanced manufacturing of nanoparticle formulations of drugs and biologics using microfluidics.

Numerous innovative nanoparticle formulations of drugs and biologics, named nano-formulations, have been developed in the last two decades. However, methods for their scaled-up production are still lagging, as the amount needed for large animal tests and clinical trials is typically orders of magnitude larger. This manufacturing challenge poses a critical barrier to successfully translating various nano-formulations. This review focuses on how microfluidics technology has become a powerful tool to overcome this challenge by synthesizing various nano-formulations with improved particle properties and product purity in large quantities. This microfluidic-based manufacturing is enabled by microfluidic mixing, which is capable of the precise and continuous control of the synthesis of nano-formulations. We further discuss the specific applications of hydrodynamic flow focusing, a staggered herringbone micromixer, a T-junction mixer, a micro-droplet generator, and a glass capillary on various types of nano-formulations of polymeric, lipid, inorganic, and nanocrystals. Various separation and purification microfluidic methods to enhance the product purity are reviewed, including acoustofluidics, hydrodynamics, and dielectrophoresis. We further discuss the challenges of microfluidics being used by broader research and industrial communities. We also provide future outlooks of its enormous potential as a decentralized approach for manufacturing nano-formulations.

Electrochemical detection and analysis of tumor-derived extracellular vesicles to evaluate malignancy of pancreatic cystic neoplasm using integrated microfluidic device.

Tumor-derived extracellular vesicles (tdEVs) are one of the most promising biomarkers for liquid biopsy-based cancer diagnostics, owing to the expression of specific membrane proteins of their cellular origin. The investigation of epithelial-to-mesenchymal transition (EMT) in cancer using tdEVs is an alternative way of evaluating the risk of malignancy transformation. An ultra-sensitive selection and detection methodology is an essential step in developing a tdEVs-based cancer diagnostic device. In this study, we developed an indium-tin-oxide (ITO) sensor integrated microfluidic device consisting of two main parts: 1) a multi-orifice flow-fractionation (MOFF) channel for extraction of pure EVs by removing blood cellular debris, and 2) an ITO sensor coupled with a geometrically activated surface interaction (GASI) channel for enrichment and quantification of tdEV. The microfluidic channel and the ITO sensors are assembled with a 3D printed magnetic housing to prevent sample leakage and to easily attach/detach the sensors to/from the microfluidic channel. The tdEVs were successfully captured on the specific antibody modified ITO surfaces in the integrated microfluidic channel. The integrated sensors showed an excellent linear response between 103 and 109 tdEVs/mL. Simultaneous evaluation of the epithelial and mesenchymal markers on the tdEV surfaces successfully revealed the EMT index of the corresponding pancreatic cancer cells. Our ITO sensor integrated microfluidic device showed excellent detection in the clinically relevant tdEVs-concentration range for patients with pancreatic cystic neoplasms. Hence, this system is expected to open a new avenue for liquid biopsy-based cancer prognostics and diagnostics.

Coil spring-powered pump with inertial microfluidic chip for size-based isolation and enrichment of biological cells.

Microfluidic chips have been widely used for in vitro diagnostics using pretreatment of biological samples; however, biologists and clinical researchers have difficulties using them in resource-limited settings. Sample injection systems for microfluidic chips are bulky, expensive, electricity-powered, and complex. A coiled spring-powered device, which can be used to isolate variously sized cells with high efficiency continuously and passively, was developed for portable, low-cost, electricity-free, and simple sample injection. The flow driving power was provided by releasing the compression spring in the mechanical syringe driver with a one-click action. In general, a syringe pump generates a stable passive flow rate. However, the syringe pumps are large in size and expensive because they have many functions such as infusion/withdrawal flow injection and the use of syringes of various sizes, allowing them to be applied in a variety of applications performed in the laboratory. In addition, it is not suitable for portable devices because of the considerable amount of electric power required. To overcome these drawbacks, we developed a device prototype that sorts different-sized particles and separates rare tumor cells or blood cells from blood with high efficiency. The performance of the coiled spring-powered device was evaluated and found to be comparable with that of syringe pump-powered devices. In situations where trained personnel cannot handle microfluidic chips for isolating circulating biomarkers (CTCs, WBCs, or plasma) from blood samples, the coiled spring-powered device can provide diagnostic tools, especially in resource-limited countries.

A modular microfluidic platform for serial enrichment and harvest of pure extracellular vesicles.

Extracellular vesicles (EVs) are recognized as promising biomarkers for several diseases. However, their conventional isolation methods have several drawbacks, such as poor yields, low purity, and time-consuming operations. Therefore, a simple, low-cost, and rapid microfluidic platform has been extensively developed to meet the requirement in biomedical applications. Herein, a modular microfluidic platform is demonstrated to isolate and enrich EVs directly from plasma, in a combination of continuous capture and purification of EVs. The EVs were selectively captured by target-specific antibody-coated beads in a horseshoe-shaped orifice micromixer (HOMM) chip within 2 min. A fish-trap-shaped microfilter unit was subsequently used to elute and purify the affinity-induced captured EVs from the microbeads. The ability of the modular chip to capture, enrich, and release EVs was demonstrated in 5 min (100 µL sample) at high throughput (100 µL min-1). The two chips can be modularized or individually operated, depending on the clinical applications such as diagnostics and therapeutics. For the diagnostic applications, the EVs on microbeads can be directly subjected to the molecular analysis whereas the pure EVs should be released from the microbeads for the therapeutic treatments. This study reveals that the fabricated modular chip can be appropriately employed as a platform for EV-related research tools.

Multi-miRNA panel of tumor-derived extracellular vesicles as promising diagnostic biomarkers of early-stage breast cancer.

Extracellular vesicles (EV) have been emerging as potential biomarkers for disease monitoring. In particular, tumor-derived EV (TDE) are known to carry oncogenic miRNA, so they can be used for diagnosis of early cancer by analyzing the expression levels of EV-miRNA circulating in the blood. Here, using our novel microfluidic device, we rapidly and selectively isolate cancerous EV expressing breast cancer-derived surface markers CD49f and EpCAM within 2 minutes. Based on seven candidates of miRNA nominated from The Cancer Genome Atlas (TCGA) database, the expression levels of miRNA in TDE were validated in a total of 82 individuals, including 62 breast cancer patients and 20 healthy controls. Among seven candidates, four miRNAs (miR-9, miR-16, miR-21, and miR-429) from the EV were highly elevated in early-stage breast cancer patients compared with healthy donors. The combination of significant miRNAs from specific EV has high sensitivities of 0.90, 0.86, 0.88, and 0.84 of the area under the receiver operating characteristic curve (AUC) in each subtype (luminal A, luminal B, HER-2, and triple-negative) of early-stage breast cancer. Our results suggest that the combination of four miRNA signatures of specific EV could serve as a sensitive and specific biomarker and enable early diagnosis of breast cancer using liquid biopsy.

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.

Salivary Exosome and Cell-Free DNA for Cancer Detection.

Liquid biopsies are easier to acquire patient derived samples than conventional tissue biopsies, and their use enables real-time monitoring of the disease through continuous sampling after initial diagnosis, resulting in a paradigm shift to customized treatment according to the patient's prognosis. Among the various liquid biopsy samples, saliva is easily obtained by spitting or swab sucking without needing an expert for sample collection. In addition, it is known that disease related biomarkers that exist in the blood and have undergone extensive research exist in saliva even at a lower concentration than the blood. Thus, interest in the use of saliva as a liquid biopsy has increased. In this review, we focused on the salivary exosome and cell-free DNA (cfDNA) among the various biomarkers in saliva. Since the exosome and cfDNA in saliva are present at lower concentrations than the biomarkers in blood, it is important to separate and concentrate them before conducting down-stream analyses such as exosome cargo analysis, quantitative polymerase chain reaction (qPCR), and sequencing. However, saliva is difficult to apply directly to microfluidics-based systems for separation because of its high viscosity and the presence of various foreign substances. Therefore, we reviewed the microfluidics-based saliva pretreatment method and then compared the commercially available kit and the microfluidic chip for isolation and enrichment of the exosome and cfDNA in saliva.

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.

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.