A Novel Microfluidic Device-Based Neurite Outgrowth Inhibition Assay Reveals the Neurite Outgrowth-Promoting Activity of Tropomyosin Tpm3.1 in Hippocampal Neurons.

Overcoming neurite inhibition is integral for restoring neuronal connectivity after CNS injury. Actin dynamics are critical for neurite growth cone formation and extension. The tropomyosin family of proteins is a regarded as master regulator of actin dynamics. This study investigates tropomyosin isoform 3.1 (Tpm3.1) as a potential candidate for overcoming an inhibitory substrate, as it is known to influence neurite branching and outgrowth. We designed a microfluidic device that enables neurons to be grown adjacent to an inhibitory substrate, Nogo-66. Results show that neurons, overexpressing hTpm3.1, have an increased propensity to overcome Nogo-66 inhibition. We propose Tpm3.1 as a potential target for promoting neurite growth in an inhibitory environment in the central nervous system.

Microfluidic-SERS Technologies for CTC: A Perspective on Clinical Translation.

Enumeration and phenotypic profiling of circulating tumor cells (CTCs) provide critical information for clinical diagnosis and treatment monitoring in cancer. To achieve this goal, an integrated system is needed to efficiently isolate CTCs from patient samples and sensitively evaluate their phenotypes. Such integration would comprise a high-throughput single-cell processing unit for the isolation and manipulation of CTCs and a sensitive and multiplexed quantitation unit to detect clinically relevant signals from these cells. Surface-enhanced Raman scattering (SERS) has been used as an analytical method for molecular profiling and in vitro cancer diagnosis. More recently, its multiplexing capability and power to create distinct molecular signatures against their targets have garnered attention. Here, we share our insights into the combined power of microfluidics and SERS in realizing CTC isolation, enumeration, and detection from a clinical translation perspective. We highlight the key operational factors in CTC microfluidic processing and SERS detection from patient samples. We further discuss microfluidic-SERS integration and its clinical utility as a paradigm shift in clinical CTC-based cancer diagnosis and prognostication. Finally, we summarize the challenges and attempt to look forward to what lies ahead of us in potentially translating the technique into real clinical applications.

SERS biosensors for liquid biopsy towards cancer diagnosis by detection of various circulating biomarkers: current progress and perspectives.

Liquid biopsy has emerged as a promising non-invasive strategy for cancer diagnosis, enabling the detection of various circulating biomarkers, including circulating tumor cells (CTCs), circulating tumor nucleic acids (ctNAs), circulating tumor-derived small extracellular vesicles (sEVs), and circulating proteins. Surface-enhanced Raman scattering (SERS) biosensors have revolutionized liquid biopsy by offering sensitive and specific detection methodologies for these biomarkers. This review comprehensively examines the application of SERS-based biosensors for identification and analysis of various circulating biomarkers including CTCs, ctNAs, sEVs and proteins in liquid biopsy for cancer diagnosis. The discussion encompasses a diverse range of SERS biosensor platforms, including label-free SERS assay, magnetic bead-based SERS assay, microfluidic device-based SERS system, and paper-based SERS assay, each demonstrating unique capabilities in enhancing the sensitivity and specificity for detection of liquid biopsy cancer biomarkers. This review critically assesses the strengths, limitations, and future directions of SERS biosensors in liquid biopsy for cancer diagnosis.

Emerging integrated SERS-microfluidic devices for analysis of cancer-derived small extracellular vesicles.

Cancer-derived small extracellular vesicles (sEVs) are specific subgroups of lipid bilayer vesicles secreted from cancer cells to the extracellular environment. They carry distinct biomolecules (e.g., proteins, lipids and nucleic acids) from their parent cancer cells. Therefore, the analysis of cancer-derived sEVs can provide valuable information for cancer diagnosis. However, the use of cancer-derived sEVs in clinics is still limited due to their small size, low amounts in circulating fluids, and heterogeneous molecular features, making their isolation and analysis challenging. Recently, microfluidic technology has gained great attention for its ability to isolate sEVs in minimal volume. In addition, microfluidics allows the isolation and detection of sEVs to be integrated into a single device, offering new opportunities for clinical application. Among various detection techniques, surface-enhanced Raman scattering (SERS) has emerged as a promising candidate for integrating with microfluidic devices due to its ultra-sensitivity, stability, rapid readout, and multiplexing capability. In this tutorial review, we start with the design of microfluidics devices for isolation of sEVs and introduce the key factors to be considered for the design, and then discuss the integration of SERS and microfluidic devices by providing descriptive examples of the currently developed platforms. Lastly, we discuss the current limitations and provide our insights for utilising integrated SERS-microfluidics to isolate and analyse cancer-derived sEVs in clinical settings.

Capillary-assisted microfluidic biosensing platform captures single cell secretion dynamics in nanoliter compartments.

Cancer cells continuously secrete inflammatory biomolecules which play significant roles in disease progression and tumor metastasis toward secondary sites. Despite recent efforts to capture cancer cells' intercellular secretion heterogeneity using microfluidics, the challenges in operation of these systems as well as the complexity of designing a biosensing assay for long-term and real-time measurement of single cell secretions have become grand research barriers. Here, we present a new capillary-based microfluidic biosensing approach to easily and reliably capture ~500 single cells inside isolated dead-end nanoliter compartments using simple pipette injection, and quantify their individual secretion dynamics at the single cell resolution over a long period of culture (~16 h). We first present a detailed investigation of the fluid mechanics underlying the formation of nanoliter compartments in the microfluidic system. Based on the measurement of single cell capture efficiency, we employ a one-step FRET-based biosensor which monitors the single cancer cells' protease activity. The sensor reports the fluorescent signal as a product of amino acid chain cleavage and reduction in its quenching capability. Using the single cell protease secretion data, we identified modes of cell secretion dynamics in our cell sample. While most of the cells had low secretion levels, two other smaller and more aggressive secretion dynamics were cells with secretion modes that include sharp spikes or slow but progressive trend. The method presented here overcomes the difficulties associated with performing single cell secretion assays, enabling a feasible and reliable technique for high throughput measurement of metabolic activities in cancer cells.

Microfluidic Actuation via 3D-Printed Molds toward Multiplex Biosensing of Cell Apoptosis.

Multiplexed analysis of biochemical analytes such as proteins, enzymes, and immune products using a microfluidic device has the potential to cut assay time, reduce sample volume, realize high-throughput, and decrease experimental error without compromising sensitivity. Despite these huge benefits, the need for expensive specialized equipment and the complex photolithography fabrication process for the multiplexed devices have, to date, prevented widespread adoption of microfluidic systems. Here, we present a simple method to fabricate a new microfluidic-based multiplexed biosensing device by taking advantage of 3D-printing. The device is an integration of normally closed (NC) microfluidic valving units which offer superior operational flexibility by using PDMS membrane (E ∼ 1-2 MPa) and require minimized energy input (1-5 kPa). To systematically engineer the device, we first report on the geometrical and operational analysis of a single 3D-printed valving unit. Based on the characterization, we introduce a full prototype multiplexed chip comprising several microfluidic valves. The prototype offers-for the first time in a 3D-printed microfluidic device-the capability of on-demand performce of both a sequential and a parallel biochemical assay. As a proof of concept, our device has been used to simultaneously measure the apoptotic activity of 5 different members of the caspase protease enzyme family. In summary, the 3D-printed valving system showcased in this study overcomes traditional bottlenecks of microfabrication, enabling a new class of sophisticated liquid manipulation required in performing multiplexed sensing for biochemical assays.

Static droplet array for culturing single live adherent cells in an isolated chemical microenvironment.

We present here a new method to easily and reliably generate an array of hundreds of dispersed nanoliter-volume semi-droplets for single-cells culture and analysis. The liquid segmentation step occurs directly in indexed traps by a tweezer-like mechanism and is stabilized by spatial confinement. Unlike common droplet-based techniques, the semi-droplet wets its surrounding trap walls thus supporting the culturing of both adherent and non-adherent cells. To eliminate cross-droplet cell migration and chemical cross-talk each semi-droplet is separated from a nearby trap by an ∼80 pL air plug. The overall setup and injection procedure takes less than 10 minutes, is insensitive to fabrication defects and supports cell recovery for downstream analysis. The method offers a new approach to easily capture, image and culture single cells in a chemically isolated microenvironment as a preliminary step towards high-throughput single-cell assays.

An easily fabricated three-dimensional threaded lemniscate-shaped micromixer for a wide range of flow rates.

Mixing fluid samples or reactants is a paramount function in the fields of micro total analysis system (µTAS) and microchemical processing. However, rapid and efficient fluid mixing is difficult to achieve inside microchannels because of the difficulty of diffusive mass transfer in the laminar regime of the typical microfluidic flows. It has been well recorded that the mixing efficiency can be boosted by migrating from two-dimensional (2D) to three-dimensional (3D) geometries. Although several 3D chaotic mixers have been designed, most of them offer a high mixing efficiency only in a very limited range of Reynolds numbers (Re). In this work, we developed a 3D fine-threaded lemniscate-shaped micromixer whose maximum numerical and empirical efficiency is around 97% and 93%, respectively, and maintains its high performance (i.e., >90%) over a wide range of 1 < Re < 1000 which meets the requirements of both the µTAS and microchemical process applications. The 3D micromixer was designed based on two distinct mixing strategies, namely, the inducing of chaotic advection by the presence of Dean flow and diffusive mixing through thread-like grooves around the curved body of the mixers. First, a set of numerical simulations was performed to study the physics of the flow and to determine the essential geometrical parameters of the mixers. Second, a simple and cost-effective method was exploited to fabricate the convoluted structure of the micromixers through the removal of a 3D-printed wax structure from a block of cured polydimethylsiloxane. Finally, the fabricated mixers with different threads were tested using a fluorescent microscope demonstrating a good agreement with the results of the numerical simulation. We envisage that the strategy used in this work would expand the scope of the micromixer technology by broadening the range of efficient working flow rate and providing an easy way to the fabrication of 3D convoluted microstructures.

A rapid co-culture stamping device for studying intercellular communication.

Regulation of tissue development and repair depends on communication between neighbouring cells. Recent advances in cell micro-contact printing and microfluidics have facilitated the in-vitro study of homotypic and heterotypic cell-cell interaction. Nonetheless, these techniques are still complicated to perform and as a result, are seldom used by biologists. We report here development of a temporarily sealed microfluidic stamping device which utilizes a novel valve design for patterning two adherent cell lines with well-defined interlacing configurations to study cell-cell interactions. We demonstrate post-stamping cell viability of >95%, the stamping of multiple adherent cell types, and the ability to control the seeded cell density. We also show viability, proliferation and migration of cultured cells, enabling analysis of co-culture boundary conditions on cell fate. We also developed an in-vitro model of endothelial and cardiac stem cell interactions, which are thought to regulate coronary repair after myocardial injury. The stamp is fabricated using microfabrication techniques, is operated with a lab pipettor and uses very low reagent volumes of 20 µl with cell injection efficiency of >70%. This easy-to-use device provides a general strategy for micro-patterning of multiple cell types and will be important for studying cell-cell interactions in a multitude of applications.