Studying neurotoxicological responses in a physiologically relevant and translatable manner remains a major challenge in biomedical research. Consequently, there has been a major push toward establishing human tissue-native approaches in research and diagnostic pipelines. Here, we present a transparent microfluidic lab-on-a-chip platform integrating an embedded array of enzymatic electrochemical glutamate sensors for real-time monitoring of extracellular neurotransmitter dynamics in human induced pluripotent stem cell-derived (hiPSC) neuronal cell cultures. The system enables continuous, multimodal-compatible interrogation of cellular responses under controlled microenvironmental conditions. We validated the platform by measuring glutamate dynamics in under baseline conditions and following exposure to the environmental neurotoxins methylmercury (MeHg) and manganese (Mn); both known to alter glutamate dynamics. The sensors exhibited stable operation over more than one week in culture and reliably detected glutamate transients with concentrations up to 120 μM glutamate. MeHg exposure resulted in significant alterations in extracellular glutamate relative to control conditions, indicating disrupted glutamate homeostasis. Similarly, neuronal cultures exposed to 500 μM Mn for 24 h demonstrated significantly altered glutamate uptake dynamics. These results validate the proposed platform as a robust tool for investigating neurotoxin-induced perturbations in glutamatergic signaling and demonstrate the feasibility of integrating electrochemical enzymatic sensing into microfluidic systems for neurotoxicity research and discovery.

Micro and nano-scale colloidal particles can be rapidly assembled at electrode-electrolyte interfaces in highly organized structures. These particles aggregate through various forces which are, among others, chemical, electrical, and thermal in nature. Patterning biological cells in such structures has high impact applications but it remains a challenge due to their limited viability under said manipulation forces. Rapid electrokinetic patterning (REP) uses AC electrothermal micro-vortices to aggregate both synthetic particles and biological cells. In this work, we explore the effects of a DC offset on a REP trap performance in bio-relevant isotonic medium. REP traps were characterized by measuring the inter-particle distance under different DC offsets, using a Delaunay triangulation. The inter-particle distance was measured in a steady-state trap followed by the disassembly of the aggregate as the REP vortex was turned off. DC offset enhanced the trapping performance for micro-particles suspended in a sugar-based isotonic medium. It was observed that an increasingly negative DC offset increased the steady-state inter-particle distance and reduced the rate of disassembly. However, no such trend was found with positive DC offsets. Changing the offset in small steps (<500 mV) affected only the inter-particle distance whereas, larger steps significantly affected the trap stability and size.

Microfluidics has enabled the analysis of several biochemical phenomena crucial to life as we know it. Encapsulation of target molecules, cells, or synthetic particles in micro and nanodroplets enables sensitive single-cell analysis. Droplet microfluidic technologies reduce the quantities of the required target molecules, which are often limited, whereas digital microfluidic technologies enable easy and repeatable manipulation of these droplets. Various mass spectrometry techniques such as electrospray ionization (ESI) and matrix-assisted or surface-assisted laser desorption-ionization (MALDI or SALDI) have resulted in pioneering research in the conception and detection of novel chemical compounds, proteins, peptides, and metabolites. Biological cell-sorting technologies such as fluorescence-activated cell sorting (FACS) have promoted multiparameter cell detection and sorting. Micromanipulation techniques such as rapid electrokinetic patterning (REP), optoelectrowetting (OEW), and others employ noninvasive optically activated electrokinetic traps to capture, transport, sort, and deposit synthetic particles and biological cells. Integration of these technologies with droplet and digital microfluidics has extended the frontiers of many fields with biochemical applications ranging from (but not limited to) pharmaceutics and sustainable agriculture to multiomics and CRISPR technologies. This chapter discusses many such microfluidic tools advancing the fields of mass spectrometry, FACS, and noninvasive micromanipulation.

Non-contact micro-manipulation tools have enabled invasion-free studies of fragile synthetic particles and biological cells. Rapid electrokinetic patterning (REP) traps target particles/cells, suspended in an electrolyte, on an electrode surface. This entrapment is electrokinetic in nature and thus depends strongly on the suspension medium's properties. REP has been well characterized for manipulating synthetic particles suspended in low concentration salt solutions (~ 2 mS/m). However, it is not studied as extensively for manipulating biological cells, which introduces an additional level of complexity due to their limited viability in hypotonic media. In this work, we discuss challenges posed by isotonic electrolytes and suggest solutions to enable REP manipulation in bio-relevant media. Various formulations of isotonic media (salt and sugar-based) are tested for their compatibility with REP. REP manipulation is observed in low concentration salt-based media such as 0.1× phosphate buffered saline (PBS) when the device electrodes are passivated with a dielectric layer. We also show manipulation of murine pancreatic cancer cells suspended in a sugar-based (8.5% w/v sucrose and 0.3% w/v dextrose) isotonic medium. The ability to trap mammalian cells and deposit them in custom patterns enables high-impact applications such as determining their biomechanical properties and 3D bioprinting for tissue scaffolding.

Trapping, sorting, transportation, and manipulation of synthetic microparticles and biological cells enable investigations in their behavior and properties. Microfluidic techniques like rapid electrokinetic patterning (REP) provide a non-invasive means to probe into the nature of these micro and nanoparticles. The opto-electrically induced nature of a REP micro vortex allows tuning of the trap characteristics in real-time. In this work, we studied the effects of transient optical heating on the induced electrothermal vortex using micro-particle image velocimetry (mu-PIV) and computational modeling. A near infra-red (980 nm) laser beam was focused on a colloidal suspension of 1 mu m polystyrene beads sandwiched between two parallel-plate electrodes. The electrodes were subjected to an AC current. The laser spot was scanned back-and-forth in a line, at different frequencies, to create the transient vortex. This phenomenon was also studied with a computational model made using COMSOL Multiphysics. We visualize fluid flow in custom-shaped REP traps by superposing multiple axisymmetric (spot) vortices and discuss the limitations of using superposition in dynamically changing traps.

Colloidal particles like polystyrene beads and metallic micro and nanoparticles are known to assemble in crystal-like structures near an electrode surface under both DC and AC electric fields. Various studies have shown that this self-assembly is governed by a balance between an attractive electrohydrodynamic (EHD) force and an induced dipole-dipole repulsion (Trau et al., 1997). The EHD force originates from electrolyte flow caused by interaction between the electric field and the polarized double layers of both the particles and the electrode surface. The particles are found to either aggregate or repel from each other on application of electric field depending on the mobility of the ions in the electrolyte (Woehl et al., 2014). The particle motion in the electrode plane is studied well under various conditions however, not as many references are available in the literature that discuss the effects of the AC electric field on their out-of-plane motion, especially at high frequencies (>10 kHz). Haughey and Earnshaw (1998), and Fagan et al. (2005) have studied the particle motion perpendicular to the electrode plane and their average height from the electrode mostly in presence of DC or low frequency AC (<1 kHz) electric field. However, these studies do not provide enough insight towards the effects of high frequency (>10 kHz) electric field on the particles’ motion perpendicular to the electrode plane.

Precise manipulation of micro and nanosized particles has enabled investigations into various applications ranging from mechanobiology of biomolecules and cells to self-assembly of two-dimensional colloids. This work is focussed on studying the nature of a noninvasive electrothermal vortex based micro-manipulation tool called rapid electrokinetic patterning (REP). Using the equipartition method, we show that a REP trap is Hookean in nature and has an ultralow trap stiffness on the order of femtonewtons/\ensuremath{\mu}m. The dynamic tunability of an optically induced REP trap makes it a versatile tool for various biophysical applications.