Electrokinetic Desalting and Salting of Water-in-Oil Droplets.

Droplet-based microfluidic platforms demand modifications to the droplet composition to facilitate reactions and analyses. However, limited techniques exist to modify the droplet contents post their generation. Here, ion transport across two ion-exchange membranes possessing distinct selectivity is employed to introduce ions into (salt) or extract ions from (desalt) water-in-oil droplets. The ion concentration distribution and transport mechanisms are visualized using a precipitation reaction and a charged fluorescent tracer. Furthermore, current measurements reveal characteristic regimes in desalting and salting modes and demonstrate that the rates of ion transport linearly correlate with applied voltage and the ionic strength of the droplets. Importantly, up to 98% desalting efficiency is achieved. This technique advances droplet-based sample preparation through the straightforward manipulation of droplet contents.

Label-Free, Non-Optical Readout of Bead-Based Immunoassays with and without Electrokinetic Preconcentration.

In this article, we report a microfluidic bead-based lateral flow immunoassay (LFIA) with a novel sensing mechanism for label-free, non-optical detection of protein binding. This device comprises two packed beds of microbeads: first, bioconjugated microbeads that serve as a test line, and second, a three-dimensional (3D) electrode for sensing. As the protein target binds the bioconjugated microbeads, a shift in ionic conductivity across the bioconjugated beads is produced and can be directly measured at the surface of the 3D electrode by obtaining current-voltage curves before and after incubation of the analyte. We use a model antigen, rabbit IgG, for quantitative evaluation of this sensor, obtaining a limit of detection (LOD) of 50 nM for the LFIA. We demonstrate that this device can be used to measure binding kinetics, exhibiting a rapid (<3 min) increase in the signal after the introduction of the analyte and an exponential decay in the signal after replacing the sample with buffer only. To improve the LOD of our system, we implement an electrokinetic preconcentration technique, faradaic ion concentration polarization (fICP), to increase the local concentration of antigen available during binding as well as the time the antigen interacts with the test line. Our results indicate that this enrichment-enhanced assay (fICP-LFIA) has an LOD of 370 pM, an 135-fold improvement over the LFIA and a 7-fold improvement in sensitivity. We anticipate that this device can be readily adapted for point-of-care diagnostics and translated to any desired protein target by simply modifying the biorecognition agent on these off-the-shelf microbeads.

Parallel Dielectrophoretic Capture, Isolation, and Electrical Lysis of Individual Breast Cancer Cells to Assess Variability in Enzymatic Activity.

Tumor cell heterogeneity drives disease progression and response to therapy, and therefore, there is a need for single-cell analysis methods. In this paper, we present an integrated, scalable method to analyze enzymatic activity in many individual cancer cells at once. The reported method uses dielectrophoresis (DEP) to selectively capture tumor cells at wireless electrodes aligned to an overlying array of cell-sized micropockets. Following hydrodynamic transfer of the captured cells into microfluidic chambers, the chambers are fluidically isolated and sealed with a hydrophobic ionic liquid, which possesses sufficient conductivity to allow for subsequent electrical lysis of the cells to access their contents for enzymatic assay. The wireless electrodes have an interlocking spiral design that ensures successful electrical lysis regardless of the location of the cell within the chamber. Here, breast cancer cells are assessed for ß-galactosidase through its activation of a fluorogenic substrate. A key point is that the fluorogenic assay solution was optimized to allow for dielectrophoretic cell capture, thereby obviating the need for a solution exchange step. Our approach has several distinct advantages including a high rate of single-cell capture, a capture efficiency that is independent of the dimensions of the reaction chambers, no need for mechanical closure of reaction volumes, and no observed cross-talk. In this study, first, the steps of cell capture, transfer, and lysis are established on this platform in the presence of the optimized assay solution. We then quantify the increase in fluorescence intensity obtained over the duration of the enzymatic assay of individual cells. Finally, this method is applied to the analysis of ß-galactosidase activity in 258 individual MDA-MB-231 breast cancer cells, revealing heterogeneity in expression of this enzyme in this cell line. We expect that the adaptability of this method will allow for expanded studies of single-cell enzymatic expression and activity. This will in turn open avenues of research into cancer cell heterogeneity in metabolism, invasiveness, and drug response. The ability to study these features of cancer at the single-cell level raises the possibility for treatment plans tailored to target the specific combinations of cell subpopulations present in tumors. Furthermore, we expect that this method can be adapted to uses outside of cancer research, such as studies of neuron metabolism, pathogenesis in bacteria, and stem cell development.

In-Droplet Electromechanical Cell Lysis and Enhanced Enzymatic Assay Driven by Ion Concentration Polarization.

Droplets enable the encapsulation of cells for their analysis in isolated domains. The study of molecular signatures (including genes, proteins, and metabolites) from a few or single cells is critical for identifying key subpopulations. However, dealing with biological analytes at low concentrations requires long incubation times and amplification to achieve the requisite signal strength. Further, cell lysis requires additional chemical lysing agents or heat, which can interfere with assays. Here, we leverage ion concentration polarization (ICP) in droplets to rapidly lyse breast cancer cells within 2 s under a DC voltage bias of 30 V. Numerical simulations attribute cell lysis to an ICP-based electric field and shear stress. We further achieve up to 19-fold concentration enrichment of an enzymatic assay product resulting from cell lysis and a 3.8-fold increase in the reaction rate during enrichment. Our technique for sensitive in-droplet cell analysis provides scope for rapid, high-throughput detection of low-abundance intracellular analytes.

Concentration Enrichment, Separation, and Cation Exchange in Nanoliter-Scale Water-in-Oil Droplets.

Droplet-based techniques have had a profound impact in chemistry, owing to their ability to perform rapid and massively parallel reactions in minute fluid volumes. In many applications, concentration enrichment is required to increase the speed of reactions or the sensitivity of assays; but in-droplet concentration enrichment remains challenging. Here, we interface electrokinetic concentration polarization with droplet microfluidics to accomplish in-droplet demixing. This result is significant because the concentration of any charged species in the droplet can be enriched and the approach can be readily integrated into existing droplet workflows. Further, we show that such electrokinetic enrichment is rapid, on the order of seconds, and is robust, occurring over a wide parametric space. We further demonstrate electrokinetic separation of two anionic fluorophores within the droplet. Such a capability potentiates the droplet-templated synthesis of particles with gradient composition and the development of mobility-shift assays, which rely on discrimination of multiple species tagged with a single color fluorophore. Finally, by using a calcium-binding dye as an indicator, we demonstrate in-droplet cation exchange. This demonstration of cation exchange in droplets is significant because of its broad applicability to strategies for synthesis and bioassays. These results lay the foundation for new advanced droplet techniques with transformative applications.

Solid-Phase Microextraction Enables Isolation of BRAF V600E Circulating Tumor DNA from Human Plasma for Detection with a Molecular Beacon Loop-Mediated Isothermal Amplification Assay.

Circulating tumor DNA (ctDNA) is a promising biomarker that can provide a wealth of information regarding the genetic makeup of cancer as well as provide a guide for monitoring treatment. Methods for rapid and accurate profiling of ctDNA are highly desirable in order to obtain the necessary information from this biomarker. However, isolation of ctDNA and its subsequent analysis remains a challenge due to the dependence on expensive and specialized equipment. In order to enable widespread implementation of ctDNA analysis, there is a need for low-cost and highly accurate methods that can be performed by nonexpert users. In this study, an assay is developed that exploits the high specificity of molecular beacon (MB) probes with the speed and simplicity of loop-mediated isothermal amplification (LAMP) for the detection of the BRAF V600E single-nucleotide polymorphism (SNP). Furthermore, solid-phase microextraction (SPME) is applied for the successful isolation of clinically relevant concentrations (73.26 fM) of ctDNA from human plasma. In addition, the individual effects of plasma salts and protein on the extraction of ctDNA with SPME are explored. The performed work expands the use of MB-LAMP for SNP detection as well as demonstrates SPME as a sample preparation tool for nucleic acid analysis in plasma.

Tuning the Electrochemical Redox Potentials of Catechol with Boronic Acid Derivatives.

A strategy to control the oxidation potential of catechol using borinic acids is presented. Borinic acids reversibly bind catechol to form boron "ate" complexes (BACs) that alter the electron density on the oxygen atoms of catechol and, in turn, the propensity of the catechol toward electrochemical oxidation. The effect of different substituents on the borinic acid are investigated to determine their efficacy in tuning the electron density within the BAC and the resulting oxidation potential.

An Electrokinetic Separation Route to Source Dialysate from Excess Fluid in Blood.

To improve the health of patients with end-stage renal disease, there is a clear need for slow, continuous hemodialysis, and the primary barrier to a wearable device is the requirement of a large reservoir of dialysate. We describe an electrokinetic means of producing dialysate from the excess fluid extant in the peripheral blood of patients undergoing therapy. A critical feature of this process is the retention of essential components of blood, especially serum albumin. In progress toward this goal, we demonstrate the separation of charged from neutral species in blood plasma at a branched microchannel junction by ion concentration polarization (ICP). Further, we introduce a method that reduces the opportunity for damage to proteins and prevents electrode biofouling. The present approach results in as high as 99.7% retention of albumin and successful separation of neutral metabolites and excess fluid to be utilized as a precursor to dialysate.

Cellular dielectrophoresis coupled with single-cell analysis.

In this review, recent advances that leverage dielectrophoretic approaches to accomplish single-cell analysis (both "on-chip" and "off-chip") are discussed with special emphasis on eukaryotic cells. Dielectrophoresis as an electric-field-induced force utilized for cell manipulation can confer selectivity without labeling. Recent technical improvements have increased the volumetric throughput of the separation of cells from complex mixtures, introduced new strategies for massively parallel single-cell confinement for subsequent on-chip analysis, made possible selective transport of individual cells off-chip, and integrated preconcentration and prefocusing steps to enhance dielectrophoretic performance. Collectively, these studies potentiate all-in-one platforms capable of taking as their input complex mixtures of cells and accomplishing single-cell analysis. Assays requiring small reaction volumes (e.g., enzymatic assays, fluorescent in situ hybridization, and immunostaining) have been demonstrated. Still greater opportunities to unravel cell-to-cell variations and for point-of-care applications can be realized by making possible on-chip gene amplification, live-cell assays, and either dielectrophoretic manipulation in native media or on-chip exchange of media. We therefore conclude with a discussion of emerging capabilities in these areas.

A Self-Digitization Dielectrophoretic (SD-DEP) Chip for High-Efficiency Single-Cell Capture, On-Demand Compartmentalization, and Downstream Nucleic Acid Analysis.

The design and fabrication of a self-digitization dielectrophoretic (SD-DEP) chip with simple components for single-cell manipulation and downstream nucleic acid analysis is presented. The device employed the traditional DEP and insulator DEP to create the local electric field that is tailored to approximately the size of single cells, enabling highly efficient single-cell capture. The multistep procedures of cell manipulation, compartmentalization, lysis, and analysis were performed in the integrated microdevice, consuming minimal reagents, minimizing contamination, decreasing lysate dilution, and increasing assay sensitivity. The platform developed here could be a promising and powerful tool in single-cell research for precise medicine.

High-Throughput Selective Capture of Single Circulating Tumor Cells by Dielectrophoresis at a Wireless Electrode Array.

We demonstrate continuous high-throughput selective capture of circulating tumor cells by dielectrophoresis at arrays of wireless electrodes (bipolar electrodes, BPEs). The use of BPEs removes the requirement of ohmic contact to individual array elements, thus enabling otherwise unattainable device formats. Capacitive charging of the electrical double layer at opposing ends of each BPE allows an AC electric field to be transmitted across the entire device. Here, two such designs are described and evaluated. In the first design, BPEs interconnect parallel microchannels. Pockets extruding from either side of the microchannels volumetrically control the number of cells captured at each BPE tip and enhance trapping. High-fidelity single-cell capture was achieved when the pocket dimensions were matched to those of the cells. A second, open design allows many non-targeted cells to pass through. These devices enable high-throughput capture of rare cells and single-cell analysis.

Recent advancements in ion concentration polarization.

In this minireview, we discuss advancements in ion concentration polarization (ICP)-based preconcentration, separation, desalination, and dielectrophoresis that have been made over the past three years. ICP as a means of controlling the distribution of the ions and electric field in a microfluidic device has rapidly expanded its areas of application. Recent advancements have focused on the development of ion-permselective materials with tunable dimensions and surface chemistry, adaptation to paper microfluidics, higher-throughput device geometries, and coupling ICP with other separation (isotachophoresis and dielectrophoresis) and fluidic (valve and droplet microfluidic) strategies. These studies have made great strides toward solving real-world problems such as low-cost and rapid analysis, accessible desalination technology, and single-cell research tools.

Negative dielectrophoretic capture and repulsion of single cells at a bipolar electrode: the impact of faradaic ion enrichment and depletion.

This paper describes the dielectrophoretic (DEP) forces generated by a bipolar electrode (BPE) in a microfluidic device and elucidates the impact of faradaic ion enrichment and depletion (FIE and FID) on electric field gradients. DEP technologies for manipulating biological cells provide several distinct advantages over other cell-handling techniques including label-free selectivity, inexpensive device components, and amenability to single-cell and array-based applications. However, extension to the array format is nontrivial, and DEP forces are notoriously short-range, limiting device dimensions and throughput. BPEs present an attractive option for DEP because of the ease with which they can be arrayed. Here, we present experimental results demonstrating both negative DEP (nDEP) attraction and repulsion of B-cells from each a BPE cathode and anode. The direction of nDEP force in each case was determined by whether the conditions for FIE or FID were chosen in the experimental design. We conclude that FIE and FID zones generated by BPEs can be exploited to shape and extend the electric field gradients that are responsible for DEP force.

Improved detection by ensemble-decision aliquot ranking of circulating tumor cells with low numbers of a targeted surface antigen.

Circulating tumor cells (CTCs) are shed from a solid tumor into the bloodstream and can seed new metastases. CTCs hold promise for cancer diagnosis and prognosis and to increase our understanding of the metastatic process. However, their low numbers in blood and varied phenotypic characteristics make their detection and isolation difficult. One source of heterogeneity among CTCs is molecular: When they leave the primary tumor, these cells must undergo a molecular transition, which increases their mobility and chance of survival in the blood. During this molecular transition, the cells lose some of their epithelial character, which is manifested by the expression of the cell surface antigen known as epithelial cell adhesion molecule (EpCAM). Some tumors shed CTCs that express high levels of EpCAM; others release cells that have a low level of the antigen. Nevertheless, many CTC isolation techniques rely on the detection of EpCAM to discriminate CTCs from other cells in the blood. We previously reported a high-throughput immunofluorescence-based technology that targets EpCAM to rank aliquots of blood for the presence or absence of a CTC. This technology, termed ensemble decision aliquot ranking (eDAR), recovered spiked-in cancer cells (taken from a model EpCAM(high) cell line) from blood at an efficiency of 95%. In this paper, we evaluated eDAR for recovery of cells that have low EpCAM expression and developed an immunofluorescence labeling strategy that significantly enhances the method's performance. Specifically, we used a cocktail of primary antibodies for both epithelial and mesenchymal antigens as well as a dye-linked secondary antibody. The cocktail allowed us to reliably detect a model EpCAM(low) cell line for triple negative breast cancer, MDA-MB-231, with a recovery efficiency of 86%. Most significantly, we observed an average of 6-fold increase in the number of CTCs isolated from blood samples from breast cancer patients. These findings underscore the importance of benchmarking CTC technologies with model cell lines that express both high and low levels of EpCAM.

Establishment of a Four-Cell In Vitro Blood-Brain Barrier Model With Human Primary Brain Cells.

The blood-brain barrier (BBB) constitutes a crucial protective anatomical layer with a microenvironment that tightly controls material transit. Constructing an in vitro BBB model to replicate in vivo features requires the sequential layering of constituent cell types. Maintaining heightened integrity in the observed tight junctions during both the establishment and post-experiment phases is crucial to the success of these models. We have developed an in vitro BBB model that replicates the cellular composition and spatial orientation of in vivo BBB observed in humans. The experiment includes comprehensive procedures and steps aimed at enhancing the integration of the four-cell model. Departing from conventional in vitro BBB models, our methodology eliminates the necessity for pre-coated plates to facilitate cell adhesion, thereby improving cell visualization throughout the procedure. An in-house coating strategy and a simple yet effective approach significantly reduce costs and provides superior imaging of cells and corresponding tight junction protein expression. Also, our BBB model includes all four primary cell types that are structural parts of the human BBB. With its innovative and user-friendly features, our in-house optimized in vitro four-cell-based BBB model showcases novel methodology and provides a promising experimental platform for drug screening processes. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Coating and culture system Basic Protocol 2: Cell seeding and Transwell insert handling Basic Protocol 3: Assessment of model functionality.

New generation of ensemble-decision aliquot ranking based on simplified microfluidic components for large-capacity trapping of circulating tumor cells.

Ensemble-decision aliquot ranking (eDAR) is a sensitive and high-throughput method to analyze circulating tumor cells (CTCs) from peripheral blood. Here, we report the next generation of eDAR, where we designed and optimized a new hydrodynamic switching scheme for the active sorting step in eDAR, which provided fast cell sorting with an improved reproducibility and stability. The microfluidic chip was also simplified by incorporating a functional area for subsequent purification using microslits fabricated by standard lithography method. Using the reported second generation of eDAR, we were able to analyze 1 mL of whole-blood samples in 12.5 min, with a 95% recovery and a zero false positive rate (n = 15).

Label-Free Electrochemical Methods for Disease Detection.

Label-free electrochemical biosensing leverages the advantages of label-free techniques, low cost, and fewer user steps, with the sensitivity and portability of electrochemical analysis. In this review, we identify four label-free electrochemical biosensing mechanisms: (a) blocking the electrode surface, (b) allowing greater access to the electrode surface, (c) changing the intercalation or electrostatic affinity of a redox probe to a biorecognition unit, and (d) modulating ion or electron transport properties due to conformational and surface charge changes. Each mechanism is described, recent advancements are summarized, and relative advantages and disadvantages of the techniques are discussed. Furthermore, two avenues for gaining further diagnostic information from label-free electrochemical biosensors, through multiplex analysis and incorporating machine learning, are examined.

Electrokinetic focusing of SARS-CoV-2 spike protein via ion concentration polarization in a paper-based lateral flow assay.

The COVID-19 pandemic highlighted the importance of designing sensitive and selective point-of-care (POC) diagnostic sensors for early and rapid detection of infection. Paper-based lateral flow assays (LFAs) are easy to use, inexpensive, and rapid, but they lack sensitivity. Preconcentration techniques can improve the sensitivity of LFAs by increasing the local concentration of the analyte before detection. Here, ion concentration polarization (ICP) is used to focus the analyte, SARS-CoV-2 Spike protein (S-protein), directly over a test line composed of angiotensin converting enzyme 2 (ACE2) capture probes. ICP is the enrichment and depletion of electrolyte ions at opposing ends of an ion-selective membrane under a voltage bias. The ion depleted zone (IDZ) establishes a steep gradient in electric field strength along its boundary. Enrichment of charged species (such as a biomolecule analyte) occurs at an axial location along this electric field gradient in the presence of a fluid flow that counteracts migration of those species - a phenomenon called ICP focusing. In this paper, running buffer composition and pretreatment solutions for ICP focusing in a paper-based LFA are evaluated, and the method of voltage application for ICP-enrichment is optimized. With a power consumption of 1.8 mW, S-protein is concentrated by a factor of 21-fold, leading to a 2.9-fold increase in the signal from the LFA compared to a LFA without ICP-enrichment. The described ICP-enhanced LFA is significant because the preconcentration strategy is amenable to POC applications and can be applied to existing LFAs for improvement in sensitivity.

Electrokinetic Enrichment and Label-Free Electrochemical Detection of Nucleic Acids by Conduction of Ions along the Surface of Bioconjugated Beads.

In this paper, we report a method to integrate the electrokinetic pre-enrichment of nucleic acids within a bed of probe-modified microbeads with their label-free electrochemical detection. In this detection scheme, hybridization of locally enriched target nucleic acids to the beads modulates the conduction of ions along the bead surfaces. This is a fundamental advancement in that this mechanism is similar to that observed in nanopore sensors, yet occurs in a bed of microbeads with microscale interstices. In application, this approach has several distinct advantages. First, electrokinetic enrichment requires only a simple DC power supply, and in combination with nonoptical detection, it makes this method amenable to point-of-care applications. Second, the sensor is easy to fabricate and comprises a packed bed of commercially available microbeads, which can be readily modified with a wide range of probe types, thereby making this a versatile platform. Finally, the sensor is highly sensitive (picomolar) despite the modest 100-fold pre-enrichment we employ here by faradaic ion concentration polarization (fICP). Further gains are anticipated under conditions for fICP focusing that are known to yield higher enrichment factors (up to 100,000-fold enrichment). Here, we demonstrate the detection of 3.7 pM single-stranded DNA complementary to the bead-bound oligoprobe, following a 30 min single step of enrichment and hybridization. Our results indicate that a shift in the slope of a current-voltage curve occurs upon hybridization and that this shift is proportional to the logarithm of the concentration of target DNA. Finally, we investigate the proposed mechanism of sensing by developing a numerical simulation that shows an increase in ion flux through the bed of insulating beads, given the changes in surface charge and zeta potential, consistent with our experimental conditions.