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Ion concentration polarization (ICP) accomplishes preconcentration for bioanalysis by localized depletion of electrolyte ions, thereby generating a gradient in electric field strength that facilitates electrokinetic focusing of charged analytes by their electromigration against opposing fluid flow. Such ICP focusing has been shown to accomplish up to a million-fold enrichment of nucleic acids and proteins in single-stage preconcentrators. However, the rate at which the sample volume is swept is limited, requiring several hours to achieve these high enrichment factors. This limitation is caused by two factors. First, an ion depleted zone (IDZ) formed at a planar membrane or electrode may not extend across the full channel cross section under the flow rate employed for focusing, thereby allowing the analyte “leak” past the IDZ. Second, within the IDZ, large fluid vortices lead to mixing, which decreases the efficiency of analyte enrichment and worsens with increased channel dimensions. Here, we address these challenges with faradaic ICP (fICP) at a three-dimensional (3D) electrode comprising metallic microbeads. This 3D-electrode distributes the IDZ, and therefore, the electric field gradient utilized for counter-flow focusing across the full height of the fluidic channel, and its large area, microstructured surface supports smaller vortices. An additional bed of insulating microbeads restricts flow patterns and supplies a large area for surface conduction of ions through the IDZ. Finally, the resistance of this secondary bed enhances focusing by locally strengthening sequestering forces. This easy-to-build platform lays a foundation for the integration of enrichment with user-defined packed bed and electrode materials.

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. Expected final online publication date for the Annual Review of Analytical Chemistry, Volume 16 is June 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Dielectrophoretic capture of circulating melanoma cell (CMC) and white blood cells (WBCs) is quantified as a function of electric field frequency, and the optimized conditions are utilized to isolate CMCs from patient-derived blood samples.

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.

Extraction-based sample preparation has been recognized as a significant step in separation science. To improve the sensitivity and selectivity of the extraction, some new approaches have been introduced, including those that employ an electric field. These electric field–assisted extraction methods create another dimension for sample preparation, and are compatible with miniaturized, portable, and multifunctional analytical platlate proteins, organelles, viral capsids, and biological cells, among others.

An array of many bipolar electrodes (BPEs) can be controlled by a single pair of driving electrodes yet allows for multiplexedanalysis of many individual biomarkers or single cells at once. A wide range of bipolar electrochemical sensors have been devised, many of which operate under battery power and produce visible signals (e.g., luminescent, electrochromic) appropriate for smartphone or naked eye readout. These features of BPEs are advantageous in the context of clinical and environmental sensing applications at the point of need. However, the sensitivity of BPEs is poor in comparison to direct measurement of current at an individual electrode, and therefore, the enhancement of signals obtained at BPEs is an active area of research. Here, we describe signal amplification by redox cycling accomplished by interdigitation of each BPE in an array with a shared driving electrode. We evaluate amplification obtained for interelectrode spacing in the range of 35 𝜇m to 15 𝜇m. Each interdigitated BPE (IDBPE) in the array has an independent, reproducible, and linear response to a reversible electroactive analyte. Therefore, this universal amplification strategy allows for multiplexed or spatially resolved sensing in point-of-need applications.

In this paper, we report a method for obtaining a visual voltammogram at a linear array of closed wireless bipolar electrodes (BPEs). This advancement is significant, because the visual voltammogram captures the entire current–potential (i–E) relationship of a faradaic reaction in one image and is continuously generated over time. Therefore, we anticipate that this method will allow monitoring in redox systems that change over time. Further, the use of a linear array of BPEs eliminates the need to use a potentiostat and can be carried out with a simple DC power supply. Our experimental and numerical results demonstrate that the visual voltammogram is similar to a linear sweep voltammogram and therefore, information about the faradaic process can be extracted from the wave position, height, and shape.

Ion concentration polarization focusing (ICPF) is an electrokinetic technique, in which analytes are enriched and separated along a localized electric field gradient in the presence of a counter flow. This field gradient is generated by depletion of ions of the background electrolyte at an ion permselective junction. In this tutorial review, we summarize the fundamental principles and experimental parameters that govern selective ion transport and the stability of the enriched analyte plug. We also examine faradaic ICP (fICP), in which local ion concentration is modulated via electrochemical reactions as an attractive alternative to ICP that achieves similar performance with a decrease in both power consumption and Joule heating. The tutorial covers important challenges to the broad application of ICPF including undesired pH gradients, low volumetric throughput, samples that induce biofouling or are highly conductive, and limited approaches to on- or off-chip analysis. Recent developments in the field that seek to address these challenges are reviewed along with new approaches to maximize enrichment, focus uncharged analytes, and achieve enrichment and separation in water-in-oil droplets. For new practitioners, we discuss practical aspects of ICPF, such as strategies for device design and fabrication and the relative advantages of several types of ion selective junctions and electrodes. Lastly, we summarize tips and tricks for tackling common experimental challenges in ICPF.

Synthetic genetic circuits leverage the information processing capability of biological machinery to tackle complex sensing tasks, yet they lack many of the advantages inherent to electrical computation. Now, an interface has been designed that provides an electrical output for synthetic genetic circuits.

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.

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.

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 non-expert 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.

The field of synthetic biology has used the engineered assembly of synthetic gene networks to create a wide range of function in biological systems. As part of this work, gene circuit-based sensors have primarily used optical proteins (e.g. fluorescent, colorimetric) as reporter outputs, which has limited the potential to measure multiple distinct signals. Here we present an electrochemical interface that permits expanded multiplexed reporting for cell-free gene circuit-based sensors. We have engineered a scalable system of reporter enzymes that cleave specific DNA sequences in solution, which results in an electrochemical signal when these newly liberated strands are captured at the surface of a nanostructured microelectrode. We describe the development of this interface and show its utility using a ligand-inducible gene circuit and toehold switch-based sensors, including the detection of multiple antibiotic resistance genes in parallel. This technology has the potential to expand the field of synthetic biology by providing an interface with materials, hardware and software.

Clusters of biological cells play an important role in normal and disease states, such as in the release of insulin from pancreatic islets and in the enhanced spread of cancer by clusters of circulating tumor cells. We report a method to pattern cells into clusters having sizes correlated to the dimensions of each electrode in an array of wireless bipolar electrodes (BPEs). The cells are captured by dielectrophoresis (DEP), which confers selectivity, and patterns cells without the need for physical barriers or adhesive interactions that can alter cell function. Our findings demonstrate that this approach readily achieves fine control of cell cluster size over a broader range set by other experimental parameters. These parameters include the magnitude of the voltage applied externally to drive capture at the BPE array, the rate of fluid flow, and the time allowed for DEP-based cell capture. Therefore, the reported method is anticipated to allow the influence of cluster size on cell function to be more fully investigated.

In this presentation, we demonstrate that ion concentration polarization (ICP) in the presence of fluid flow drives focusing and efficient capture of target nucleic acids within a bed of oligoprobe-conjugated beads. A key finding is that ion conduction along the surface of the bioconjugated beads is the dominant contributor to current to the electrode that drives ICP, under the condition of ICP-induced depletion of ions from the bulk solution. Therefore, hybridization of a target nucleic acid (a polyanion) to the bead surface leads to a shift in the slope of the current-voltage curve. This approach is versatile in that a target nucleic acid can be detected electrically, in the absence of a label, or electrochemically via a label that generates an electroactive product. The resulting approach allows for a plug-and-play nucleic acid sensor using off-the-shelf bioconjugated beads and simple electronics, making it advantageous for point-of-care testing.

We present integration of selective single-cell capture at an array of wireless electrodes (bipolar electrodes, BPEs) with transfer into chambers, reagent exchange, fluidic isolation and rapid electrical lysis in a single platform, thus minimizing sample loss and manual intervention steps., We present integration of selective single-cell capture at an array of wireless electrodes (bipolar electrodes, BPEs) with transfer into chambers, reagent exchange, fluidic isolation and rapid electrical lysis in a single platform, thus minimizing sample loss and manual intervention steps. The whole process is achieved simply by exchanging the solution in a single inlet reservoir and by adjusting the applied voltage at a pair of driving electrodes, thus making this approach particularly well-suited for a broad range of research and clinical applications. Further, the use of BPEs allows the array to be scalable to increase throughput. Specific innovations reported here include the incorporation of a leak channel to balance competing flow paths, the use of ‘split BPEs’ to create a distinct recapture and electrical lysis point within the reaction chamber, and the dual purposing of an ionic liquid as an immiscible phase to seal the chambers and as a conductive medium to permit electrical lysis at the split BPEs.

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.

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.

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.

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.

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).

Bipolar electrodes (BPEs) hold enormous promise for rapid screening of electrocatalysts owing to their ability to be operated wirelessly – without direct ohmic contact to a power source – and in parallel. A BPE is a conductor, immersed in an ionically conducting phase, that facilitates coupled oxidation and reduction reactions at opposing ends when exposed to an electric field. The current arising from these coupled processes can be monitored if one of the reactions leads to a visible change, such as chemiluminescence or anodic stripping of a metal film. Therefore, an array of BPEs operated with a single pair of driving electrodes can facilitate many electrochemical measurements simultaneously. Several reports have clearly demonstrated the utility of BPEs to obtain relative catalytic activity. However, to date, the extraction of specific activity and kinetic rate constants from these measurements remains challenging. We discuss here the experimental factors that contribute to this challenge and we present initial results demonstrating alternative approaches to extract this valuable information from BPE-based screens.

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.

This paper describes the use of electro-hydrodynamic actuation to control the transition between three major flow patterns of an aqueous-oil Newtonian flow in a microchannel: droplets, beads-on-a-string (BOAS), and multi-stream laminar flow. We observed interesting transitional flow patterns between droplets and BOAS as the electric field was modulated. The ability to control flow patterns of a two-phase fluid in a microchannel adds to the microfluidic tool box and improves our understanding of this interesting fluid behavior.

In this paper we show that a microelectrochemical cell comprising two parallel microchannels spanned by a single bipolar electrode can be used to simultaneously enrich and separate both anions and cations within a single microchannel. This is possible because reduction and oxidation of water at the cathodic and anodic poles of the bipolar electrode, respectively, lead to ion depletion zones. Specifically, TrisH(+) is neutralized by OH(-) at the cathodic pole, while acetate buffer is neutralized by H(+) at the anodic pole. This action creates a local electric field gradient having both positive and negative components, and hence positive and negative ions follow their respective field gradients leading to separation. In the presence of an opposing counter-flow (pressure driven flow in this case), enrichment also occurs. In addition to separation and enrichment in a single channel, it is also possible to simultaneously enrich cations in one microchannel and anions in the other. Enrichment is achieved by controlling experimental parameters, including the type of buffer and the direction and magnitude of the opposing counter-flow.

Heterogeneity in the content and function of subcellular organelles on the intercellular and intracellular level plays an important role in determining cell fate. These variations extend to normal-state and disease-state cellular functions and responses to environmental stimuli, such as oxidative stress and therapeutic drugs. Analytical tools to characterize variation in all types of organelles are essential to provide insights that can lead to advances in medicine, such as therapies targeted to specific subcellular regions. In this review, we discuss analytical techniques for interrogating individual intact organelles (e.g. mitochondria and synaptic vesicles) and lysates in a high-throughput manner, including a recently developed nanoscale fluorescence-activated subcellular sorter and techniques based on capillary electrophoresis with laser-induced fluorescence detection. We then highlight the advantages that droplet microfluidics offers for probing subcellular heterogeneity.

We show that a label-free electrochemical method can be used to monitor the position of an enriched analyte band during bipolar electrode focusing in a microfluidic device. The method relies on formation of a depleted buffer cation region, which is responsible for concentration enrichment of the charged analyte. However, this depletion region also leads to an increase in the local electric field in the solution near a bipolar electrode (BPE), and this in turn results in enhanced faradaic reactions (oxidation and reduction of water) at the BPE. Therefore, it is possible to detect the presence of the concentrated analyte band by measuring the current passing through the BPE used for concentration enrichment, or the concentrated band can be detected at a secondary BPE dedicated to that purpose. Both experiments and simulations are presented that fully elucidate the underlying phenomenon responsible for these observations.

Here we report that pressure-driven flow alone (no external electrical energy) can be used to drive faradaic electrochemical reactions in microchannels with charged walls. Specifically, we show that solution flow can generate streaming potentials on the order of volts and that this is sufficient to carry out reactions on the anodic and cathodic poles of a bipolar electrode (BPE). The existence of faradaic reactions is proven by electrodissolution of Ag from the anodic end of the BPE.

Over the past decade, bipolar electrochemistry has emerged from relative obscurity to provide a promising new means for integrating electrochemistry into lab-on-a-chip systems. This article describes the fundamental operating principles of bipolar electrodes, as well as several interesting applications.

With global demand rising faster than availability, fresh water is quickly becoming a limited resource. In fact, the United Nations estimates one-third of the world's population is living in water stressed regions, and by 2025 this number is expected to double.[1] Seawater desalination is an attractive solution to this problem, because seawater accounts for more than 97% of the world's water supply.[2] Currently, the primary limitation preventing the widespread use of seawater desalination as a fresh water supply is the immense amount of energy required to drive the process.[3] Here, we describe a new, electrochemically-mediated desalination (EMD) method for membraneless seawater desalination.