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.

Out-of-plane faradaic ion concentration polarization: stable focusing of charged analytes at a three-dimensional porous electrode.

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 to "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.

Tutorial review: Enrichment and separation of neutral and charged species by ion concentration polarization focusing.

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.

Continuous micellar electrokinetic focusing of neutral species driven by ion concentration polarization.

Ion concentration polarization (ICP) has been broadly applied to accomplish electrokinetic focusing of charged species. However, ICP-based extraction and enrichment of uncharged (neutral) compounds, important for pharmaceutical, biological, and environmental applications, has not yet been reported. Here, we report the ICP-based continuous extraction of two neutral compounds from aqueous solution, by their partition into an ionic micellar phase. Our initial results show that the efficiency of the extraction increases with the concentration of the surfactant comprising the micellar phase, reaching 98 ± 2%, and drops precipitously when the concentration of the target compound exceeds the capacity of the micelles. As a key feature relevant to the practical application of this method, we show that focusing occurs even an order of magnitude below the critical micelle concentration through the local enrichment and assembly of surfactants into micelles, thus minimizing their consumption. To underscore the relevance of this approach to water purification, this method is applied to the extraction of pyrene, a model for polyaromatic hydrocarbons. This approach provides access to a broad range of strategies for selective separation that have been developed in micellar electrokinetic chromatography.

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.