Ali Rohani

Scaling down constriction‐based (electrodeless) dielectrophoresis devices for trapping nanoscale bioparticles in physiological media of high‐conductivity

Selective trapping of nanoscale bioparticles (size <100 nm) is significant for the separation and high‐sensitivity detection of biomarkers. Dielectrophoresis is capable of highly selective trapping of bioparticles based on their characteristic frequency response. However, the trapping forces fall steeply with particle size, especially within physiological media of high‐conductivity where the trapping can be dissipated by electrothermal (ET) flow due to localized Joule heating. Herein, we investigate the influence of device scaling within the electrodeless insulator dielectrophoresis geometry through the application of highly constricted channels of successively smaller channel depth, on the net balance of dielectrophoretic trapping force versus ET drag force on bioparticles. While higher degrees of constriction enable dielectrophoretic trapping of successively smaller bioparticles within a short time, the ETflow due to enhanced Joule heating within media of high conductivity can cause a significant dissipation of bioparticle trapping. This dissipative drag force can be reduced through lowering the depth of the highly constricted channels to submicron sizes, which substantially reduces the degree of Joule heating, thereby enhancing the range of voltages and media conductivities that can be applied toward rapid dielectrophoretic concentration enrichment of silica nanoparticles (∼50 nm) and streptavidin protein biomolecules (∼5 nm). We envision the application of these methodologies toward nanofabrication, optofluidics, biomarker discovery, and early disease diagnostics.

Memristor Crossbar-Based Hardware Implementation of the IDS Method

Ink drop spread (IDS) is the engine of an active learning method, which is the methodology of soft computing. IDS, as a pattern-based processing unit, extracts useful information from a system that is subjected to modeling. In spite of its excellent potential to solve problems such as classification and modeling compared with other soft-computing tools, finding its simple and fast hardware implementation is still a challenge. This paper describes a new hardware implementation of the IDS method that is based on the memristor crossbar structure. In addition to simplicity, being completely real time, having low latency, and the ability to continue working properly after the occurrence of power failure are some of the advantages of our proposed circuit. Moreover, some of operations in the IDS method have fuzzy nature, and as we will show at the end of this paper, updation of rules in the IDS structure and spiky neural networks are very similar. Therefore, IDS can be considered as a new fuzzy implementation of artificial spiky neural networks as well.

Review: Microbial analysis in dielectrophoretic microfluidic systems

Infections caused by various known and emerging pathogenic microorganisms, including antibiotic-resistant strains, are a major threat to global health and well-being. This highlights the urgent need for detection systems for microbial identification, quantification and characterization towards assessing infections, prescribing therapies and understanding the dynamic cellular modifications. Current state-of-the-art microbial detection systems exhibit a trade-off between sensitivity and assay time, which could be alleviated by selective and label-free microbial capture onto the sensor surface from dilute samples. AC electrokinetic methods, such as dielectrophoresis, enable frequency-selective capture of viable microbial cells and spores due to polarization based on their distinguishing size, shape and sub-cellular compositional characteristics, for downstream coupling to various detection modalities. Following elucidation of the polarization mechanisms that distinguish bacterial cells from each other, as well as from mammalian cells, this review compares the microfluidic platforms for dielectrophoretic manipulation of microbials and their coupling to various detection modalities, including immuno-capture, impedance measurement, Raman spectroscopy and nucleic acid amplification methods, as well as for phenotypic assessment of microbial viability and antibiotic susceptibility. Based on the urgent need within point-of-care diagnostics towards reducing assay times and enhancing capture of the target organism, as well as the emerging interest in isolating intact microbials based on their phenotype and subcellular features, we envision widespread adoption of these label-free and selective electrokinetic techniques.

Electrical tweezer for highly parallelized electrorotation measurements over a wide frequency bandwidth

Electrorotation (ROT) is a powerful tool for characterizing the dielectric properties of cells and bioparticles. However, its application has been somewhat limited by the need to mitigate disruptions to particle rotation by translation under positive DEP and by frictional interactions with the substrate. While these disruptions may be overcome by implementing particle positioning schemes or field cages, these methods restrict the frequency bandwidth to the negative DEP range and permit only single particle measurements within a limited spatial extent of the device geometry away from field nonuniformities. Herein, we present an electrical tweezer methodology based on a sequence of electrical signals, composed of negative DEP using 180‐degree phase‐shifted fields for trapping and levitation of the particles, followed by 90‐degree phase‐shifted fields over a wide frequency bandwidth for highly parallelized electrorotation measurements. Through field simulations of the rotating electrical field under this wave‐sequence, we illustrate the enhanced spatial extent for electrorotation measurements, with no limitations to frequency bandwidth. We apply this methodology to characterize subtle modifications in morphology and electrophysiology of Cryptosporidium parvum with varying degrees of heat treatment, in terms of shifts in the electrorotation spectra over the 0.05–40 MHz region. Given the single particle sensitivity and the ability for highly parallelized electrorotation measurements, we envision its application toward characterizing heterogeneous subpopulations of microbial and stem cells.

Quantifying spatio-temporal dynamics of biomarker pre-concentration and depletion in microfluidic systems by intensity threshold analysis

Microfluidic systems are commonly applied towards pre-concentration of biomarkers for enhancing detection sensitivity. Quantitative information on the spatial and temporal dynamics of pre-concentration, such as its position, extent, and time evolution are essential towards sensor design for coupling pre-concentration to detection. Current quantification methodologies are based on the time evolution of fluorescence signals from biomarkers within a statically defined region of interest, which does not offer information on the spatial dynamics of pre-concentration and leads to significant errors when the pre-concentration zone is delocalized or exhibits wide variations in size, shape, and position over time under the force field. We present a dynamic methodology for quantifying the region of interest by using a statistical description of particle distribution across the device geometry to determine the intensity thresholds for particle pre-concentration. This method is applied to study the delocalized pre-concentration dynamics under an electrokinetic force balance driven by negative dielectrophoresis, for aligning the pre-concentration and detection regions of neuropeptide Y, and for quantifying the polarizability dispersion of silica nano-colloids with frequency of the force field. We envision the application of this automated methodology on data from 2D images and 3D Z-stacks for quantifying pre-concentration dynamics over delocalized regions as a function of the force field.

Nanoslit design for ion conductivity gradient enhanced dielectrophoresis for ultrafast biomarker enrichment in physiological media

Selective and rapid enrichment of biomolecules is of great interest for biomarker discovery, protein crystallization, and in biosensing for speeding assay kinetics and reducing signal interferences. The current state of the art is based on DC electrokinetics, wherein localized ion depletion at the microchannel to nanochannel interface is used to enhance electric fields, and the resulting biomarker electromigration is balanced against electro-osmosis in the microchannel to cause high degrees of biomarker enrichment. However, biomarker enrichment is not selective, and the levels fall off within physiological media of high conductivity, due to a reduction in ion concentration polarization and electro-osmosis effects. Herein, we present a methodology for coupling AC electrokinetics with ion concentration polarization effects in nanoslits under DC fields, for enabling ultrafast biomarker enrichment in physiological media. Using AC fields at the critical frequency necessary for negative dielectrophoresis of the biomarker of interest, along with a critical offset DC field to create proximal ion accumulation and depletion regions along the perm-selective region inside a nanoslit, we enhance the localized field and field gradient to enable biomarker enrichment over a wide spatial extent along the nanoslit length. While enrichment under DC electrokinetics relies solely on ion depletion to enhance fields, this AC electrokinetic mechanism utilizes ion depletion as well as ion accumulation regions to enhance the field and its gradient. Hence, biomarker enrichment continues to be substantial in spite of the steady drop in nanostructure perm-selectivity within physiological media.

Label-Free Quantification of Intracellular Mitochondrial Dynamics Using Dielectrophoresis

Mitochondrial dynamics play an important role within several pathological conditions, including cancer and neurological diseases. For the purpose of identifying therapies that target aberrant regulation of the mitochondrial dynamics machinery and characterizing the regulating signaling pathways, there is a need for label-free means to detect the dynamic alterations in mitochondrial morphology. We present the use of dielectrophoresis for label-free quantification of intracellular mitochondrial modifications that alter cytoplasmic conductivity, and these changes are benchmarked against label-based image analysis of the mitochondrial network. This is validated by quantifying the mitochondrial alterations that are carried out by entirely independent means on two different cell lines: human embryonic kidney cells and mouse embryonic fibroblasts. In both cell lines, the inhibition of mitochondrial fission that leads to a mitochondrial structure of higher connectivity is shown to substantially enhance conductivity of the cell interior, as apparent from the significantly higher positive dielectrophoresis levels in the 0.5–15 MHz range. Using single-cell velocity tracking, we show ∼10-fold higher positive dielectrophoresis levels at 0.5 MHz for cells with a highly connected versus those with a highly fragmented mitochondrial structure, suggesting the feasibility for frequency-selective dielectrophoretic isolation of cells to aid the discovery process for development of therapeutics targeting the mitochondrial machinery.

Tracking Inhibitory Alterations during Interstrain Clostridium difficile Interactions by Monitoring Cell Envelope Capacitance

Global threats arising from the increasing use of antibiotics coupled with the high recurrence rates of Clostridium difficile (C. difficile) infections (CDI) after standard antibiotic treatments highlight the role of commensal probiotic microorganisms, including nontoxigenic C. difficile (NTCD) strains in preventing CDI due to highly toxigenic C. difficile (HTCD) strains. However, optimization of the inhibitory permutations due to commensal interactions in the microbiota requires probes capable of monitoring phenotypic alterations to C. difficile cells. Herein, by monitoring the field screening behavior of the C. difficile cell envelope with respect to cytoplasmic polarization, we demonstrate that inhibition of the host-cell colonization ability of HTCD due to the S-layer alterations occurring after its co-culture with NTCD can be quantitatively tracked on the basis of the capacitance of the cell envelope of co-cultured HTCD. Furthermore, it is shown that effective inhibition requires the dynamic contact of HTCD cells with freshly secreted extracellular factors from NTCD because contact with the cell-free supernatant causes only mild inhibition. We envision a rapid method for screening the inhibitory permutations to arrest C. difficile colonization by routinely probing alterations in the HTCD dielectrophoretic frequency response due to variations in the capacitance of its cell envelope.

Single-cell electro-phenotyping for rapid assessment of Clostridium difficile heterogeneity under vancomycin treatment at sub-MIC (minimum inhibitory concentration) levels

Current methods for measurement of antibiotic susceptibility of pathogenic bacteria are highly reliant on microbial culture, which is time consuming (requires > 16 hours), especially at near minimum inhibitory concentration (MIC) levels of the antibiotic. We present the use of single-cell electrophysiology-based microbiological analysis for rapid phenotypic identification of antibiotic susceptibility at near-MIC levels, without the need for microbial culture. Clostridium difficile (C. difficile) is the single most common cause of antibiotic-induced enteric infection and disease recurrence is common after antibiotic treatments to suppress the pathogen. Herein, we show that de-activation of C. difficile after MIC-level vancomycin treatment, as validated by microbiological growth assays, can be ascertained rapidly by measuring alterations to the microbial cytoplasmic conductivity that is gauged by the level of positive dielectrophoresis (pDEP) and the frequency spectra for co-field electro-rotation (ROT). Furthermore, this single-cell electrophysiology technique can rapidly identify and quantify the live C. difficile subpopulation after vancomycin treatment at sub-MIC levels, whereas methods based on measurement of the secreted metabolite toxin or the microbiological growth rate can identify this persistent C. difficile subpopulation only after 24 hours of microbial culture, without any ability to quantify the subpopulation. The application of multiplexed versions of this technique is envisioned for antibiotic susceptibility screening.