Zachary Gagnon

Cellular dielectrophoresis: applications to the characterization, manipulation, separation and patterning of cells.

Over the past decade, dielectrophoresis (DEP) has evolved into a powerful, robust and flexible method for cellular characterization, manipulation, separation and cell patterning. It is a field with widely varying disciplines, as it is quite common to see DEP integrated with a host of applications including microfluidics, impedance spectroscopy, tissue engineering, real‐time PCR, immunoassays, stem‐cell characterization, gene transfection and electroporation, just to name a few. The field is finally at the point where analytical and numerical polarization models can be used to adequately describe and characterize the dielectrophoretic behavior of cells, and there is ever increasing evidence demonstrating that electric fields can safely be used to manipulate cells without harm. As such, DEP is slowly making its way into the biological sciences. Today, DEP is being used to manipulate individual cells to specific regions of space for single‐cell assays. DEP is able to separate rare cells from a heterogeneous cell suspension, where isolated cells can then be characterized and dynamically studied using nothing more than electric fields. However, there is need for a critical report to integrate the many new features of DEP for cellular applications. Here, a review of the basic theory and current applications of DEP, specifically for cells, is presented.

Dielectrophoretic detection and quantification of hybridized DNA molecules on nano-genetic particles

DNA–DNA hybridization reactions on 100 nm oligonucleotide‐functionalized silica nanoparticles are found to sensitively affect the amplitude and direction of the dielectrophoretic mobility of the particles at nanomolar target ssDNA concentrations. Such sensitivity permits visual detection of the hybridization event without fluorescent labeling and confocal microscopy by imaging the cross‐over frequency (cof) of the particle suspension on a quadrupole electrode array. Strong correlation with effective particle radius and zeta‐potential measurements suggests that the dielectrophoretic cof offers not just sensitive signatures for successful functionalization and hybridization but also those for three distinct DNA surface conformations that appear at different surface densities of hybridized DNA. A properly normalized cof calibration chart allows simplified quantification of the target ssDNA concentrations. These results provide a simple, rapid and portable genetic detection method compatible for use outside the laboratory.

Dielectrophoretic discrimination of bovine red blood cell starvation age by buffer selection and membrane cross-linking

We report an interesting buffer electric relaxation time tuning technique, coupled with a glutaraldehyde cross-linking cell fixation reaction, which allows for sensitive dielectrophoretic analysis and discrimination of bovine red blood cell (bRBC) starvation age. The buffer composition is selected such that two easily accessible dielectrophoretic crossover frequencies (cof) exist. Low concentration glutaraldehyde fixation was observed to produce a threefold decrease in the higher cof with a comparable increase in the lower cof also witnessed. More importantly, increased glutaraldehyde fixation concentration significantly increased the higher cof by a factor found to be sensitive to the bRBC starvation age.

Bovine red blood cell starvation age discrimination through a glutaraldehyde-amplified dielectrophoretic approach with buffer selection and membrane cross-linking.

We report a novel buffer electric and dielectric relaxation time tuning technique, coupled with a glutaraldehyde (Glt.) cross‐linking cell fixation reaction that allows for sensitive dielectrophoretic analysis and discrimination of bovine red blood cells of different starvation age. Guided by a single‐shell oblate spheroid model, a zwitterion buffer composition is selected to ensure that two measurable crossover frequencies (cofs) near 500 kHz exist for dielectrophoresis (DEP) within a small range of each other. It is shown that the low cof is sensitive to changes in the cell membrane dielectric constant, in which cross‐linking by Glt. reduces the dielectric constant of the cell membrane from 10.5 to 3.8, while the high cof is sensitive to cell cytoplasm conductivity changes. We speculate that this enhanced particle polarizability that results from the cross‐linking reaction is because younger (reduced starvation time) cells possess more amino groups that the reaction can release to enhance the cell interior ionic strength. Such sensitive discrimination of cells with different age (surface protein density) by DEP is not possible without the zwitterion buffer and cleavage by Glt. treatment. It is then expected that rapid identification and sorting of healthy from diseased cells can be similarly sensitized.

Optimized DNA hybridization detection on nanocolloidal particles by dielectrophoresis

Oligonucleotides of varying surface coverage are functionalized onto the surface of 100 nm silica particles and the corresponding hybridization reaction with target ssDNA is studied using dielectrophoresis (DEP). The measured DEP cross‐over frequency (cof) is found to be sensitive to the oligonucleotide surface conformation. Zeta potential and particle size measurements suggest that at low oligo surface concentrations, non‐specific binding of oligo to the particle surface prevents efficient hybridization. At high surface coverage, steric hindrance due to the fully stretched, tightly packed oligo conformation prevents diffusion of DNA molecules to the particle surface. The optimum surface coverage exists at intermediate coverage where the particle is found to be the least electrically conductive, and hence exhibits the lowest measured cof. A simple DEP cof measurement hence allows one to determine the optimal oligo surface coverage for increased hybridization efficiency and detection sensitivity.

Glutaraldehyde enhanced dielectrophoretic yeast cell separation

We introduce a method for improved dielectrophoretic (DEP) discrimination and separation of viable and nonviable yeast cells. Due to the higher cell wall permeability of nonviable yeast cells compared with their viable counterpart, the cross-linking agent glutaraldehyde (GLT) is shown to selectively cross-link nonviable cells to a much greater extent than viable yeast. The DEP crossover frequency (cof) of both viable and nonviable yeast cells was measured over a large range of buffer conductivities (22 muScm-400 muScm) in order to study this effect. The results indicate that due to selective nonviable cell cross-linking, GLT modifies the DEP cof of nonviable cells, while viable cell cof remains relatively unaffected. To investigate this in more detail, a dual-shelled oblate spheroid model was evoked and fitted to the cof data to study cell electrical properties. GLT treatment is shown to minimize ion leakage out of the nonviable yeast cells by minimizing changes in cytoplasm conductivity over a large range of ionic concentrations. This effect is only observable in nonviable cells where GLT treatment serves to stabilize the cell cytoplasm conductivity over a large range of buffer conductivity and allow for much greater differences between viable and nonviable cell cofs. As such, by taking advantage of differences in cell wall permeability GLT magnifies the effect DEP has on the field induced separation of viable and nonviable yeasts.

Electrothermal ac electro-osmosis

Two ac polarization mechanisms, charge accumulation due to electrode double layer charging and bulk permittivity/conductivity gradients generated by Joule heating, are combined in the double layer by introducing zwitterions to produce a new ac electrokinetic pump with the largest velocity (>1 mm/s) and flow penetration depth (100 μm) reported for low-conductivity fluids. The large fluid velocity is due to a quartic scaling with respect to voltage, as is true of electrothermal flow, but exhibits a clear maximum at a frequency corresponding to the electrode double layer inverse RC time.

Fluidic dielectrophoresis: The polarization and displacement of electrical liquid interfaces

Traditional particle‐based dielectrophoresis has been exploited to manipulate bubbles, particles, biomolecules, and cells. In this work, we investigate analytically and experimentally how to utilize Maxwell–Wagner polarization to initiate fluidic dielectrophoresis (fDEP) at electrically polarizable aqueous liquid–liquid interfaces. In fDEP, an AC electric field is applied across a liquid electrical interface created between two coflowing fluid streams with different electrical properties. When potentials as low as 2 volts are applied, we observe a frequency‐dependent interfacial displacement that is dependent on the relative differences in the electrical conductivity ( Δσ) and dielectric constant ( Δɛ) between the two liquids. At low frequency this deflection is independent of dielectric constant, while at high frequency it is independent of electrical conductivity. At intermediate frequencies, we observe an fDEP cross‐over frequency that is independent of applied voltage, sensitive to both fluid electrical properties, and where no displacement is observed. An analytical fDEP polarization model is presented that accurately predicts the liquid interfacial cross‐over frequency, the dependence of interfacial displacement on liquid electrical conductivity and dielectric constant, and accurately scales the measured fDEP displacement data. The results show that miscible aqueous liquid interfaces are capable of polarizing under AC electric fields, and being precisely deflected in a direction and magnitude that is dependent on the applied electric field frequency.

Label-free biomolecular detection at electrically displaced liquid interfaces using interfacial electrokinetic transduction (IET).

Biosensors require a biorecognition element that specifically binds to a target analyte, and a signal transducer, which converts this targeted binding event into a measurable signal. While current biosensing methods are capable of sensitively detecting a variety of target analytes in a laboratory setting, there are inherent difficulties in developing low-cost portable biosensors for point-of-care diagnostics using traditional optical, mass, or electroanalytical-based signal transducers. It is therefore important to develop new biosensing transducer elements for recognizing binding events at low cost and in portable environments. Here, we demonstrate a novel electrokinetic liquid biosensing method for the sensitive label-free detection of a model biomolecule against a background of serum protein. The biosensor is based on the motion of a microfluidic-generated electrical liquid interface when subjected to an external alternating current electrical field. We demonstrate that the electric field-induced motion of the interface can be used as a sensitive and specific transducer for the detection of avidin at femtomolar concentrations in solution. This new detection strategy does not require surface functionalization or fluorescent labels, and has the potential to serve as a sensitive low-cost method for portable biomarker detection.

Microfluidics made easy: A robust low-cost constant pressure flow controller for engineers and cell biologists

Over the last decade, microfluidics has become increasingly popular in biology and bioengineering. While lab-on-a-chip fabrication costs have continued to decrease, the hardware required for delivering controllable fluid flows to the microfluidic devices themselves remains expensive and often cost prohibitive for researchers interested in starting a microfluidics project. Typically, microfluidic experiments require precise and tunable flow rates from a system that is simple to operate. While many labs use commercial platforms or syringe pumps, these solutions can cost thousands of dollars and can be cost prohibitive. Here, we present an inexpensive and easy-to-use constant pressure system for delivering flows to microfluidic devices. The controller costs less than half the price of a single syringe pump but can independently switch and deliver fluid through up to four separate fluidic inlets at known flow rates with significantly faster fluid response times. It is constructed of readily available pressure regulators, gauges, plastic connectors and adapters, and tubing. Flow rate is easily predicted and calibrated using hydraulic circuit analysis and capillary tubing resistors. Finally, we demonstrate the capabilities of the flow system by performing well-known microfluidic experiments for chemical gradient generation and emulsion droplet production.