Harnessing Joule heating in microfluidic thermal gel electrophoresis to create reversible barriers for cell enrichment

Abstract

Gel electrophoresis is a ubiquitous bioanalytical technique used to characterize the components of cell lysates. However, analyses of bulk lysates sacrifice detection sensitivity because intracellular biomolecules become diluted, and the liberation of proteases and nucleases can degrade target analytes. This report describes a method to enrich cells directly within a microfluidic gel as a first step toward online measurement of trace intracellular biomolecules with minimal dilution and degradation. Thermal gels were employed as the gel matrix because they can be reversibly converted between liquid and solid phases as a function of temperature. Rather than fabricate costly heating elements into devices to control temperature—and thus the phase of the gel—Joule heating was used instead. Adjoining regions of liquid-phase and solid-phase gel were formed within microfluidic channels by selectively inducing localized Joule heat. Cells migrated through the liquid gel but could not enter the solid gel—accumulating at the liquid-solid gel boundary—whereas small molecule contaminants passed through to waste. Barriers were then liquified on-demand by removing Joule heat to collect the purified, non-lysed cells for downstream analyses. Using voltage-controlled Joule heating to regulate the phase of thermal gels is an innovative approach to facilitate in-gel cell enrichment in low-cost microfluidic devices.

1. Introduction

Cells are the foundational blocks of larger biological organisms. Different cell types have evolved to synergistically perform physiological tasks to maintain the health of the larger organism. A primary focus of modern biomedical research is determining the biomolecular compositions of cells, especially in the context of pathogenesis. Cellular expression changes in response to disease, and elucidating these changes is key to rationally designing drugs to target the underlying cause of the condition [1]. The research workflow typically involves culturing cells, followed by lysis and analysis of intracellular contents by gel electrophoresis to confirm the presence of the target biomolecule(s) within the sample [2]. Despite the ubiquity of this approach, its analytical performance is limited. Cell lysates suffer from inherent dilution because intracellular biomolecules become dispersed into relatively large volumes of lysis buffer. This dilution can prevent detection of low-abundance analytes. Additionally, cell lysis can bias measurements due to the degradation of target analytes. Lysates contain nucleases and proteases that are released from cells along with the target nucleic acid or protein analytes. Enzymatic digestion of the target molecules—in the absence of enzyme inhibitors—can preclude their detection and subsequent isolation for use in future studies. Analytes can also undergo covalent modifications due to oxidation or reactions with other species [3]. Consequently, methods are needed to directly couple cell lysis with separation to minimize potential issues that can bias conclusions of biological studies.

Microfluidic technologies provide alternative approaches for analyzing cell samples. Low-volume microchannels are adept at handling samples with minimal dilution following cell lysis. Microfluidic devices also have the potential to couple cell enrichment with a subsequent separation to improve detection sensitivity of target biomolecules. Numerous enrichment techniques have been developed to concentrate cells on-chip including acoustophoresis [4,5], dielectrophoresis [6–8], or centrifugal microfluidics [9–11]. However, the experimental complexity of these methods is relatively high because they require specialized external equipment and elaborate microfluidic devices. Additionally, although these techniques effectively collect cells, they are not amenable to direct cell lysis and electrophoretic analysis following enrichment. Alternative strategies have been developed to analyze cells on-chip without an enrichment step. Microchip electrophoresis can introduce single cells into a microchannel for lysis and immediate separation of small molecules [12,13]. This approach requires the cell handling and separation be conducted in a liquid phase background electrolyte, which limits separation resolution of macromolecules compared to gel electrophoresis. Gel electrophoresis methods have also been developed to analyze single cells [14,15], but their complexity is relatively high. Because cells cannot migrate through the small, rigid pores of polyacrylamide gels, specialized loading techniques were required to introduce cells into wells. For an analytical technique to be widely adopted by biological research laboratories, new methods must minimize complexity and cost. To that end, cell analyses would benefit from a gel electrophoresis technique that effectively couples on-chip cell handling, cell lysis, and high-resolution gel separations.

Thermal gel electrophoresis (TGE) is well positioned to address these demanding analytical requirements. Thermal gels are solutions of thermally responsive polymers that undergo a change in viscosity in response to changing temperature. These gels have been used as a gel separations matrix to attain high-resolution separations of proteins [16–19], nucleic acids [20–22], and small molecules [23]. Pluronic F-127 (PF-127), for example, is a biocompatible triblock copolymer composed of polyethylene oxide and polypropylene oxide that forms micelles in aqueous solutions. PF-127 thermal gel transitions from a liquid phase to a solid phase with increasing temperature as polyethylene oxide chains between micelles entangle more strongly [22,24]. This behavior decreases the intermicelle distance and increases micelle packing density, [25,26], leading to temperature-dependent viscosity increases of > 1000-fold [27,28]. The ability to dynamically control the phase of PF-127 through a thermal dimension affords an unprecedented opportunity to manipulate whole cells in a gel electrophoresis format.

Maintaining distinct temperatures within a microfluidic device can localize thermal gel solidification, which has the potential to facilitate cell handling and analysis on-chip. Cells could migrate through liquid-phase gel (e.g., 10°C) but would enrich upon encountering a solid gel barrier (e.g., 25°C). To demonstrate that this proposed strategy can concentrate cells, discrete in-channel temperatures must be established. Previous studies accomplished this effect on-chip by fabricating heating elements into microfluidic devices [29]. Chemical vapor deposition was used to deposit material for the heaters onto a glass substrate, which were then manually aligned over etched microchannels with micron-precision. Although the heaters regulated temperature and locally solidified thermal gel to direct fluid flow, devices could not be operated for extended time because heat radiated throughout the entire device, which caused gel to solidify over an unintended, wide area. Also, the technical expertise required to perform such complex microfabrication limits its accessibility to other research laboratories and increases the cost of analysis.

We hypothesize that Joule heat produced during TGE could serve as a cost-free mechanism to control the temperature—and, consequently, the phase—of thermal gel inside of microfluidic channels. Joule heating is the resistive heating of background electrolyte solution that is produced as current passes through a system when voltage is applied [30,31]. Joule heating is characterized by Eq. (1):

$$Q=\sigma E^2,$$ (1)

where the heat generated (Q) is proportional to the conductivity of the solution (σ) and the applied electric field (E) [32]. Although Joule heating is universally considered detrimental to electrophoretic analyses, it also has the potential to facilitate control over thermal gels. Voltage-induced Joule heating would enable targeted spatial control over gelation in low-cost microfluidic devices that do not require elaborate fabrication (e.g., chemical vapor deposition, channel alignment). Voltage can be precisely controlled using a commercially available electrophoresis power supply. Furthermore, rational application of different voltages across different channels is expected to generate localized Joule heat to maintain distinct temperatures and provide discontinuous phases of thermal gel within a device. Spatial precision of temperature control is expected to be higher than that of heaters because Joule heat is generated within the fluid rather than in the device itself. The potential to induce targeted, reversible solidification of thermal gel using voltage presents a unique opportunity to analyze cells, which cannot be accomplished with standard gel materials (e.g., polyacrylamide).

This report describes the development of an on-chip sample preparation method to handle and enrich cells using gel electrophoresis. Joule heating-induced solidification of PF-127 thermal gel was first characterized using fluorescent beads as model cells. Electrophoretic mobilities of individual beads were found to decrease with increasing device temperature, electric field strength, and gel conductivity due to solidification of the thermal gel. In-channel temperature measurements verified that Joule heating effectively regulated channel temperature—and the gel phase—with response times of <1 s. Joule heating-controlled gelation was then used to enrich cells against a thermal gel barrier and filter excess staining dye to waste. The barrier prevented cells from entering the high-field region to protect them until electrical lysis was desired. Purified, non-lysed cells could also be collected by deactivating the barrier by removing the Joule heat. The results of our studies demonstrate that reversible, voltage-actuated thermal gels facilitate cell handling on-chip using standard electrophoresis equipment. This low-complexity experimental approach to enrich cells will be translated in the future for the analysis of low-concentration intracellular analytes.

2. Materials and Methods

2.1. Chemical reagents

Pluronic F-127, rhodamine B, and sodium chloride were purchased from Millipore Sigma (Burlington, MA). Fluo-Spheres carboxylate-modified microspheres (2 μm diameter), 1 M Tris-HCl, 10× PBS, DMEM growth medium, and PrestoBlue Cell Viability Reagent were obtained from ThermoFisher (Waltham, MA). All solutions were prepared using 18.2 MΩ·cm ultrapure water from an ELGA LabWater Purelab Classic (High Wycombe, UK).

2.2. Microfluidic device fabrication and operation

Microfluidic devices were fabricated out of PDMS molded from 4-inch silicon wafers (University Wafer, South Boston, MA). Standard photolithography was used to create channel features on master wafers using SU-8 2015 negative photoresist (MicroChem, Westborough, MA) [33]. Two photoreticles (Photronics, Brookfield, CT) were used to create devices with either 3 cm straight channels or simple-t configurations with 1 cm sidearms and a 3 cm separation channel (Supporting Information Fig. S1). A profilometer (Mitutoyo, Aurora, IL) was used to measure the final height (20 μm) and width (100 μm) of the raised features. A 10:1 mixture of degassed PDMS elastomer base and curing agent (Ellsworth Adhesives, Germantown, WI) was poured onto the wafers and baked for 2 h at 70°C. Individual devices were diced from the cured PDMS, and reservoirs were created using a 3 mm biopsy punch (Ted Pella, Redding, CA). PDMS channel layers were placed onto glass microscope slides (AmScope, Irvine, CA) to complete the devices.

Thermal gel stock solution was prepared to contain 33.3% (w/v) Pluronic F-127 in 50 mM Tris-HCl. Fluosphere beads were sonicated and then diluted into 50 mM Tris-HCl. Analytical samples were prepared by combining beads 1:9 with thermal gel stock to yield final concentrations of 30% (w/v) PF-127 and 6000 beads/μL for analysis. All thermal gel solutions were prepared at 5°C to maintain the gel in its liquid state. Microfluidic channels were filled with thermal gel at 5°C using vacuum. Once the channel(s) was filled, 5 μL of thermal gel was added to the reservoirs. The microfluidic device was then placed on a custom temperature-controlled microscope stage for 2 min to equilibrate at the desired temperature. A Peltier (TEC1-12730, Amazon.com) and resistance temperature detector (Omega Engineering, Norwalk, CT) were used to regulate stage temperature. These components were controlled by a thermoelectric controller (Wavelength Electronics, Bozeman, MT) operated through a custom LabVIEW program (National Instruments, Austin, TX). A four-channel high voltage power supply (Ultravolt Inc., Ronkonkoma, NY) was then used to apply voltage across the microfluidic device and measure the separation current. Videos of bead migration were captured using an AZ100 epifluorescent microscope (Nikon Instruments Inc., Melville, NY) equipped with a 12-bit Andor ZylasCMOS camera (Oxford Instruments, Concord, MA). A SOLA Light Engine (Lumencor, Beaverton, OR) with a Texas Red filter cube (560/630 nm) was used to illuminate the beads at an intensity of 5 mW/mm². Images were acquired using μ Manager at 500 ms intervals with exposure times of 150 ms at 2.4× magnification [34].

In-channel temperatures during analyses were determined by measuring the temperature-dependent fluorescence of rhodamine B. Channels and reservoirs were loaded with 5 μM of dye in 30% (w/v) PF-127 and placed on the thermally controlled microscope stage. An initial image of the device was acquired at 10°C to serve as a control image to field-flatten the fluorescence intensities in the channel(s) [35,36]. A calibration curve was generated by measuring field-flattened fluorescence versus stage temperature (see Supporting Information Figs. S2 and S3). Voltage experiments were then conducted using rhodamine B fluorescence to measure the Joule heating-induced temperature increases in the channels. False color images from these experiments were created in FIJI [37].

2.3. Data processing

Fluorescence images collected during TGE studies were processed in FIJI using the 2D/3D Particle Tracker program (MOSAIC Group, Dresden, Germany). The particle tracker traced migration paths from individual beads to measure their distances traveled. Beads that were in-frame for 30 s were included in the analysis. The sample particle concentration was selected to achieve a bead density that enabled the program to precisely track individual particles with negligible overlap or misassignment. All experimental conditions were evaluated using n = 4 replicate devices with an average of 100 beads tracked per device. The electrophoretic mobilities of individual particles within each device were calculated by dividing the measured bead velocities by the applied electric field. The mean particle mobilities between replicate devices was averaged to produce the data shown in the subsequent figures. Error bars in the data figures represent ±1 standard deviation from n = 4 devices per each condition evaluated.

2.4. Cell culture

HEK293T cells (Lenti-X, Takara Bio, Mountain View, CA) were cultured in DMEM growth medium (high glucose, 10% fetal bovine serum, penicillin/streptomycin). An aliquot of cells was then pelleted via centrifugation and reconstituted to create a 4 × 10⁷ cells/mL sample. PrestoBlue was added to the cells at a 1× concentration and incubated for 2 h. Once the cells were stained, the sample was centrifuged to pellet the cells (100× g, 5 min). Cells were washed three times with 1× PBS to remove excess dye following the same procedure. Stained cells were then resuspended in PBS and diluted 1:9 into thermal gel for a final concentration of 4 × 10⁶ cells/mL in 30% PF-127. Samples were loaded into microfluidic devices and analyzed via TGE using identical epifluorescence microscope settings as with the bead studies.

3. Results and Discussion

3.1. Characterizing thermal gel solidification

Initial TGE experiments were conducted in single-channel microfluidic devices (Supporting Information Fig. S1A). Devices were created by molding a channel into PDMS and placing it on a glass microscope slide. No complex microfabrication procedures or device alignment was required, which minimized experimental complexity. Fluorescent beads were used as a model system to simulate cells. Beads were anticipated to migrate below the PF-127 gelation temperature but slow at warmer temperatures as the gel began to solidify. Both liquid and solid gel phases of PF-127 thermal gel are optically transparent to facilitate optical detection regardless of gel phase [24].

Beads were loaded into thermal gel and electrophoresed at 333 V/cm over a series of temperatures regulated by the thermal stage. The electrophoretic mobilities of individual particles were found to decrease linearly from 5°C to 20°C (Fig. 1, black) at which point movement halted entirely. A further increase in temperature did not affect particle migration as beads were embedded in a solid gel and their mobilities remained at zero. These results are opposite of the behavior in conventional electrophoresis where analyte mobilities increase with increasing temperature [38], which indicates the prominent effect of thermal gel solidification on the experimental outcomes. Separation current increased linearly over the temperature range evaluated (Fig. 1, gray) including after bead migration had stopped. This response indicates that ions freely move through the gel—regardless of its phase—unlike beads. The increase in current with increasing temperature was attributed to the direct proportionality between temperature (i.e., power) and current at a constant voltage. These experiments demonstrate that bead migration can serve as an indicator of gelation in microfluidic channels and verifies our expectation that mobility decreases with increasing temperature. It should be noted that appreciable bead-to-bead variations were observed within a single device at a single temperature (RSD = 16–30%). Although the manufacturer stated that the beads are uniform in size, the high variance in mobility suggests that the surface charge on individual beads is heterogeneous.

Figure 1.

The electrophoretic mobilities of beads (black circles) and separation current in the device (gray triangles) were measured over a range of stage setpoint temperatures at a field strength of 333 V/cm. Error bars represent ±1 standard deviation from n = 4 devices per temperature. Some error bars are smaller than the data point markers.

The significant impact of temperature on bead migration demonstrates that binary mobilities (i.e., stop or go) can be attained in thermal gels by selecting the appropriate temperatures (e.g., 5°C and 25°C). This result represents the first step toward achieving our goal of in-gel cell enrichment. Next, validation was required to determine whether Joule heat could be used to control solidification of the thermal gel. A pilot experiment was first conducted in which a series of gels was made with increasing concentrations of sodium chloride. These higher conductivity gels were expected to experience increased Joule heating during electrophoresis (see Eq. (1)) and prevent bead migration. Gels were loaded into single-channel devices at 5°C. All gels were liquid at this temperature regardless of salt content. Devices were then placed on the microscope stage thermostatted at 5°C, and electrophoresis was conducted. Increasing salt concentrations reduced bead mobilities until migration was entirely halted (Fig. 2, black). The immobility of beads at 150 mM sodium chloride indicated that the thermal gel solidified immediately when voltage was applied through this highly conductive gel. The separation current in the system was found to increase linearly with ionic strength across the range (Fig. 2, gray) and did not track with particle mobility. Ohm’s law predicts that as current increases at a constant voltage, resistance in the channel would decrease. This reduced resistance should result in higher electrophoretic mobility; however, the opposite trend was observed, which was attributed to solidification of the gel. Although the small ions that generate the separation current were unimpeded by the solid gel, large beads experienced a significant reduction in mobility in the solid. This result demonstrates that the phase of the gel is the predominant factor governing bead mobility while the influence of electrical resistance is negligible.

Figure 2.

Bead mobilities (black circles) and separation current (gray triangles) were measured over a range of added sodium chloride concentrations. Devices were operated at 333 V/cm at 5°C.

The reduced mobilities at higher ionic strengths can be rationalized by two complementary factors. First, Joule heating became more prominent at higher solution conductivities. The larger currents passing through the device increased temperature, which resulted in solidification of the thermal gel even at relatively low voltage. Second, the gelation temperatures of PF-127 thermal gels shift to lower values with increasing salt concentrations [25,39]. Solidification occurs as water molecules preferentially solvate the salt ions, which results in greater entanglement of the PF-127 polymer chains, causing the gels to solidify at lower temperatures. Because all gels were liquid at 5°C when being loaded into our microchannels, a combination of Joule heating and gelation temperature depression was likely responsible for solidification of the gel. However, the positive correlation between Joule heating and thermal gelation affirmed the predicted response of the pilot study, warranting further evaluations of Joule heating-controlled in-gel cell enrichment (described in the next sections). Furthermore, the data in Fig. 2 demonstrate that TGE can analyze biological saline samples with the gel in its liquid phase. Our standard sample preparation procedure dilutes sample 1:9 into thermal gel. A 150 mM saline sample, therefore, results in the equivalent of 15 mM NaCl added to the analytical sample. At this salt concentration, a reduction in mobility was observed from the control sample, but bead mobility remained relatively high in the liquid gel. Preserving liquid-state gels at biologically relevant salt concentrations suggests that cells can be handled on-chip using TGE.

3.2. Characterizing Joule heating-mediated gelation

The studies above demonstrated that setpoint stage temperature and solution ionic strength influence bead mobility and suggest Joule heating can influence the solidification of thermal gel. To directly evaluate the effect of Joule heating on particle migration, beads were electrophoresed over a range of electric field strengths (see Eq. (1)). A setpoint temperature of 10°C was employed to maintain the thermal gel in its liquid state during analysis in single-channel microfluidic devices. Beads would be expected to migrate freely under these conditions unless the increased electric field caused Joule heating-induced gel solidification. Although maintaining temperatures at 10°C is not suitable for our long-term goal of developing a low-complexity TGE analysis system, PF-127 was carried forward in these studies because of its prevalence in the literature. Future experiments will use a thermal gel with a higher gelation temperature (e.g., PF-68) to enable analyses to be carried out at room temperature [40].

Results from the electric field study showed statistically insignificant deviations in bead mobility when applying between 333 and 833 V/cm (Fig. 3A, black). However, mobilities decreased sharply at 1000 V/cm and reached zero by 1333 V/cm. These results indicate a phase change of the thermal gel occurred at higher electric fields due to Joule heating. It is interesting to note that the reduction in mobility is not linear with voltage. The mobility versus electric field curve mirrors previously reported PF-127 viscosity versus temperature curves [27,41]. The sinusoidal shape suggests that bead mobility is stable at lower fields and then abruptly decreases as gel viscosity abruptly increases. This behavior is attributed to the ability of PF-127 to act as a non-Newtonian fluid that differently impacts bead migration depending on the force applied [40]. Separation currents increased linearly with increasing electric fields (Fig. 3A, gray), disconnected from the abrupt changes in particle mobility, but consistent with the response predicted by Ohm’s law. This observation shows that electrical resistance through the gel does not change at higher electric fields despite the gel solidifying. Additionally, runaway current characteristic of problematic Joule heating was not observed despite operating at voltages significantly higher than those typically used in electrophoresis conducted in PDMS devices. This result demonstrates that PF-127 thermal gel significantly attenuates current to prevent catastrophic Joule heating and air bubble formation, which make it an ideal matrix for gel electrophoresis.

Figure 3.

(A) Bead mobilities (black circles) and separation currents (gray triangles) and (B) internal channel temperatures (blue squares) were measured as a function of electric field strength at a setpoint stage temperature of 10°C. Increased temperature was observed at higher field strengths, which solidified the gel and reduced bead mobilities.

To conclusively verify that Joule heating caused temperature to increase in our devices, in-channel temperatures were determined using rhodamine B. Rhodamine B is a thermally responsive dye whose fluorescence is inversely proportional to temperature (Supporting Information Fig. S2) [42,43]. The emission intensity of the dye was used to measure temperature within our channels. Microfluidic devices were filled with dye-containing thermal gel and placed on the microscope stage at 10°C. Voltage was then applied and the fluorescence measured to determine the channel temperature and assess Joule heating (Fig. 3B). Only the lowest field strength (333 V/cm) exhibited a channel temperature matching the 10°C setpoint. Increases in field strength increased channel temperature by 1°C per 66 V/cm—over the range from 667 to 1667 V/cm—despite actively thermostatting devices at 10°C. Comparing bead mobility (Fig. 3A, black) to channel temperature (Fig. 3B) revealed that mobility was similar at electric fields between 333 and 667 V/cm because the temperatures were similar. The thermal gel was in its liquid state at both temperatures, so bead migration was relatively unaffected. At 1000 V/cm, the in-channel temperature was 17°C, and a combination of moving and stationary beads was observed within individual devices. The heterogeneous particle migration was attributed to nonuniform gel solidification across the channel. At 1333 V/cm, the channel temperature was 21°C, which solidified the gel and halted bead mobility entirely. Beads also remained stationary at higher fields. These results demonstrate that electric field-dependent Joule heating increased temperature in the channel, which affected the phase of the thermal gel. Based on the bead mobility data, the gelation temperature of 30% (w/v) PF-127 in our system was determined to be 18°C, which is consistent with previous literature [27].

These voltage-dependent outcomes validated our hypothesis that Joule heating could be used to control temperature within the device, and thus control the phase of the thermal gel. We then sought to characterize the rate of PF-127 gelation by stepping voltage up or down during an analysis to solidify or liquify the thermal gel in situ. Beads were loaded into the channel and analyzed at 10°C as before. Particle mobilities were measured over 1 s intervals to track migration with sufficient temporal resolution to characterize gel behavior. A low field strength (333 V/cm) was first maintained for 15 s where beads migrated with similar mobilities as reported above (Fig. 4A, black). Voltage was then increased to a high field strength (1333 V/cm) causing bead mobilities to decrease to zero within 1 s of this voltage step. These results indicate that PF-127 thermal gel micelles pack from their liquid state into their solid state in <1 s to halt bead migration. This rapid rate of thermal gel solidification is consistent with prior literature that used microfabricated heaters to regulate temperature [29]. The constant supply of Joule heat during voltage application could not be effectively dissipated by the thermal stage despite being set 8°C below the gelation temperature. Rhodamine B was then measured to determine in-channel temperatures (Fig. 4A, blue) using the identical voltage program as in the bead experiments. These measurements validate the observed changes in bead mobility as temperatures rose from below the gelation temperature to above it after the voltage step. The response time of the temperature increase was also <1 s, confirming that the rapid rise in temperature is responsible for changing the phase of the gel and consequently stopping bead mobility.

Figure 4.

Bead mobilities (black circles) and in-channel temperatures (blue squares) were measured as voltage was adjusted within an analysis with the stage held at 10°C. (A) Field strengths were increased from 333to 1333 V/cm or (B) decreased from 1333 to 333 V/cm at t = 15 s.

After characterizing gel solidification, experiments were conducted to evaluate the rate of gel liquification in situ. A high field strength (1333 V/cm) was first applied within the device at a setpoint temperature of 10°C. The abundant Joule heating caused the gel to solidify immediately following voltage application (Fig. 4B, black), which further confirms the rapid rate of thermal gel solidification. After 15 s, voltage was decreased to 333 V/cm. Beads gained mobility within the first 2 s following the voltage step decrease, but average mobilities were negative (i.e., moved backwards) and highly variable. Between 3 and 5 s after the voltage decrease, beads regained a positive mobility suggesting that the gel continued to transition into its liquid phase. After 5 s, a steady-state mobility was reached similar to the expected values at 333 V/cm. In-channel temperature measurements revealed that temperature dropped within 1 s of the voltage reduction, quickly reaching a steady state (Fig. 4B, blue). These results demonstrate that gel liquification begins as the temperature decreases, but bead mobilities do not reach a steady state as quickly as does the temperature. Water must repack between PF-127 micelles to gain fluidity, so we posit that 5 s is required to fully rehydrate the polymer and enable beads to uniformly migrate through. However, it is unclear why beads initially moved backwards after the voltage step. This behavior may be an artifact of our imaging—all images were collected in the center of the device—and mobilities may be heterogeneous across the length of the channel during the rehydration period. However, because the steady-state bead migration was not affected, we did not investigate this further. The primary conclusion from these studies is that that Joule heating can initiate a reversible gel phase change in situ by altering the channel temperature.

3.3. Harnessing joule heating for cell enrichment and purification

The rapid rate of Joule heating-induced gel solidification and liquification demonstrate the feasibility of using thermal gels to create reversible barriers in microfluidic channels. This foundational work was translated to implement voltage-actuated barriers to first enrich cells in-gel and then introduce the purified sample into the analysis channel. Cells were cast into thermal gel and loaded in the top channel of a simple-t microfluidic device while blank thermal gel filled the other channels (Supporting Information Fig. S1B). The stage was set to 10°C, and a high electric field was applied across the horizontal channel while a low field was applied across the vertical channel (Supporting Information Table S1). This voltage scheme provided sufficient Joule heat to solidify the thermal gel in the horizontal channel but insufficient heat to gel the vertical channel (see the Supporting Information for further details). Cells migrated freely through the liquid-phase gel in the top channel upon voltage application (Fig. 5A). However, cells were unable to enter the channel intersection because of the solidified thermal gel barrier in the horizontal channel, resulting in their collection over time (Fig. 5A, 1 and 2.5 min). A video of cell enrichment is shown in Supporting Information Video S1. Rhodamine B was used to determine in-channel temperatures during the enrichment step, and a false color map was generated to illustrate the distinct temperatures maintained in the two channels (Fig. 5B, Enrich). These results validate that temperature was low in the vertical channel, which enabled the thermal gel to remain liquid and cells to migrate through. Temperature in the horizontal channel was above the gelation temperature and was thus solidified to serve as an enrichment barrier. Because the solid gel stopped cell migration, cells did not enter the high-field region, which preserved cell integrity as evidenced by the intracellular dye remaining confined within the cells. To initiate lysis, cells were introduced into the channel intersection where the electric field was high to electrically lyse cells and liberate their internal contents (Supporting Information Fig. S4). Cells were also introduced into lower channel for analysis by deactivating voltage in the horizontal channel to remove the Joule heat (Fig. 5A, Collect). The low voltage applied during the collection step enabled the temperature to remain low and preserve a liquid state gel (Fig. 5B, Collect).

Figure 5.

(A) Fluorescence images depicting enrichment of cells (white) over a 2.5-min duration using reversible voltage-mediated thermal gel barriers. Lines were added to the images to indicate the channel boundaries (yellow). (B) A false color heat map illustrates the in-channel temperature during cell concentration.

The samples used to demonstrate cell enrichment required multiple washing and centrifugation steps to remove excess dye from the sample prior to loading into microfluidic devices. Although this washing procedure is standard practice in biological research laboratories, it is tedious and reduces research throughput. We sought to demonstrate that thermal gel barriers can expedite sample preparation by purifying cells on-chip. Unwashed cell samples were cast into thermal gel and loaded into the top channel of simple-t microfluidic devices. Fluorescence from the excess dye was initially seen throughout the device because dye in the liquid state gel had diffused into the other channels (Fig. 6, 0 min). Voltage was then applied to actuate the thermal gel barrier and initiate cell enrichment. Dye readily passed through the semipermeable gel barrier and was directed to the waste reservoir (Fig. 6, 1 min) akin to a gated injection scheme. Cells, however, could not pass through the solidified gel and concentrated at the barrier. Excess dye was removed from the sample channel within 2.5 min. These proof-of-concept demonstrations illustrate how Joule heating can be harnessed in thermal gels to enrich and purify cells by creating semipermeable microfluidic barriers. These results also build toward our long-term goal of collecting cells for lysis, and then transferring intracellular biomolecules into the bottom separation channel for gel electrophoresis analysis.

Figure 6.

Fluorescence images depicting cell purification. Excess small molecule dye passes through the thermal gel barrier to waste to facilitate cell washing and concurrent enrichment. Lines were added to the images to indicate the channel boundaries.

4. Concluding Remarks

Joule heating is universally considered to be a detriment to electrophoresis, but this report demonstrates that Joule heat can be utilized in TGE to attain unique analytical functionalities. Voltage-controlled Joule heating was used to control the phase of thermal gels. This heating impacted bead mobility with effects accentuated at higher applied electric fields and in higher conductivity solutions. The electrophoretic mobility of fluorescent beads was found to be a simple, effective indicator of thermal gel solidification as temperature in the system was altered. Thermal gel solidification and liquification had response times of <1 s, which illustrates the potential for rapid actuation of reversible thermal gel barriers. Proof-of-concept experiments demonstrated that thermal gels accommodated enrichment and purification of cells using standard geometry microfluidic devices made from inexpensive PDMS substrates. Controlling voltage—and consequently temperature and gel phase—within low complexity microfluidic devices provides a facile means of handling cells on-chip using widely available electrophoresis power supplies, which increases the accessibility of our methods. Future work will use a thermal gel with a higher gelation temperature to enable analyses to be conducted under ambient conditions to further enhance translation of our methods to other labs. Future studies will integrate in-gel cell handling with electrophoretic separations to analyze intracellular cargo.

Abbreviations:

PF-127

Pluronic F-127

TGE

thermal gel electrophoresis