Controlling Dispersion during Single-Cell Polyacrylamide-Gel Electrophoresis in Open Microfluidic Devices

Abstract

New tools for measuring protein expression in individual cells complement single-cell genomics and transcriptomics. To characterize a population of individual mammalian cells, hundreds to thousands of microwells are arrayed on a polyacrylamide-gel-coated glass microscope slide. In this "open" fluidic device format, we explore the feasibility of mitigating diffusional losses during lysis and polyacrylamide-gel electrophoresis (PAGE) through spatial control of the pore-size of the gel layer. To reduce in-plane diffusion-driven dilution of each single-cell lysate during in-microwell chemical lysis, we photopattern and characterize microwells with small-pore-size sidewalls ringing the microwell except at the injection region. To reduce out-of-plane-diffusion-driven-dilution-caused signal loss during both lysis and single-cell PAGE, we scrutinize a selectively permeable agarose lid layer. To reduce injection dispersion, we photopattern and study a stacking-gel feature at the head of each <1 mm separation axis. Lastly, we explore a semienclosed device design that reduces the cross-sectional area of the chip, thus reducing Joule-heating-induced dispersion during single-cell PAGE. As a result, we observed a 3-fold increase in separation resolution during a 30 s separation and a >2-fold enhancement of the signal-to-noise ratio. We present well-integrated strategies for enhancing overall single-cell-PAGE performance.

Figure 1.

Benchmarking to ascertain effectiveness of dispersion-reduction strategies in scPAGE. (A) Schematic of the dispersion-control condition (Disc⁺), which comprises a uniform PA gel for scPAGE, with two regions of interest: (i) a dense PA-gel sidewall partially ringing each microwell to define an injector region in the PAGE-separation axis and (ii) a stacking gel defined by a step increase in gel density down the separation axis to reduce sample-injection dispersion during scPAGE. The lysis step prior to separation is indicated by t = 0; scPAGE duration is indicated by t = t_s. (B) Schematic of the benchmark condition incorporating no dispersion control (None). The benchmark None condition comprises a uniform 10% T PA gel with open microwells. (C) Disc⁺ system setup: enclosed device that incorporates an agarose-hydrogel lid. This design is reduces protein loss during cell lysis and protein diffusion during separation by mitigating Joule heating. (D) None-system setup: open-chamber device with electrophoresis buffer filled to 10 mm. (E) Reduction of protein diffusion during cell lysis resulting in narrower injection width. Data shows representative tGFP-concentration profiles at the initiation of cell lysis measured at the cross-sections of microwells, each containing a single U251 GFP human glioblastoma cell. (F) Concentration profiles of separated FITC-BSA* and FITC-OVA* during scPAGE, illustrating the impacts of diffusion and Joule-heating mitigation on separation performance. The Disc⁺ condition is the combination of the patterned PA gel with dense sidewalls, the stacking gel, and the enclosed PAGE device. The open-chamber condition comprises a uniform PA gel in an open-chamber device.

Figure 2.

Grayscale photopatterning creating nonuniform gel density around the microwells, defining an injector region and stacking gel for scPAGE. (A) Schematic of grayscale photopatterning to create nonuniform PA gels aligned to each scPAGE microwell. The grayscale photomask consists of arrays of transparent and opaque squares (5–20 μm in length). Squares are not to scale. (B) Calibration of the relationship between the photomask grayscale values and PA-gel density assessed by electrophoretic mobility. For patterning a 6% T stacking gel and a 10% T separation gel, 60 and 40% grayscale patterns were used, respectively. (C) PA gel density changes along the separation axis at the interface of stacking gel and separation gel (higher signal correlates with higher percent T). Fluorescence micrograph of an FITC-decorated allylamine gel reports nonuniform gel density around the microwell periphery and into the separation axis (right-hand side of the image).

Figure 3.

Nonuniform-pore-size PA gels reducing analyte dispersion during cell lysis and sample injection. (A) Estimated diffusion time for lysis buffer and lysate molecules through various agarose-layer thicknesses. Small lysis-buffer components (ions and micelles) can diffuse through agarose, whereas larger species, including proteins, cannot diffuse as readily. The inset shows a wider y-axis to indicate the BSA*-diffusion time scale. (B) Viable single U251-GFP cells after encapsulation by agarose. (C) Epifluorescence micrographs reporting the GFP distribution at completion of a 30 s cell-lysis period. The patterned PA gel with the agarose lid (Disc⁺) and the uniform 10% T PA gel with open microwells (None) are shown. (D) Monitoring of fluorescence intensity during lysis of U251-GFP cells in microwells with high-density PA-gel sidewalls and an agarose lid vs in microwells of a uniform gel, open fluidic device. Error bars are standard deviations (n = 3). (E) Representative epifluorescence micrographs and accompanying concentration profiles for in situ single-cell lysis and protein injection in two conditions: the patterned-PA-gel and agarose-lid configuration (Disc⁺) and the uniform 10% T PA gel and open-microwell configuration (None). The analyte distribution is shown after 30 s of the cell-lysis period and 1 s after injection. Microwell edges are highlighted for clarity.

Figure 4.

Reduction of Joule heating during scPAGE in the enclosed device. (A) Local temperature measured by an IR sensor and electrical current during scPAGE in open and enclosed scPAGE devices (t_PAGE = 35 s, E = 50 V/cm). (B) Signal-to-noise ratio (SNR) measured for PAGE of OVA* in an open chamber and in the Disc⁺ format (c_OVA*=0.5 μM, E = 50 V/cm). Error bars are standard deviations (n = 3).

Figure 5.

Injection dispersion impacts scPAGE SR and detection sensitivity. (A) Inverted grayscale epifluorescence micrographs of in situ U251-GFP cell lysis and subsequent PAGE of GFP in a dispersion-control device and in an open fluidic device. (B) Inverted grayscale epifluorescence micrographs of stacking of BSA* and OVA* in the Disc⁺ device, compared with those of a uniform 10% T gel in an open-chamber configuration (E = 50 V/cm). (C) Fluorescence intensity along the separation path in (B). (D) SNR of OVA* and SR of scPAGE in (B). Error bars are 1 standard deviation (n = 3).