Controlled Nanoconfinement in a Microfluidic Modular Bead Array Device via Elastomeric Diaphragm Collapse for Enhancing Biomolecular Binding Kinetics

Abstract

Nanoscale confinement strategies alleviate diffusional transport limitations to enhance target binding kinetics with receptors, motivating their utilization for screening and selecting receptors based on binding affinities with target molecules. Herein, a modular and multiplexed device for creating nanoconfinement is presented through the collapse of an elastomeric diaphragm onto microbead arrays immobilized with biomolecules, followed by repeated diaphragm withdrawal to promote bulk transport, thereby enhancing receptor binding kinetics. To repeatedly create controlled nanoconfinement over large spatial extents on the bead, the diaphragm is integrated on its top side with a strain sensor for modulating vertical displacement, while microfabricated nanoposts (≈500 nm depth) on its bottom side control the lateral extent. The modular platform enables facile assembly of beads, each immobilized with different targets into eight microwells for multiplexed screening of receptors, and facile disassembly for quantifying DNA-binding on each bead by downstream q-PCR. Nanoconfinement enhances biomolecular binding at 1 Hz diaphragm pressurization, as validated by rapid DNA immobilization (time constant of ≈6 min vs >60 min under no confinement) and through saturating the binding of target molecules with optimal aptamer candidates (88% site occupancy vs 5% under no confinement at 10 nm levels), thereby screening candidate receptors based on binding affinity parameters.

Keywords: aptamers; binding affinities; binding kinetics; diaphragms; microfluidics; nanofluidics.

Figure 1

A) Schematic of the bead with immobilized target molecules as the binding reactor to screen aptamer receptors through creating nanoconfinement in the micro‐2‐nano (µ2n) platform. B) i) Diaphragm deformation in balloon shape within obstacle‐free space versus ii) around microbead topography in a tent‐shape, which requires the integration of strain sensors to control displacement level. iii) Top view of 3D nanoconfinement around the bead. C) Cross‐sectional view of nanoconfinement around the bead using 500 nm depth posts (nanoposts) integrated with liquid metal (EGaIn) sensor on the diaphragm to control the depth and lateral extent of confinement to reduce diffusion times. D) Overall automated system for creating and controlling nanoconfinement in the µ2n platform. E) Operation and detection: i) 8 multiplexed bead reaction chambers within the µ2n platform, each immobilized with specific target and control molecules on the bead. ii) repeated diaphragm switching on the µ2n platform between nanoconfinement and bulk sample transport mode. iii) bead removal from the reaction chamber for off‐chip real‐time q‐PCR measurement of DNA binding.

Figure 2

A) i) Cross‐section showing assembly of each layer of the multilayer µ2n platform; ii) Detailed design of each layer (1–7). iii) Scanning Electron Microscopy (SEM) image of nano‐posts (500 nm height, 20 µm diameter, and 40 µm spacing) used to control the lateral extent of nanoconfinement. iv) Nano‐post depth is validated with white light interferometry (WLI). B) Top‐view photograph of the µ2n platform showing the 8 microwells to each hold a bead (red dashed circles), fluid inlet reservoir (orange dashed circle), fluid outlet (light blue circle), and air pressure control (royal blue circle) for creating nanoconfinement that is detected by a strain sensor (image in the inset) using liquid metal (EgaIn) injected into a channel on the diaphragm.

Figure 3

A) Fluidic implementation of the µ2n platform for enhancing binding kinetics: i) Sample is introduced into the reservoir (sample loading inlet), while the liquid outlets and the air inlet remain closed. ii) Negative pressure is then applied to the liquid outlets to draw in the sample from the reservoir into each µbead chamber, with the air inlet closed at this stage. iii) The air inlet is opened to pressurize diaphragms in each chamber to create nano‐confinement around the beads, with the excess liquid sample flowing to the reservoir for mixing. The liquid inlet and outlets are closed at this stage. B) i) Finite element simulations of the fluid velocity magnitude profile in the µ2n platform under confined conditions. ii) Simulations of the velocity field and flow profile in the µ2n platform under applied pressure, leading to diaphragm bulging and the establishment of nanoconfined conditions. iii) Simulations of the velocity field and flow profile when the applied pressure is released, allowing the diaphragm to revert to its original relaxed state, thereby inducing reverse flow conditions. Photo of the µ2n platform in: C) the relaxed state (sample loaded into each bead chamber from the reservoir) versus D) the nanoconfined state (diaphragms with nanoposts displaced into the bead chamber to create nanoconfinement around the beads, with excess liquid back to the reservoir for mixing). Uneven color in some microwells is an optical effect from the 3 M tape to temporarily seal the inlet.

Figure 4

A) Finite element simulation of tent‐shaped diaphragm displacement around the bead under: i) deformed (ΔP = 60 kPa) conditions, with an enlarged view of dotted box region in (ii), and bead in the designed microwell under relaxed diaphragm in (iii). B) Confocal images to quantify confinement level (%D) measured based on the surface area of the bead (S) confined by the diaphragm in the µ2n platform. %D = S/S_Total (S_Total is bead surface area or πd² ). The green color is from the fluorescein‐containing sample and the dark color represents fluorescence‐blocking regions, such as the bead and diaphragm, with the red dashed line and polygons indicating the confining diaphragm with nanoposts. i) No nanoconfinement at ΔP = 0 kPa, with no diaphragm covering the bead (%D = 0). ii) Moderate confinement level at ΔP = 30 kPa, with the diaphragm covering ≈20% of the total bead area (%D = 20%). iii) High confinement level at ΔP = 60 kPa, with diaphragm covering ≈45% of the total bead area (%D = 45%). C) These %D levels determined from confocal imaging are used to calibrate the resistance change of the integrated EGaIn‐based strain sensor at corresponding pressure levels, based on the ratio of R _o (altered resistance) to R _i (initial resistance) (number of iterations, n = 10). The insets (i–iii) show photographs of the diaphragm surrounding the beads at the respective confinement levels. The scale bar represents 250 µm for all the images.

Figure 5

A) Scheme for immobilization of modified thrombin aptamer (so‐called HT5‐R9) that is composed of a 15‐nucleotide sequence (HT5) linked to primer sites (R9), with a thiolated poly‐AT linker (24‐AT‐SH) for binding to Au‐coated beads on thiol‐gold interaction for optimizing the nanoconfinement parameters. B) Comparison of DNA binding kinetics on beads based on thiol‐gold interaction (quantified by q‐PCR) using the µ2n platform without confinement versus with confinement (ΔP = 60 kPa on diaphragm applied at 1 Hz), with 100 nm concentration of HT5‐R9R in 1 m NaCl solution at different reaction times. C) Quantification of DNA binding on beads within 10 min of immobilization based on thiol‐gold interaction in the µ2n platform, with confinement (ΔP = 60 kPa to diaphragm) for 100 nm concentration of HT5‐R9R in 1 m NaCl solution at different confinement frequency (0.1–1 Hz). D) Comparison of DNA binding after 10 min without confinement versus with confinement using different concentrations of HT5‐R9 in 1 m NaCl solution. E) Enhancement of DNA‐thiol binding kinetics under nanoconfinement (100 nm concentration of HT5‐R9R in 1 m NaCl solution per B) based on time constant (τ) for 63% adsorption site occupancy (τ ≈ 6 min) versus τ >60 min under the absence of confinement. The drop of unoccupied sites follows the exponential drop (per the indicated fit). Error bars from measurements on 4 beads from each microwell are reported.

Figure 6

A) Scheme for quantifying DNA aptamer interactions with immobilized protein target by off‐chip q‐PCR of beads. Using beads in the µ2n platform immobilized with a specific target (4 beads immobilized with HT or human thrombin) and non‐specific target (4 beads immobilized with HSA or human serum albumin), the binding of HT3‐R9R aptamers are quantified at: B) 100 nm and C) 10 nm aptamer concentration levels for 5 min reaction times under no confinement versus with confinement (ΔP = 60 kPa to diaphragm at 1 Hz frequency).