We investigate the use of periodic micropillar arrays produced by high-fidelity
microfabrication with cyclic olefin polymers for solid-phase immunoassays. These
three-dimensional (3D) templates offer higher surface-to-volume ratios than
two-dimensional substrates, making it possible to attach more antibodies and so increase
the signal obtained by the assay. Micropillar arrays also provide the capacity to induce
wicking, which is used to distribute and confine antibodies on the surface with spatial
control. Micropillar array substrates are modified by using oxygen plasma treatment,
followed by grafting of (3-aminopropyl)triethoxysilane for binding proteins covalently
using glutaraldehyde as a cross-linker. The relationship between microstructure and
fluorescence signal was investigated through variation of pitch (10–50 μm),
pillar diameter (5–40 μm), and pillar height (5–57 μm). Our
findings suggest that signal intensity scales proportionally with the 3D surface area
available for performing solid-phase immunoassays. A linear relationship between
fluorescence intensity and microscale structure can be maintained even when the aspect
ratio and pillar density both become very high, opening the possibility of tuning assay
response by design such that desired signal intensity is obtained over a wide dynamic
range compatible with different assays, analyte concentrations, and readout instruments.
We demonstrate the versatility of the approach by performing the most common immunoassay
formats—direct, indirect, and sandwich—in a qualitative fashion by using
colorimetric and fluorescence-based detection for a number of clinically relevant
protein markers, such as tumor necrosis factor alpha, interferon gamma (IFN-γ),
and spike protein of severe acute respiratory syndrome coronavirus 2. We also show
quantitative detection of IFN-γ in serum using a fluorescence-based sandwich
immunoassay and calibrated samples with spike-in concentrations ranging from 50 pg/mL to
5 μg/mL, yielding an estimated limit of detection of ∼1 pg/mL for arrays
with high micropillar density (11561 per mm
2
) and aspect ratio (1:11.35).