Eliminating Size-Associated Diffusion Constraints for Rapid On-Surface Bioassays with Nanoparticle Probes

Abstract

Nanoparticle probes enable implementation of advanced on-surface assay formats, but impose often underappreciated size-associated constraints, in particular on assay kinetics and sensitivity. The present study highlights substantially slower diffusion-limited assay kinetics due to the rapid development of a nanoprobe depletion layer next to the surface, which static incubation and mixing of bulk solution employed in conventional assay setups often fail to disrupt. In contrast, cyclic solution draining and replenishing yields reaction-limited assay kinetics irrespective of the probe size. Using common surface bioassays, enzyme-linked immunosorbent assays and immunofluorescence, this study shows that this conceptually distinct approach effectively "erases" size-dependent diffusion constraints, providing a straightforward route to rapid on-surface bioassays employing bulky probes and procedures involving multiple labeling cycles, such as multicycle single-cell molecular profiling. For proof-of-concept, the study demonstrates that the assay time can be shortened from hours to minutes with the same probe concentration and, at a typical incubation time, comparable target labeling can be achieved with up to eight times lower nanoprobe concentration. The findings are expected to enable realization of novel assay formats and stimulate development of rapid on-surface bioassays with nanoparticle probes.

Keywords: assay kinetics; bioassays; diffusion limitation; molecular profiling; nanoparticle probes; on-surface bioassays.

Figure 1

Schematic illustration of the cyclic draining-replenishing (CDR) technology. (a) Conventional surface-based assays typically employ static incubation for probe binding to surface-immobilized targets. As a result of fast binding kinetics and slow diffusion, probes get rapidly depleted from the volume adjacent to the surface, yielding a substantially lower effective probe concentration and limiting further target labeling kinetics by slow mass transfer from the bulk solution. Improving bulk fluid exchange by rocking, stirring, and shaking has little effect on depletion layer near the surface. (b) CDR method enables rapid target labeling by eliminating the probe diffusion limitation. The probe depletion layer is quickly removed by complete draining of the staining solution, rather than speeding up probe diffusion in the solution. Subsequent refilling of the surface with the same bulk solution keeps the probe concentration near the surface constant and equivalent to bulk solution, thus maintaining fast target labeling kinetics.

Figure 2

Kinetics of rapid ELISA diagnostics. (a) Assay performed with Poly-HRP probes exhibited slow mass transfer-limited kinetics under static incubation (t½ = 36 min) and rotary shaking (t½ = 43 min) conditions, but demonstrated a dramatically improved performance with CDR (t½ = 5 min). Compared to standard ELISA protocols where incubation typically takes 1 h, CDR reaches the same staining intensity in 7 minutes. (b) Smaller mono-HRP probes exhibited faster kinetics under static incubation (t½ = 26 min) and rotary shaking (t½ = 28 min) conditions, featuring further improvement in assay speed with CDR (t½ = 12 min). Solution absorbance at 450 nm with respect to assay time is shown and fitted by an exponential curve. Background absorbance by the TMB substrate alone was subtracted from all measurements. Error bars represent one standard deviation from triplicate assays.

Figure 3

Rapid immunofluorescence staining with QDot probes. (a-d) Characterization of QDot-1'Ab and dye-labeled 1'Ab probes targeting Lamin A. One-step immunofluorescence images obtained with (a) QDot-1’Ab and (b) dye-labeled 1’Ab probes produced staining patterns consistent with the nuclear membrane localization of Lamin A and (c,d) results obtained with QDot-2'Ab in a conventional two-step staining procedure (positive Lamin A staining is shown in (c) and control lacking 1’Ab incubation in (d)). Scale bar, 50 μm. (e) Quantitative evaluation of staining intensity with respect to staining time achieved via CDR, static incubation, and rotary shaking techniques. Notably, CDR achieved comparable staining intensity 6 times faster than conventional methods, producing detectable signal within the first 10 minutes of staining. Error bars represent one standard deviation of an average Lamin A staining intensity from four different fields of view. (f) Representative cell staining intensity maps obtained after 10-min CDR in comparison to 60-min static and rotary shaking incubation. All images were normalized and color-coded with a heat map for direct comparison of staining pattern and intensities. Scale bar, 50 μm.

Figure 4

Background-free immunofluorescence staining via CDR. (a-d) Representative fluorescence intensity maps obtained after a 1-hour incubation with (a) 7 nM QDot-1'Ab (1× [probe]) under rotary shaking and (b) 0.3 nM (0.04× [probe]), (c) 0.9 nM (0.13× [probe]), and (d) 1.5 nM (0.21× [probe]) QDot-1'Ab using CDR procedure. Images were normalized and color-coded with a heat map. Scale bar, 250 μm. (e) Average fluorescence intensities of Lamin A staining achieved after a 1-hour incubation under rotary shaking with 1× [probe] and CDR with 0.04×, 0.13×, and 0.21× QDot-1'Ab concentration. Consistent with qualitative observations in (a-d), quantitative analysis demonstrated that comparable staining could be obtained with ~8-times lower probe concentration via CDR methodology. Error bars represent one standard deviation of an average Lamin A staining intensity from four different fields of view. (f) A strong background fluorescence originating from the QDots in 1× [probe] bulk solution used for staining under rotary shaking required extensive specimen washing prior to imaging. At the same time, 0.13× [probe] solution employed with CDR methodology featured nearly no background, enabling real-time monitoring of staining evolution. Scale bar, 50 μm.

Figure 5

Rapid multicycle immunofluorescence staining. Five sequential cycles of single-color staining and imaging were performed on the same cell subpopulation for multiplexed detection of five molecular targets (Lamin A, HSP90, Ki-67, Cox-4 and β-tubulin) using self- assembled QDot-SpA-Ab probes. During each cycle, labeling was done via either a 10-minute CDR (a) or 1-hour rotary shaking (b) incubation. Both methods produced highly specific staining patterns with no carry-over fluorescence, build-up of background fluorescence, or cross-staining between cycles, yielding accurate 5-target imaging and analysis. However, CDR methodology enabled a substantial reduction in the overall assay time due to a dramatically improved labeling kinetics. Composite 5-target images were generated from false-colored, aligned, and cropped images of individual targets. Scale bar, 50 μm.