The retention and elution of proteins in ion-exchange chromatography is routinely controlled by adjusting the mobile phase salt concentration. It has repeatedly been observed, as judged from adsorption isotherms, that the apparent heterogeneity of adsorption is lower at more-eluting, higher ionic strength. Here, we present an investigation into the mechanism of this phenomenon using a single-molecule, super-resolution imaging technique called motion-blur Points Accumulation for Imaging in Nanoscale Topography (mbPAINT). We observed that the number of functional adsorption sites was smaller at high ionic strength and that these sites had reduced desorption kinetic heterogeneity, and thus narrower predicted elution profiles, for the anion-exchange adsorption of α-lactalbumin on an agarose-supported, clustered-charge ligand stationary phase. Explanations for the narrowing of the functional population such as inter-protein interactions and protein or support structural changes were investigated through kinetic analysis, circular dichroism spectroscopy, and microscopy of agarose microbeads, respectively. The results suggest the reduction of heterogeneity is due to both electrostatic screening between the protein and ligand and tuning the steric availability within the agarose support. Overall, we have shown that single molecule spectroscopy can aid in understanding the influence of ionic strength on the population of functional adsorbent sites participating in the ion-exchange chromatographic separation of proteins.

Sample delivery is a crucial aspect of point-of-care applications where sample volumes need to be low and assay times need to be short, while providing high analytical and clinical sensitivity. In this paper, we explore the influence of the factors surrounding sample delivery on analyte capture in an immunoassay-based sensor array manifold of porous beads resting in individual wells. We model using computational fluid dynamics and a flow-through device containing beads sensitized specifically to C-reactive protein (CRP) to explore the effects of volume of sample, rate of sample delivery, and use of recirculation vs. unilateral delivery on the effectiveness of the capture of CRP on and within the porous bead sensor. Rate of sample delivery lends to the development of a time-dependent, shrinking depletion region around the bead exterior. Our findings reveal that at significantly high rates of delivery, unique to porous bead substrates, capture at the rim of the bead is reaction-limited, while capture in the interior of the bead is transport-limited. While the fluorescence signal results from the aggregate of captured material throughout the bead, multiple kinetic regimes exist within the bead. Further, under constant pressure conditions dictated by the array architecture, we reveal the existence of an optimal flow rate that generates the highest signal, under point-of-care constraints of limited-volume and limited-time. When high sensitivity is needed, recirculation can be implemented to overcome the analyte capture limitations due to volume and time constraints. Computational simulations agree with experimental results performed under similar conditions.

This article reports on the fabrication of a disposable bio-nano-chip (BNC), a microfluidic device composed of polydimethylsiloxane (PDMS) and thiolene-based optical epoxy which is both cost-effective and suitable for high performance immunoassays. A novel room temperature (RT) bonding technique was utilized so as to achieve irreversible covalent bonding between PDMS and thiolene-based epoxy layers, while at the same time being compatible with the insertion of agarose bead sensors, selectively arranged in an array of pyramidal microcavities replicated in the thiolene thin film layer. In the sealed device, the bead-supporting epoxy film is sandwiched between two PDMS layers comprising of fluidic injection and drain channels. The agarose bead sensors used in the device are sensitized with anti-C-reactive protein (CRP) antibody, and a fluorescent sandwich-type immunoassay was run to characterize the performance of this device. Computational fluid dynamics (CFD) was used based on the device specifications to model the bead penetration. Experimental data revealed analyte penetration of the immunocomplex to 100 μm into the 280 μm diameter agarose beads, which correlated well with the simulation. A dose–response curve was obtained and the linear dynamic range of the assay was established over 1 ng/mL to 50 ng/mL with a limit of detection less than 1 ng/mL.

Measuring low concentrations of clinically-important biomarkers using porous bead-based lab-on-a-chip (LOC) platforms is critical for the successful implementation of point-of-care (POC) devices. One way to meet this objective is to optimize the geometry of the bead holder, referred to here as a micro-container. In this work, two geometric micro-containers were explored, the inverted pyramid frustum (PF) and the inverted clipped pyramid frustum (CPF). Finite element models of this bead array assay system were developed to optimize the micro-container and bead geometries for increased pressure, to increase analyte capture in porous bead-based fluorescence immunoassays. Custom micro-milled micro-container structures containing an inverted CPF geometry resulted in a 28% reduction in flow-through regions from traditional anisotropically-etched pyramidal geometry derived from Si-111 termination layers. This novel “reduced flow-through” design resulted in a 33% increase in analyte penetration into the bead and twofold increase in fluorescence signal intensity as demonstrated with C-Reactive Protein (CRP) antigen, an important biomarker of inflammation. A consequent twofold decrease in the limit of detection (LOD) and the limit of quantification (LOQ) of a proof-of-concept assay for the free isoform of Prostate-Specific Antigen (free PSA), an important biomarker for prostate cancer detection, is also presented. Furthermore, a 53% decrease in the bead diameter is shown to result in a 160% increase in pressure and 2.5-fold increase in signal, as estimated by COMSOL models and confirmed experimentally by epi-fluorescence microscopy. Such optimizations of the bead micro-container and bead geometries have the potential to significantly reduce the LODs and reagent costs for spatially programmed bead-based assay systems of this type.