Enhancement of performance in porous bead-based microchip sensors: Effects of chip geometry on bio-agent capture.

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.

Hot embossed polyethylene through-hole chips for bead-based microfluidic devices.

Over the past decade, there has been a growth of interest in the translation of microfluidic systems into real-world clinical practice, especially for use in point-of-care or near patient settings. While initial fabrication advances in microfluidics involved mainly the etching of silicon and glass, the economics of scaling of these materials is not amendable for point-of-care usage where single-test applications force cost considerations to be kept low and throughput high. As such, materials base more consistent with point-of-care needs is required. In this manuscript, the fabrication of a hot embossed, through-hole low-density polyethylene ensembles derived from an anisotropically etched silicon wafer is discussed. This semi-opaque polymer that can be easily sterilized and recycled provides low background noise for fluorescence measurements and yields more affordable cost than other thermoplastics commonly used for microfluidic applications such as cyclic olefin copolymer (COC). To fabrication through-hole microchips from this alternative material for microfluidics, a fabrication technique that uses a high-temperature, high-pressure resistant mold is described. This aluminum-based epoxy mold, serving as the positive master mold for embossing, is casted over etched arrays of pyramidal pits in a silicon wafer. Methods of surface treatment of the wafer prior to casting and PDMS casting of the epoxy are discussed to preserve the silicon wafer for future use. Changes in the thickness of polyethylene are observed for varying embossing temperatures. The methodology described herein can quickly fabricate 20 disposable, single use chips in less than 30 min with the ability to scale up 4 times by using multiple molds simultaneously. When coupled as a platform supporting porous bead sensors, as in the recently developed Programmable Bio-Nano-Chip, this bead chip system can achieve limits of detection, for the cardiac biomarker C-reactive protein, of 0.3 ng/mL, thereby demonstrating that the approach is compatible with high performance, real-world clinical measurements in the context of point-of-care testing.

Porous bead-based diagnostic platforms: bridging the gaps in healthcare.

Advances in lab-on-a-chip systems have strong potential for multiplexed detection of a wide range of analytes with reduced sample and reagent volume; lower costs and shorter analysis times. The completion of high-fidelity multiplexed and multiclass assays remains a challenge for the medical microdevice field; as it struggles to achieve and expand upon at the point-of-care the quality of results that are achieved now routinely in remote laboratory settings. This review article serves to explore for the first time the key intersection of multiplexed bead-based detection systems with integrated microfluidic structures alongside porous capture elements together with biomarker validation studies. These strategically important elements are evaluated here in the context of platform generation as suitable for near-patient testing. Essential issues related to the scalability of these modular sensor ensembles are explored as are attempts to move such multiplexed and multiclass platforms into large-scale clinical trials. Recent efforts in these bead sensors have shown advantages over planar microarrays in terms of their capacity to generate multiplexed test results with shorter analysis times. Through high surface-to-volume ratios and encoding capabilities; porous bead-based ensembles; when combined with microfluidic elements; allow for high-throughput testing for enzymatic assays; general chemistries; protein; antibody and oligonucleotide applications.

Effects of sample delivery on analyte capture in porous bead sensors.

Sample delivery is a crucial aspect of point-of-care applications where sample volumes need to be low and assay times 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.

Modeling analyte transport and capture in porous bead sensors.

Porous agarose microbeads, with high surface to volume ratios and high binding densities, are attracting attention as highly sensitive, affordable sensor elements for a variety of high performance bioassays. While such polymer microspheres have been extensively studied and reported on previously and are now moving into real-world clinical practice, very little work has been completed to date to model the convection, diffusion, and binding kinetics of soluble reagents captured within such fibrous networks. Here, we report the development of a three-dimensional computational model and provide the initial evidence for its agreement with experimental outcomes derived from the capture and detection of representative protein and genetic biomolecules in 290 µm porous beads. We compare this model to antibody-mediated capture of C-reactive protein and bovine serum albumin, along with hybridization of oligonucleotide sequences to DNA probes. These results suggest that, due to the porous interior of the agarose bead, internal analyte transport is both diffusion and convection based, and regardless of the nature of analyte, the bead interiors reveal an interesting trickle of convection-driven internal flow. On the basis of this model, the internal to external flow rate ratio is found to be in the range of 1:170 to 1:3100 for beads with agarose concentration ranging from 0.5% to 8% for the sensor ensembles here studied. Further, both model and experimental evidence suggest that binding kinetics strongly affect analyte distribution of captured reagents within the beads. These findings reveal that high association constants create a steep moving boundary in which unbound analytes are held back at the periphery of the bead sensor. Low association constants create a more shallow moving boundary in which unbound analytes diffuse further into the bead before binding. These models agree with experimental evidence and thus serve as a new tool set for the study of bioagent transport processes within a new class of medical microdevices.

A disposable bio-nano-chip using agarose beads for high performance immunoassays.

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.

Location of biomarkers and reagents within agarose beads of a programmable bio-nano-chip.

The slow development of cost-effective medical microdevices with strong analytical performance characteristics is due to a lack of selective and efficient analyte capture and signaling. The recently developed programmable bio-nano-chip (PBNC) is a flexible detection device with analytical behavior rivaling established macroscopic methods. The PBNC system employs ≈300 µm-diameter bead sensors composed of agarose "nanonets" that populate a microelectromechanical support structure with integrated microfluidic elements. The beads are an efficient and selective protein-capture medium suitable for the analysis of complex fluid samples. Microscopy and computational studies probe the 3D interior of the beads. The relative contributions that the capture and detection of moieties, analyte size, and bead porosity make to signal distribution and intensity are reported. Agarose pore sizes ranging from 45 to 620 nm are examined and those near 140 nm provide optimal transport characteristics for rapid (<15 min) tests. The system exhibits efficient (99.5%) detection of bead-bound analyte along with low (≈2%) nonspecific immobilization of the detection probe for carcinoembryonic antigen assay. Furthermore, the role analyte dimensions play in signal distribution is explored, and enhanced methods for assay building that consider the unique features of biomarker size are offered.