Affordable Microfluidic Bead-Sorting Platform for Automated Selection of Porous Particles Functionalized with Bioactive Compounds

Abstract

The ability to rapidly and accurately evaluate bioactive compounds immobilized on porous particles is crucial in the discovery of drugs, diagnostic reagents, ligands, and catalysts. Existing options for solid phase screening of bioactive compounds, while highly effective and well established, can be cost-prohibitive for proof-of-concept and early stage work, limiting its applicability and flexibility in new research areas. Here, we present a low-cost microfluidics-based platform enabling automated screening of small porous beads from solid-phase peptide libraries with high sensitivity and specificity, to identify leads with high binding affinity for a biological target. The integration of unbiased computer assisted image processing and analysis tools, provided the platform with the flexibility of sorting through beads with distinct fluorescence patterns. The customized design of the microfluidic device helped with handling beads with different diameters (~100–300 µm). As a microfluidic device, this portable novel platform can be integrated with a variety of analytical instruments to perform screening. In this study, the system utilizes fluorescence microscopy and unsupervised image analysis, and can operate at a sorting speed of up to 125 beads/hr (~3.5 times faster than a trained operator) providing >90% yield and >90% bead sorting accuracy. Notably, the device has proven successful in screening a model solid-phase peptide library by showing the ability to select beads carrying peptides binding a target protein (human IgG).

Purification of polyclonal immunoglobulin G from human serum using peptide‐based adsorbents

“…The LigaGuard™ adsorbent has been originally developed for purifying monoclonal antibodies from Chinese Hamster Ovary (CHO) cell culture supernatants in flow‐through mode 27,40,41 . The resin is functionalized with an ensemble of peptide ligands that capture a broad spectrum of protein impurities differing by composition, posttranslational modification, size, and titer, while allowing the antibody product to flow through unbound.…”

“…The LigaGuard™ adsorbent has been originally developed for purifying monoclonal antibodies from Chinese Hamster Ovary (CHO) cell culture supernatants in flow-through mode. 27,40,41 The resin is At the same time, the higher complexity of plasma, where the ratio of Ig vs. non-Ig proteins is about 2 Â 10 5 ppm, 42 compared to that of recombinant sources, where the same ratio varies between 1 and 2 Â 10 5 ppm, [43][44][45] poses the need to optimize both the composition of the LigaGuard™ ligands and the chromatographic protocol.…”

Multiplexed Competitive Screening of One-Bead-One-Component Combinatorial Libraries Using a ClonePix 2 Colony Sorter

“…Most notably, the selected HCP-binding peptides showed considerable sequence homology, grouped in multipolar and hydrophobic positive sequences [19], and afforded specific capture of HCPs with minimal binding of IgG. While positive controls were not explicitly analyzed with the ClonePix 2 platform in this work, the selection of an antibody-binding peptide (HWRGWV) from a mixture of peptide-functionalized beads (HWRGWV-ChemMatrix and a portion of a combinatorial OBOP library) has previously been demonstrated using this multiplexed approach implemented on a microfluidic bead-sorting platform [14].…”

“…A myriad of approaches and instruments have been introduced to increase throughput and decrease the frequency of false positives. Notable examples include the expansion of selection criteria in the COPAS Flow Pilot system [3,4,12], the Pickoscreen confocal nanoscanning and bead-picking platform (CONA) [13], library screening via microfluidic sorting [14], bead blot approaches [15], methods to reduce interference from non-specific binding [16,17], and the emergence of low-cost automated systems for library screening by wide-field fluorescence microscopy [18]. While great progress has been achieved in throughput and the ability to modulate the binding strength of the identified leads to match target specifications, current techniques rarely focus on tailoring binding selectivity based on the complexity of the intended application.…”

Purification of polyclonal Immunoglobulin G from human serum using LigaGuardTM and LigaTrapTM adsorbents

“…The LigaGuard TM adsorbent has been originally developed by our team as a combination of resins functionalized with multi-modal peptides for purifying monoclonal antibodies (mAbs) in flow-through mode from recombinant sources [ [44][45][46] ]. The ensemble of ligands is capable of capturing a broad spectrum of protein impurities while allowing the antibody product to flow through unbound.…”