Sophia Kenrick

Multitarget Dielectrophoresis Activated Cell Sorter

The ability to rapidly and efficiently isolate specific viruses, bacteria, or mammalian cells from complex mixtures lies at the heart of biomedical applications ranging from in vitro diagnostics to cell transplantation therapies. Unfortunately, many current selection methods for cell separation, such as magnetic activated cell sorting (MACS), only allow the binary separation of target cells that have been labeled via a single parameter (e.g., magnetization). This limitation makes it challenging to simultaneously enrich multiple, distinct target cell types from a multicomponent sample. We describe here a novel approach to specifically label multiple cell types with unique synthetic dielectrophoretic tags that modulate the complex permittivities of the labeled cells, allowing them to be sorted with high purity using the multitarget dielectrophoresis activated cell sorter (MT-DACS) chip. Here we describe the underlying physics and design of the MT-DACS microfluidic device and demonstrate approximately 1000-fold enrichment of multiple bacterial target cell types in a single-pass separation.

Bacterial display enables efficient and quantitative peptide affinity maturation

A quantitative screening method was developed to enable isolation and affinity maturation of peptide ligands specific for a given target from peptide libraries displayed on the outer surface of Escherichia coli using multi-parameter flow cytometry. From a large, random 15-mer peptide library, screening identified a core motif of W-E/D-W-E/D that conferred binding to vascular endothelial growth factor (VEGF). One cycle of affinity maturation resulted in the identification of several families of VEGF-binding peptides having distinct consensus sequences, from which a preferred disulfide constraint emerged. In the second affinity maturation cycle, high affinity peptides were favored by the addition of a decoy protein that bound an adjacent epitope on the display scaffold. The decoy apparently reduced rebinding or avidity effects, and the resulting peptides exhibited consensus at 12 of 19 amino acid positions. Peptides identified and affinity matured using bacterial display were remarkably similar to the best affinity matured using phage display and exhibited comparable dissociation constants (within 2-fold; K(D) = 4.7 x 10(-7) M). Screening of bacterial-displayed peptide libraries using cytometry enabled optimization of screening conditions to favor affinity and specificity and rapid clonal characterization. Bacterial display thus provides a new quantitative tool for the discovery and evolutionary optimization of protein-specific peptide ligands.

Characterization of Protein-Protein Interactions via Static and Dynamic Light Scattering

Light scattering in its various flavors constitutes a label-free, non-destructive probe of macromolecular interactions in solution, providing a direct indication of the formation or dissociation of complexes by measuring changes in the average molar mass or molecular radius as a function of solution composition and time. It is a first-principles technique, thoroughly grounded in thermodynamics, permitting quantitative analysis of key properties such as stoichiometry, equilibrium association constants, and reaction rate parameters.

Flow Cytometric Sorting of Bacterial Surface‐Displayed Libraries

The protocols herein detail methods for isolating binding peptides from a combinatorial library displayed on the surface of bacterial cells. These methods are appropriate for a variety of display scaffolds and a large range of library sizes, up to ∼5 × 109 or more. Instructions have been provided for isolating peptides that bind to both proteins and non‐protein targets, such as whole cells or inorganic particles. Qualitative analysis by flow cytometry can be exploited for bacterial libraries to characterize a displayed peptides binding properties with a target of interest, and sorting conditions can be tuned to maximize binding affinity. Curr. Protocol. Cytom. 42:4.6.1‐4.6.27.

Cell-Based Protein Sensor Array Using Genetically Engineered Microbes

A cell-based protein-detecting array was developed on which genetically engineered microbes were spatially organized as sensor elements in a microfluidic device. Whole E. coli cells displaying protein-binding peptide ligands at high density on their outer membrane served as effective capture reagents. Cells were immobilized on gold electrodes using dielectrophoresis, thus allowing each sensor element to be electrically addressable. On-chip fluorescence analysis and flow cytometry were used to confirm highly specific capture of target molecules. The use of dielectrophoresis in microfluidic devices enabled efficient utilization of whole-cell capture reagents for a variety of protein detection and diagnostic applications.