Controlling the selection stringency of phage display using a microfluidic device

Neuroscience Research Institute, University of California, Santa Barbara, CA93106, USA.
Lab on a Chip (Impact Factor: 6.12). 05/2009; 9(8):1033-6. DOI: 10.1039/b820985e
Source: PubMed


We report the utilization of microfluidic technology to phage selection and demonstrate that accurate control of washing stringency in our microfluidic magnetic separator (MMS) directly impacts the diversity of isolated peptide sequences. Reproducible generation of magnetic and fluidic forces allows controlled washing conditions that enable rapid convergence of selected peptide sequences. These findings may provide a foundation for the development of automated microsystems for rapid in vitro directed evolution of affinity reagents.

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Available from: Frank V Cochran, Jan 20, 2014
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    • "Recently a new cell-surface biopanning method based on the microfluidics technology was proposed. The accurate control of washing stringency in the microfluidic magnetic separator (MMS) circumvents cell loss and allows efficient removal of weakly or nonspecifically-bound phages [31]. The small numbers of cells required constitutes an advantage, rendering MMS a promising tool for the discovery of biomarkers from patient samples. "
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    ABSTRACT: Since the advent of phage display technology, dating back to 1985, antibody libraries displayed on filamentous phage surfaces have been used to identify specific binders for many different purposes, including the recognition of tumors. Phage display represents a high-throughput technique for screening billions of random fusion antibodies against virtually any target on the surface or inside cancer cells, or even soluble markers found in patient serum. Many phage display derived binders targeting important tumor markers have been identified. Selection directed to tumoral cells' surfaces lead to the identification of unknown tumoral markers. Also the improvement of methods that require smaller amounts of cells has opened the possibility to use this approach on patient samples. Robust techniques combining an antibody library displayed on the phage surface and protein microarray allowed the identification of auto antibodies recognized by patient sera. Many Ab molecules directly or indirectly targeting angiogenesis have been identified, and one of them, ramucirumab, has been tested in 27 phase I-III clinical trials in a broad array of cancers. Examples of such antibodies will be discussed here with emphasis on those used as probes for molecular imaging and other clinical trials.
    International Journal of Molecular Sciences 12/2012; 13(5):5420-40. DOI:10.3390/ijms13055420 · 2.86 Impact Factor
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    • "The micromagnetic separation (MMS) chip was fabricated and assembled according to a process previously described by our group (11–13). Briefly, the device was fabricated on borosilicate glass substrates with 25 -µm-thick double-coated tape (3M). "
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    ABSTRACT: Multivalent molecular interactions can be exploited to dramatically enhance the performance of an affinity reagent. The enhancement in affinity and specificity achieved with a multivalent construct depends critically on the effectiveness of the scaffold that joins the ligands, as this determines their positions and orientations with respect to the target molecule. Currently, no generalizable design rules exist for construction of an optimal multivalent ligand for targets with known structures, and the design challenge remains an insurmountable obstacle for the large number of proteins whose structures are not known. As an alternative to such design-based strategies, we report here a directed evolution-based method for generating optimal bivalent aptamers. To demonstrate this approach, we fused two thrombin aptamers with a randomized DNA sequence and used a microfluidic in vitro selection strategy to isolate scaffolds with exceptionally high affinities. Within five rounds of selection, we generated a bivalent aptamer that binds thrombin with an apparent dissociation constant (K(d)) <10 pM, representing a ∼200-fold improvement in binding affinity over the monomeric aptamers and a ∼15-fold improvement over the best designed bivalent construct. The process described here can be used to produce high-affinity multivalent aptamers and could potentially be adapted to other classes of biomolecules.
    Nucleic Acids Research 10/2012; 40(22). DOI:10.1093/nar/gks899 · 9.11 Impact Factor
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    ABSTRACT: Sample preparation is often the most tedious and demanding step in an assay, but it also plays an essential role in determining the quality of results. As biological questions and analytical methods become increasingly sophisticated, there is a rapidly growing need for systems that can reliably and reproducibly separate cells and particles with high purity, throughput and recovery. Microfluidics technology represents a compelling approach in this regard, allowing precise control of separation forces for high performance separation in inexpensive, or even disposable, devices. In addition, microfluidics technology enables the fabrication of arrayed and integrated systems that operate either in parallel or in tandem, in a capacity that would be difficult to achieve in macro-scale systems. In this report, we use recent examples from our work to illustrate the potential of microfluidic cell- and particle-sorting devices. We demonstrate the potential of chip-based high-gradient magnetophoresis that enable high-purity separation through reversible trapping of target particles paired with high-stringency washing with minimal loss. We also describe our work in the development of devices that perform simultaneous multi-target sorting, either through precise control of magnetic and fluidic forces or through the integration of multiple actuation forces into a single monolithic device. We believe that such devices may serve as a powerful "front-end" module of highly integrated analytical platforms capable of providing actionable diagnostic information directly from crude, unprocessed samples - the success of such systems may hold the key to advancing point-of-care diagnostics and personalized medicine.
    Journal of the Association for Laboratory Automation 12/2009; 14(6):331-340. DOI:10.1016/j.jala.2009.06.003 · 1.50 Impact Factor
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