Robert J. Meagher

Peptidomimetic polymers for antifouling surfaces

Exposure of therapeutic and diagnostic medical devices to biological fluids is often accompanied by interfacial adsorption of proteins, cells, and microorganisms. Biofouling of surfaces can lead to compromised device performance or increased cost and in some cases may be life-threatening to the patient. Although numerous antifouling polymer coatings have enjoyed short-term success in preventing protein and cell adsorption on surfaces, none have proven ideal for conferring long-term biofouling resistance. Here we describe a new biomimetic antifouling N-substituted glycine polymer (peptoid) containing a C-terminal peptide anchor derived from residues found in mussel adhesive proteins for robust attachment of the polymer onto surfaces. The methoxyethyl side chain of the peptoid portion of the polymer was chosen for its chemical resemblance to the repeat unit of the known antifouling polymer poly(ethylene glycol) (PEG), whereas the composition of the 5-mer anchoring peptide was chosen to directly mimic the DOPA- and Lys-rich sequence of a known mussel adhesive protein. Surfaces modified with this biomimetic peptide-peptoid conjugate exhibited dramatic reduction of serum protein adsorption and resistance to mammalian cell attachment for over 5 months in an in vitro assay. These new synthetic peptide based antifouling polymers may provide long-term control of surface biofouling in the physiologic, marine, and industrial environments.

Ligase detection reaction for the analysis of point mutations using free-solution conjugate electrophoresis in a polymer microfluidic device

We have developed a new method for the analysis of low abundant point mutations in genomic DNA using a combination of an allele‐specific ligase detection reaction (LDR) with free‐solution conjugate electrophoresis (FSCE) to generate and analyze the genetic products. FSCE eliminates the need for a polymer sieving matrix by conjugating chemically synthesized polyamide “drag‐tags” onto the LDR primers. The additional drag of the charge‐neutral drag‐tag breaks the linear scaling of the charge‐to‐friction ratio of DNA and enables size‐based separations of DNA in free solution using electrophoresis with no sieving matrix. We successfully demonstrate the conjugation of polyamide drag‐tags onto a set of four LDR primers designed to probe the K‐ras oncogene for mutations highly associated with colorectal cancer, the simultaneous generation of fluorescently labeled LDR/drag‐tag conjugate (LDR‐dt) products in a multiplexed, single‐tube format with mutant:WT ratios as low as 1:100, respectively, and the single‐base, high‐resolution separation of all four LDR‐dt products. Separations were conducted in free solution with no polymer network using both a commercial capillary array electrophoresis (CAE) system and a PMMA microchip replicated via hot‐embossing with only a Tris‐based running buffer containing additives to suppress the EOF. Typical analysis times for LDR‐dt were 11 min using the CAE system and as low as 85 s for the PMMA microchips. With resolution comparable to traditional gel‐based CAE, FSCE along with microchip electrophoresis decreased the separation time by more than a factor of 40.

Completely monodisperse, highly repetitive proteins for bioconjugate capillary electrophoresis: development and characterization.

Protein-based polymers are increasingly being used in biomaterial applications because of their ease of customization and potential monodispersity. These advantages make protein polymers excellent candidates for bioanalytical applications. Here we describe improved methods for producing drag-tags for free-solution conjugate electrophoresis (FSCE). FSCE utilizes a pure, monodisperse recombinant protein, tethered end-on to a ssDNA molecule, to enable DNA size separation in aqueous buffer. FSCE also provides a highly sensitive method to evaluate the polydispersity of a protein drag-tag and thus its suitability for bioanalytical uses. This method is able to detect slight differences in drag-tag charge or mass. We have devised an improved cloning, expression, and purification strategy that enables us to generate, for the first time, a truly monodisperse 20 kDa protein polymer and a nearly monodisperse 38 kDa protein. These newly produced proteins can be used as drag-tags to enable longer read DNA sequencing by free-solution microchannel electrophoresis.