Kjell E. Nelson

Microfluidic diagnostic technologies for global public health

The developing world does not have access to many of the best medical diagnostic technologies; they were designed for air-conditioned laboratories, refrigerated storage of chemicals, a constant supply of calibrators and reagents, stable electrical power, highly trained personnel and rapid transportation of samples. Microfluidic systems allow miniaturization and integration of complex functions, which could move sophisticated diagnostic tools out of the developed-world laboratory. These systems must be inexpensive, but also accurate, reliable, rugged and well suited to the medical and social contexts of the developing world.

Chimeric Peptides of Statherin and Osteopontin That Bind Hydroxyapatite and Mediate Cell Adhesion

Extracellular matrix proteins play key roles in controlling the activities of osteoblasts and osteoclasts in bone remodeling. These bone-specific extracellular matrix proteins contain amino acid sequences that mediate cell adhesion, and many of the bone-specific matrix proteins also contain acidic domains that interact with the mineral surface and may orient the signaling domains. Here we report a fusion peptide design that is based on this natural approach for the display of signaling peptide sequences at biomineral surfaces. Salivary statherin contains a 15-amino acid hydroxyapatite binding domain (N15) that is loosely helical in solution. To test whether N15 can serve to orient active peptide sequences on hydroxyapatite, the RGD and flanking residues from osteopontin were fused to the C terminus. The fusion peptides bound tightly to hydroxyapatite, and the N15-PGRGDS peptide mediated the dose-dependent adhesion of Moαv melanoma cells when immobilized on the hydroxyapatite surface. Experiments with an integrin-sorted Moαv subpopulation demonstrated that the αvβ3 integrin was the primary receptor target for the fusion peptide. Solid state NMR experiments showed that the RGD portion of the hydrated fusion peptide is highly dynamic on the hydroxyapatite surface. This fusion peptide framework may thus provide a straightforward design for immobilizing bioactive sequences on hydroxyapatite for biomaterials, tissue engineering, and vaccine applications.

Surface plasmon resonance measurement of binding and dissociation of wild-type and mutant streptavidin on mixed biotin-containing alkylthiolate monolayers

Abstract The attachment of recognition elements to surfaces through streptavidin–biotin links has potential applications in sensor technology. Surface plasmon resonance (SPR) was used to monitor the kinetics of binding of streptavidin (SA) to mixed biotin-containing alkylthiolate monolayers (BTMs) on gold and the subsequent competitive desorption kinetics. Various binary compositions of BTMs were prepared from a mixture of biotinylated alkylthiol (BAT) and a poly(ethylene oxide) (PEO) alkylthiol. Differences in both the amount of adsorbed protein and the relative desorption rates between a low-binding affinity mutant (W120A) and wild-type streptavidin (WT) were observed as a function of the composition of the BTM. The rates of WT and W120A desorption from these mixed BTMs were correlated with the amount of biotin present on the surface. The results confirm the specific nature of the bonds which attach streptavidin to the biotinylated surface in the mixed BAT/PEO monolayer. However, when the PEO-terminated alkylthiolate was replaced with a simple methyl-terminated alkylthiolate, non-specific adsorption of the streptavidin dominates.

SPR imaging-based salivary diagnostics system for the detection of small molecule analytes

Abstract:  Saliva is an underused fluid with considerable promise for biomedical testing. Its potential is particularly great for monitoring small‐molecule analytes since these are often present in saliva at concentrations that correlate well with their free levels in blood. We describe the development of a prototype diagnostic device for the rapid detection of the antiepileptic drug (AED) phenytoin in saliva. The multicomponent system includes a hand‐portable surface plasmon resonance (SPR) imaging instrument and a disposable microfluidic assay card.

Modeling of a competitive microfluidic heterogeneous immunoassay: sensitivity of the assay response to varying system parameters.

We present a fractional sensitivity analysis of a competitive microfluidic heterogeneous immunoassay for a small molecule analyte. A simple two-dimensional finite element model is used to determine the fractional sensitivity of the assay signal with respect to analyte concentration, flow rate, initial surface density of binding sites, and antibody concentration. The fractional sensitivity analysis can be used to identify (1) the system parameters for which it is most crucial to control or quantify the variability between assays and (2) operating ranges for these parameters that improve assay sensitivity (within the constraints of the experimental system). Experimental assay results for the drug phenytoin, obtained using surface plasmon resonance imaging, are shown to be consistent with the predictions of the model.

Conditioning saliva for use in a microfluidic biosensor

This report details an approach to saliva conditioning for compatibility of raw patient samples with microfluidic immunoassay components, principally biosensor surfaces susceptible to fouling. Stimulated whole human saliva spiked with a small molecule analyte (phenytoin, 252 Da) was first depleted of cells, debris and high molecular weight glycoproteins (mucins) using membrane filtration. This process significantly reduced but did not eliminate fouling of biosensor surfaces exposed to the sample. An H-filter, which separates solutes from mixed samples based on their diffusion in laminar flow, was used to extract the analyte from the remaining large molecular weight species in the filtered saliva sample. Patient samples treated in this way retained 23% of the analyte with 97% and 92% reduction in glycoproteins and proteins, respectively, and resulted in 3.6 times less surface fouling than either untreated or filtered saliva alone. These sample conditioning steps will enable the use of fouling-sensitive detection techniques in future studies using clinical saliva samples.

Constrained Cell Recognition Peptides Engineered into Streptavidin

Streptavidin is widely used as an adaptor molecule in diagnostics, separations, and laboratory assay applications. We have here engineered cell adhesive peptides into the three‐dimensional scaffolding of streptavidin to convert streptavidin into a functional protein. The mutations did not alter refolding or tetramer assembly and the slow biotin dissociation rate of wild‐type streptavidin was retained. The peptide targets were hexapeptide sequences derived from osteopontin and fibronectin that contain the RGD cell adhesion sequence. Cell binding assays directly demonstrated that rat aortic endothelial cells and human melanoma cells adhered to surfaces coated with either of the two RGD streptavidin mutants in a dose‐dependent fashion. Wild‐type streptavidin displayed no significant cell binding activity. Inhibition studies with soluble RGD peptides confirmed that the cell adhesion was RGD mediated. Further inhibition studies with antibodies directed against αvβ3 demonstrated that the RGD−streptavidin interaction was primarily mediated by this integrin with melanoma cells. These results demonstrate that peptide recognition sequences can be engineered into accessible surface regions of streptavidin without disrupting biotin binding properties. This approach to introducing secondary functional activities into streptavidin may improve streptavidins utility in existing applications or provide new technology opportunities.

Structural characterization and comparison of RGD cell-adhesion recognition sites engineered into streptavidin

The RGD (arginine-glycine-aspartic acid) sequence is found in several important extracellular matrix proteins and serves as an adhesion ligand for members of the integrin family of cell-surface receptors. This sequence and flanking residues from fibronectin or osteopontin have been engineered into an accessible surface loop of streptavidin to create two new streptavidin variants (FN-SA or OPN-SA, respectively) that bind cells through the alpha(v)beta(3) and/or alpha(5)beta(1) integrin receptors. Their crystal structures confirm the design and construction of the mutants and provide evidence about the conformational dynamics of the RGD loops. The loops in the isomorphous crystal structures are involved in crystal-packing interactions and this stabilizes their structures. Even so, the loop in OPN-SA is slightly disordered and two of the residues are not seen in difference electron-density maps. Comparison with other experimentally determined structures of RGD loops in cell-adhesion molecules shows that these loops occupy a large subset of conformational space. This is consistent with the view that RGD loops, at least those involved in cell adhesion, sample a number of structures dynamically, a few of which display high affinity for appropriate receptors.

Chapter 10:SPR Imaging for Clinical Diagnostics

Surface plasmon resonance (SPR)-based detection for clinical applications has attracted significant interest in recent years. Studies have demonstrated the detection of clinically relevant analytes such as prostate-specific antigen [1], C-reactive protein [2] and human ferritin [3] at physiologicall...

Biomaterials that talk

Summary form only given. We are working to develop a variety of biomaterials that are designed to communicate with biomolecules, cells, and tissues. Here we report the joint use of protein engineering and microfabrication approaches to generate surfaces that control cell phenotype in confined dimensions on device surfaces, and the solid-state NMR characterization of peptide and protein conformation on hydroxyapatite and hydrophobic biomaterial surfaces that interact with cell surface receptors.