Publications

A novel conductimetric sensor, capable of detecting and continuously monitoring the concentration of a specific nonionic microsolute (glucose) in a multicomponent aqueous solution (e.g., whole blood or plasma) is described, and preliminary experimental evidence supporting the validity of the sensor concept is presented. Detection is based upon the use of a glucose-selective complexing agent (a boronic acid immobilized in a hydrogel) which liberates a mobile microion (hydrogen ion) when it binds a glucose molecule The change in ionic conductivity of the hydrogel resulting from the increase in ion concentration is thus directly related to the ambient glucose concentration. Confinement of the liberated ions within the hydrogel, and prevention of entry of extraneous electrolytes present in the test solution, is necessary to make the conductimetric measurement meaningful. This is achieved by encapsulating the hydrogel within a bipolar ion exchange membrane impermeable to ions but freely permeable to glucose (and other nonionic microsolutes). A physicochemical model of the complexation equilibrium, kinetics of solute transport through the membrane and hydrogel phase, and their impact upon the ionic conductivity of the hydrogel is presented, which supports the utility of this sensor concept as a potentially reliable and sensitive glucose monitor. Its generic utility for monitoring nonionic microsolutes in multicomponent aqueous solutions is suggested.



"Chiral Ligand Exchange Adsorbents for Amines and Underivatized Amino Acids: 'Bait-and-Switch' Molecular Imprinting,"  F. H. Arnold, S. Striegler and V. Sundaresan, ACS Symposium Ser., 703, 109-118 (1998).

Ligand-exchange adsorbents for the chiral separation of underivatized a-amino acids and amine analogs have been prepared by molecular imprinting with a polymerizable copper complex and the amino acid as template.  Because the complexation equilibria in these mixed-ligand systems involve numerous species, it is extremely important to characterize the template:monomer assemblies prior to polymerization.  Isothermal titration calorimetry and classical species distribution diagrams have been used to determine the conditions under which the amino acids (phenylalanine) form strong 1:1 mixed-ligand complexes with the copper-iminodiacetate (CuIDA) functional monomer.  In contrast, the diamine template ethylenediamine does not form 1:1 complexes with CuIDA; the diamine instead competes with IDA for the metal ion, and a stronger chelating group is required.  Adsorbents prepared using enantiomers of phenylalanine as template show enantioselectivity for phenylalanine and tyrosine as well as for a chiral amine analog of phenylalanine, alpha-methylphenethylamine.  The use of the amino acid as the template in this 'bait-and-switch' imprinting approach allows creation of chirally selective binding sites suitable for separations of chiral amines.


"Molecularly Imprinted Ligand Exchange Adsorbents for Chiral Separations of Underivatized Amino acids," S. Vidyasankar, M. Ru and F. H. Arnold, J. Chromatography, 775, 51-63 (1997). 

  

Ligand-exchange adsorbents that are enantioselective for underivatized alpha-amino acids have been synthesized by molecular imprinting using only achiral monomers. Bulk polymers were prepared by allowing the functional monomer, Cu(II)-N-(4-vinylbenzyl)iminodiacetic acid, to form complexes with the template amino acid in solution, followed by crosslinking with ethylene glycol dimethacrylate. To make supports suitable for chromatography, the imprinted polymer was grafted to derivatized silica particles. Racemic mixtures of various underivatized alpha-amino acids are resolved on the imprinted adsorbents. Adsorbents prepared from amino acids with larger, aromatic side chains exhibit the highest selectivities (alpha = 1.65 for the enantioresolution of d,l-phenylalanine). Cross-selectivity for similar amino acids also depends on side chain size: materials templated with l- or d-phenylalanine exhibit good enantioselectivity when challenged with racemic tyrosine (alpha ~ 1.4) and much reduced enantioselectivities towards d,l-tryptophan or aliphatic amino acids. Materials imprinted with alanine show no selectivity. The ability of a material imprinted with an amino acid enantiomer to resolve an analogous chiral amine is also demonstrated. The mechanisms underlying the observed enantioselectivity are discussed in light of the three-point interaction model for conventional chiral ligand exchange separations. 

"A Glucose-Sensing Polymer," G. Chen, V. Sundaresan and F. H. Arnold, Proceedings of the ACS Division of Polymeric Materias: Science and Engineering, 76, 378-379 (1997).  

"A Glucose-Sensing Polymer," G. Chen, Z. Guan, C.-T.Chen, D. Lee, L. Fu and F. H. Arnold, Nature Biotechnology, 15, 354-357 (1997). 

  

We have prepared a robust polymer that can be used to measure glucose concentrations in complex biological media. At alkaline pH, this metal-complexing polymer binds glucose and instantly release protons in proportion to the glucose concentration over a clinically relevant range (0-25 mM). The inexpensive polymer is sufficiently selective to provide an easily measurable response to glucose in porcine plasma. The polymer's ability to function at nonphysiological pH's (at which the buffer capacity of biological samples is small) makes it possible to design simple and inexpensive sensing devices based on measurement of changes in proton concentration. 

"Substrate Selectivity of Molecularly Imprinted Polymers Incorporating a Rigid Chelating Monomer, Bis-Methacrylato-(4-methyl, 4'-vinyl)2,2'-bipyridine Cu(II)," P. K. Dhal and F. H. Arnold, New J. Chemistry, 20, 695-698 (1996). 

The substrate binding selectivities of imprinted polymers prepared using bis-methacrylato (4-methyl, 4'-vinyl) 2,2'-bipyridine Cu(II) (1) as the metal-chelating monomer have been investigated. This monomer was designed to minimize the flexibility of the metal ions in the binding sites formed by molecular imprinting. A series of bisimidazole derivatives (3-5) were used as the templates for the preparation of imprinted polymers. Formation of monomer:template assemblies in solution was investigated by visible and ESR spectroscopy. Polymerization of these assemblies with ethylene glycol dimethacrylate yielded macroporous, insoluble polymers. In competitive rebinding experiments, the resulting polymers showed greater selectivity for their bisimidazole templates than imprinted polymers prepared previously using an iminodiacetate metal-chelating monomer (2). 

"Polymeric Sensor Materials for Glucose," C.-T. Chen, G. Chen, D. Lee and F. H. Arnold, Polymer Preprint, 37, 217-218 (1996). 

"Multipoint Binding in Metal Affinity Chromatography II. Effect of pH and Imidazole on Chromatographic Retention of Engineered Histidine-Containing Cytochromes c," R. D. Johnson, R. J. Todd and F. H. Arnold, Chromatography A, 725, 225 (1996). 

"Surface Site Hetereogeneity and Lateral Interactions in Multipoint Protein Adsorption," R. D. Johnson, Z.-G. Wang and F. H. Arnold, J. Phys. Chem., 100, 5134- 5139 (1996). 

Studies carried out using engineered proteins have demonstrated that protein adsorption to functional surfaces involves multiple interactions between specific groups on the protein and complementary binding sites distributed on the surface. A consequence of multipoint interactions is that protein binding affinity should depend strongly on the distribution of surface binding sites. In this investigation we present a thermodynamic framework for multipoint protein binding to a random arrangement of surface binding sites that also includes lateral interactions among adsorbed protein molecules. This framework results in reversible adsorption behavior analogous to that predicted by the Temkin model and chromatographic behavior analogous to that predicted by the "stoichiometric displacement" model (SDM). Using this framework we can now interpret the semi-empirical parameters obtained using these models for protein binding in chromatographic systems in terms of thermodynamic parameters for protein-surface interactions. We show a correlation between Temkin model parameters for a series of cytochrome c variants in immobilized metal affinity chromatography (IMAC) that is consistent with protein adsorption to a nonuniform arrangement of surface binding sites. Lateral interactions among adsorbed protein molecules are shown to be insignificant for this system. 

"Multipoint Binding and Heterogeneity in Immobilized Metal-Affinity Chromatography," R. D. Johnson and F. H. Arnold, Biotechnology & Bioengineering, 48, 437-443 (1995). 

Studies carried out using engineered proteins clearly demonstrate that adsorption to derivatized surfaces involves multiple interactions between functional groups on the protein and complementary sites distributed on the surface. The fact that adsorption involves multipoint interactions has important implications for the design of separations processes and for the interpretation of heterogeneity in biological recognition phenomena. Increasing the density of surface metal sites (immobilized copper ions) is found to be functionally equivalent to increasing the number of metal-coordinating groups on the protein (histidines and dep rotonated amines), in that both processes increase the likelihood of simultaneous interactions between the protein and the surface. A consequence of multiple-site interactions is a significant increase in protein binding affinity that depends on the arrangement of surface sites. A protein will show the highest affinity for arrangements of surface sites which best match its own pattern of functional groups and will show lower affinity for less optimal arrangements, resulting in binding that is inherently heterogeneous. We have found that reversible protein adsorption in immobilized metal affinity chromatography (IMAC) is described by the Temkin model, which characterizes binding heterogeneity by a uniform distribution of binding energies over the population of surface binding sites. 

"The Temkin Isotherm Describes Heterogeneous Protein Adsorption," R. D. Johnson and F. H. Arnold, Biochim. Biophys. Acta., 1247, 293-297 (1995). 

"Surface Grafting of Functional Polymers to Macroporous Poly(Trimethylolpropane Trimethacrylate)," P. K. Dhal, S. Vidyasankar and F. H. Arnold, Chemistry of Materials 7, 154-162 (1995). 

"Surface Modification with Molecularly-Imprinted Polymers for Selective Recognition," F. H. Arnold, S. Plunkett, P. K. Dhal and V. Sundaresan, Polymer Preprint s 36, 97-98 (1995). 

"Molecularly-Imprinted Polymers on Silica: Selective Supports for High Performance Ligand-Exchange Chromatography," S. Plunkett and F. H. Arnold, J. Chromatography, A. 708, 19-29 (1995). 

Thin coatings of molecularly-imprinted, metal-complexing polymers have been grafted to activated silica beads suitable for high performance liquid chromatography (HPLC). Propylmethacrylate-activated silica particles were coated by copolymerization with a metal-chelating monomer, Cu2+-[N-(4-vinylbenzyl)-imino]diacetic acid (1), a metal-coordinating (imidazole) template (4 - 7), and ethylene glycol dimethacrylate. After extraction to remove the template and re-loading with metal, the composite materials re-bind the templates with which they were prepared and exhibit selectivities comparable to bulk-polymerized imprinted materials. The strong Cu2+-imidazole interaction, desirable for creating a high-fidelity imprint, leads to excessive retention in elution chromatography. By replacing the copper in the imprinted metal-complexing polymers with weaker-binding Zn2+, these novel ligand-exchange supports can effect partial to complete chromatographic separation of their bis-imidazole templates from other, highly similar imidazole-containing substrates. This 'bait and switch' approach can significantly enhance the performance of molecularly-imprinted materials. Scatchard plots of equilibrium binding data show a significant degree of heterogeneity in the imprinted binding sites of material prepared with a bis-imidazole template, but not a mono-imidazole template. The best chromatographic separations are observed with small sample sizes, where the substrates occupy the strongest (highest-fidelity) sites. 

"Molecular Imprinting: Selective Materials for Separations, Sensors and Catalysis," S. Vidyasankar and F. H. Arnold, Current Opinion in Biotechnology 6, 218-22 4 (1995). 

"Selective Ligand-Exchange Adsorbents Prepared by Template Polymerization," V. Sundaresan, P. K. Dhal, S. D. Plunkett and F. H. Arnold, Biotechnology & Bioengineering, 48, 431-436 (1995). 

Highly selective ligand-exchange adsorbents have been prepared by template polymerization, a process in which the target molecule serves as a template for assembly of specific recognition sites. In an effort to develop materials suitable for chromatographic separations, thin coatings of the selective templated polymers have been grafted to two reactive macroporous supports, poly(trimethylolpropane trimethacrylate) (TRIM) and propylmethacrylate-derivatized silica beads. The precursor polymer prepared from the trifunctional TRIM monomer is macroporous and highly crosslinked, providing a stable structure for surface grafting. The TRIM precursor polymer and various surface-grafted copolymers have been characterized by scanning electron microscopy (SEM) and IR, 13C NMR and XPS spectroscopic techniques. Composite adsorbents have also been prepared using propylmethacrylate-modified silica particles. While equilibrium rebinding selectivities for both types of surface-templated materials are similar to those reported previously for bulk-polymerized template polymers, the composite materials are far better suited to chromatographic separations. Highly similar bis-imidazole substrates can be separated by ligand-exchange chromatography on these new templated adsorbe nts. 

"Synthetic Bis-Metal Ion Receptors for Bis-Imidazole 'Protein Analogs'," S. Mallik, R. D. Johnson, and F. H. Arnold, J. Am. Chem. Soc., 116 (20), 8902-8911 (1994). 

"Towards Materials for the Specific Recognition and Separation of Proteins," S.JMallik, S. D. Plunkett, P. K. Dhal, R. D. Johnson, D. Pack, D. Shnek and F. H. A rnold, New J. Chemistry 18, 299-304 (1994). 

"Multiple-site Binding Interactions in Metal-Affinity Chromatography. I. Equilibrium Binding of Engineered Histidine-Containing Cytochromes c," R. J. Todd, R . D. Johnson and F. H. Arnold, J. Chromatography 662, 13-26 (1994). 

"Template Polymerization using Metal Ion Coordination: Metal Replacement to Optimize Templating and Substrate Re-Binding" F. H. Arnold, S. Plunkett, V. Sundares an, Polymer Preprints 35, 996-997 (1994). 

"Selective Recognition of Bis-Imidazoles by Complementary Bis-Metal Ion Complexes," S. Mallik, R. D. Johnson, and F. H. Arnold, J. Am. Chem. Soc. 115, 2518-25 20 (1993). 

"Metal-Coordination Interactions in the Template-Mediated Synthesis of Substrate-Selective Polymers: Recognition of Bis(imidazole) Substrates by Cu(II)-Complex ing Polymers," P. K. Dhal and F. H. Arnold, Macromolecules 25, 7051-7059 (1992). 

"Metal-Affinity Separations: A New Dimension in Protein Bioprocessing," F. H. Arnold, Bio/Technology 9, 151-156 (1991). 

"Template-Mediated Synthesis of Metal-Complexing Polymers for Molecular Recognition," P. K. Dhal and F. H. Arnold, J. Am. Chem. Soc. 113, 7417-7418 (1991). 

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