Resistive-pulse studies of proteins and protein/antibody complexes using a conical nanotube sensor

There is increasing interest in using nanopores in synthetic membranes as resistive-pulse sensors for molecular and macromolecule analytes. In general, this method entails measuring current pulses associated with translocation of the analyte through the nanopore sensor element. A key challenge for this sensing paradigm is building selectivity into the protocol so that the current pulses for the target analyte can be distinguished from current pulses for other species that might be present in the sample. We show here that this can be accomplished with a protein analyte by adding to the solution an antibody that selectively binds the protein. We demonstrate this concept using bovine serum albumin (BSA) and a Fab fragment from a BSA-binding polyclonal antibody. Because the complex formed upon binding of the Fab to BSA is larger than the free BSA molecule, the current-pulse signature for the BSA/Fab complex can be easily distinguished from the free BSA. Furthermore, the BSA/Fab pulses can be easily distinguished from the pulses obtained for the free Fab and from pulses obtained for a control protein that does not bind to the Fab. Finally, we also show that the current-pulse signature for the BSA/Fab complex can provide information about the size and stoichiometry of the complex.     

Modelling of the impedimetric responses of an aflatoxin B1 immunosensor prepared on an electrosynthetic polyaniline platform

Aflatoxins are a group of mycotoxins that have deleterious effects on humans and are produced during fungal infection of plants or plant products. An electrochemical immunosensor for the determination of aflatoxin B₁ (AFB₁) was developed with AFB₁antibody (AFB₁-Ab) immobilized on Pt electrodes modified with polyaniline (PANi) and polystyrene sulphonic acid (PSSA). Impedimetric analysis shows that the electron transfer resistances of the Pt/PANi–PSSA electrode, the Pt/PANi–PSSA/AFB₁-Ab immunosensor and Pt/PANi–PSSA/AFB₁-Ab incubated in bovine serum albumin (BSA) were 0.458, 720 and 1,066 kΩ, respectively. These results indicate that electrochemical impedance spectroscopy (EIS) is a suitable method for monitoring the change in electron transfer resistance associated with the immobilization of the antibody. Modelling of EIS data gave equivalent circuits which showed that the electron transfer resistance increased from 0.458 kΩ for the Pt/PANi–PSSA electrode to 1,066 kΩ for the Pt/PANi–PSSA/AFB₁-Ab immunosensor, indicating that immobilization of the antibody and incubation in BSA introduced an electron transfer barrier. The AFB₁ immunosensor had a detection limit of 0.1 mg/L and a sensitivity of 869.6 kΩ L/mg.     

Parallel DFT gradients using the Fourier Transform Coulomb method

The recently described Fourier Transform Coulomb (FTC) algorithm for fast and accurate calculation of Density Functional Theory (DFT) gradients (Füsti-Molnar, J Chem Phys 2003, 119, 11080) has been parallelized. We present several calculations showing the speed and accuracy of our new parallel FTC gradient code, comparing its performance with our standard DFT code. For that part of the total derivative Coulomb potential that can be evaluated in plane wave space, the current parallel FTC gradient algorithm is up to 200 times faster in total than our classical all-integral algorithm, depending on the system size and basis set, with essentially no loss in accuracy. Proposed modifications should further improve the overall performance relative to the classical algorithm.     

Completely aqueous procedure for the growth of polymer brushes on polymeric substrates

The growth of polymer brushes on polymer substrates is often challenging because of substrate incompatibility with the organic solvents used for initiator attachment. This letter reports the use of layer-by-layer adsorption of macroinitiators and subsequent aqueous ATRP from these immobilized initiators to prepare polymer brushes on polymeric substrates. Polyethersulfone (PES) films and porous membranes were modified with polyelectrolyte multilayer films, and a previously developed polycationic initiator, poly(2-(trimethylammonium iodide)ethyl methacrylate-co-2-(2-bromoisobutyryloxy)ethyl acrylate), was then electrostatically adsorbed onto these polyelectrolyte films. The immobilized macroinitiator is very efficient in initiating the growth of polymer brushes on PES, as demonstrated by aqueous syntheses of poly(2-hydroxyethyl methacrylate) (PHEMA) and poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) films. PHEMA (250 nm thick) and PDMAEMA (40 nm thick) brushes were grown in 2 h from surfaces modified with polycationic initiators. Moreover, this procedure is effective for growing brushes in the pores of PES membranes.     

Comparison of dansylated aminopropyl controlled pore glass solvated by molecular and ionic liquids

We compare how (i) four ionic liquids (ILs) (1-butyl-3-methylimidazolium tetrafluoroborate ([C4mim][BF4]), 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([C4mim][Tf2N]), 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide ([C4mpy][Tf2N]), and trihexyltetradecylphosphonium bis(trifluoromethylsulfonyl)imide ([P(C6)3C14][Tf2N])) and (ii) two conventional molecular liquids (methanol and 1-octanol) solvate/wet luminescent organic moieties that are covalently attached to the surface of silica controlled pore glass (CPG). A series of aminopropyl CPG particles that have been covalently tagged with the solvatochromic fluorescent probe group dansyl were used in this study. The results demonstrate that ILs solvate/wet the silica surface differently in comparison to molecular liquids (MLs). Specifically, when comparing ILs and MLs that appear to solvate the free probe, dansylpropylsulfonamide (DPSA), equally in solution, we find that ILs do not solvate/wet the silica surfaces as well as the corresponding MLs. The cation component in these ILs is the significant factor in how the ILs solvate/wet silica surfaces. Solvation/wetting of surface-bound species at a silica surface depends on the cation size. Chlorosilane end-capping of the surface silanol and amine residues attenuates the cation's affects.     

The binding avidity of a nanoparticle-based multivalent targeted drug delivery platform

Dendrimer-based anticancer nanotherapeutics containing approximately 5 folate molecules have shown in vitro and in vivo efficacy in cancer cell targeting. Multivalent interactions have been inferred from observed targeting efficacy, but have not been experimentally proven. This study provides quantitative and systematic evidence for multivalent interactions between these nanodevices and folate-binding protein (FBP). A series of the nanodevices were synthesized by conjugation with different amounts of folate. Dissociation constants (K(D)) between the nanodevices and FBP measured by SPR are dramatically enhanced through multivalency ( approximately 2,500- to 170,000-fold). Qualitative evidence is also provided for a multivalent targeting effect to KB cells using flow cytometry. These data support the hypothesis that multivalent enhancement of K(D), not an enhanced rate of endocytosis, is the key factor resulting in the improved biological targeting by these drug delivery platforms.     

Adaptive multilevel finite element solution of the Poisson–Boltzmann equation II. Refinement at solvent‐accessible surfaces in biomolecular systems

We apply the adaptive multilevel finite element techniques (Holst, Baker, and Wang 21 ) to the nonlinear Poisson–Boltzmann equation (PBE) in the context of biomolecules. Fast and accurate numerical solution of the PBE in this setting is usually difficult to accomplish due to presence of discontinuous coefficients, delta functions, three spatial dimensions, unbounded domains, and rapid (exponential) nonlinearity. However, these adaptive techniques have shown substantial improvement in solution time over conventional uniform‐mesh finite difference methods. One important aspect of the adaptive multilevel finite element method is the robust a posteriori error estimators necessary to drive the adaptive refinement routines. This article discusses the choice of solvent accessibility for a posteriori error estimation of PBE solutions and the implementation of such routines in the “Adaptive Poisson–Boltzmann Solver” (APBS) software package based on the “Manifold Code” (MC) libraries. Results are shown for the application of this method to several biomolecular systems. © 2000 John Wiley & Sons, Inc. J Comput Chem 21: 1343–1352, 2000     

Adaptive multilevel finite element solution of the Poisson–Boltzmann equation I. Algorithms and examples

This article is the first of two articles on the adaptive multilevel finite element treatment of the nonlinear Poisson–Boltzmann equation (PBE), a nonlinear eliptic equation arising in biomolecular modeling. Fast and accurate numerical solution of the PBE is usually difficult to accomplish, due to the presence of discontinuous coefficients, delta functions, three spatial dimensions, unbounded domain, and rapid (exponential) nonlinearity. In this first article, we explain how adaptive multilevel finite element methods can be used to obtain extremely accurate solutions to the PBE with very modest computational resources, and we present some illustrative examples using two well‐known test problems. The PBE is first discretized with piece‐wise linear finite elements over a very coarse simplex triangulation of the domain. The resulting nonlinear algebraic equations are solved with global inexact Newton methods, which we have described in an article appearing previously in this journal. A posteriori error estimates are then computed from this discrete solution, which then drives a simplex subdivision algorithm for performing adaptive mesh refinement. The discretize–solve–estimate–refine procedure is then repeated, until a nearly uniform solution quality is obtained. The sequence of unstructured meshes is used to apply multilevel methods in conjunction with global inexact Newton methods, so that the cost of solving the nonlinear algebraic equations at each step approaches optimal O(N) linear complexity. All of the numerical procedures are implemented in MANIFOLD CODE (MC), a computer program designed and built by the first author over several years at Caltech and UC San Diego. MC is designed to solve a very general class of nonlinear elliptic equations on complicated domains in two and three dimensions. We describe some of the key features of MC, and give a detailed analysis of its performance for two model PBE problems, with comparisons to the alternative methods. It is shown that the best available uniform mesh‐based finite difference or box‐method algorithms, including multilevel methods, require substantially more time to reach a target PBE solution accuracy than the adaptive multilevel methods in MC. In the second article, we develop an error estimator based on geometric solvent accessibility, and present a series of detailed numerical experiments for several complex biomolecules. © 2000 John Wiley & Sons, Inc. J Comput Chem 21: 1319–1342, 2000     

Constrained optimization in delocalized internal coordinates

Using the recently introduced delocalized internal coordinates, in conjunction with the classical method of Lagrange multipliers, an algorithm for constrained optimization is presented in which the desired constraints do not have to be satisfied in the starting geometry. The method used is related to a previous algorithm by the same author for constrained optimization in Cartesian coordinates [J. Comput. Chem., 13 , 240 (1992)], but is simpler and far more efficient. Any internal (distance or angle/torsion) constraint can be imposed between any atoms in the system whether or not the atoms involved are formally bonded. Imposed constraints can be satisfied exactly. © 1997 John Wiley & Sons, Inc. J Comput Chem 18 :1079–1095, 1997