This paper discusses the methodology and effectiveness of silanizing surfaces with TCVS, coating with PEO-PPO-PEO block polymers, and γ-irradiating the coatings to covalently attach the PEO block polymers. Significant findings include very stable layers produced by γ-irradiation of vinyl groups with adsorbed Pluronics, and higher protein repulsion by layers irradiated in the presence of excess Pluronic (vs. only water). This method is the basis for our past TCVS-silica microsphere-F108-nisin studies.[M.Ryder/Rat]

Description of synthesis and characterization of end-group-activated Pluronics (EGAP). Contains details of synthesis and use for tether immobilization of sulfhydryl-containing molecules by hydrophobic association of central PPO block with hydrophobic surfaces. [rat]

Synthesis and use of metal-chelating nitrilotriacetic acid (NTA) terminated Pluronic F108 to immobilize firefly luciferase. This is the mechanism to produce EGAP-NTA. [rat]

Comparison of vinyl layers on cellulose membranes produced by trichlorovinylsilane (TCVS) and allyldimethylchlorosilane (ADCS). Both silanes produced vinyl layers, but the ADCS chemistry was more well-behaved. This is because ADCS has only one reactive chlorine (vs. 3 for TCVS). In addition, the allyl group contains an extra carbon vs. TCVS. This motivated our attempts to use ADCS for γ-immobilization of Pluronics. However, our findings indicate the rougher layer produced by TCVS is actually more effective for F108 immobilization. [rat]

This paper discusses the methodology and effectiveness of silanizing surfaces, coating with PEO block polymers and γ-irradiation to covalently attach the PEO block polymers. [M.Ryder]

Synthesis and immobilization of vinyl-containing PEO-PBD-PEO triblocks on dimethylsilane surfaces by γ-irradiation. Uses urethane linkage between hydroxylated PBD centerblock and PEO side-chains; characterization by ¹H-NMR (spectrum) and size-exclusion. [J.McGuire]

… contrary to what is more commonly observed for PEO in normal situations… the PEO chains are actually not ‘hydrophilic’ when they exist as polymer brush chains… the PEO brush layer exhibits positive surface pressures, because the hydrophobicity of the PEO brush chains (which favors compression) is insufficient to overcome the opposing effect of the chain conformational entropy (which resists compression). [rat]

Detailed theoretical discussion of models of protein adsorption at brushes; theoretical thermodynamics, kinetics, etc. of polymer brushes; predicts repulsion of large proteins, and integration of small proteins into protein brushes. End-groups affect the properties and stability of the brush (and locate at ~70% height of the brush). [J.McGuire]

Guidelines for polymer size and spacing necessary for protein repulsion; adsorption/desorption kinetics depends on relationship between protein size and brush layer thickness; entrapment of proteins within brushes of height > protein diameter greatly slows desorption; theoretical modeling of brush-protein interactions and predictions of time to adsorb; once the brush engulfs a protein, its length does not matter for equilibrium adsorbed amount, only the desorption time (kinetics) is affected. [J.McGuire]

Epoxy-functionalized glass grafted with amino-polystyrene, interpenetration of more flexible polymers (see Gast and Leibler equation) into brush is nearly the same as onto a bare surface. Use of a poor solvent and high concentration increase grafting density. High-temperature annealing prior to rinsing and concentrations > dilute-semidilute concentration limit increase surface density; the optimum concentration decreases as ~log Mn, although the tethered mass is ~constant. Use of ellipsometry on equivalent flat surfaces to verify layer thickness. End-capping for (TGA-validated) quantification with TCA isocyanate, with solution depletion adsorption calibrated with non-reactive polymer. Claimed surface loading of 2.7/nm² epoxide on 10 µm non-porous spheres. [rat]

Theoretical development of a random sequential adsorption (RSA) model for proteins on PEO layers. Their model fitted various experimental data in the literature. They claim that all brush layers formed by processes in which surface rearrangement of the brush polymers cannot occur are inherently full of holes with no protein-repellent behavior. As they state: “Protein size strongly affects adsorption; non-spherical proteins preferentially adsorb in initial orientations that minimize the required open space, and smaller proteins preferentially adsorb from protein mixtures (e.g., serum). For a given protein size and PEO chain length, the PEO chain density is a critical parameter displaying a sharp transition between significant adsorption and almost complete resistance to protein adsorption.” [rat]

Free polystyrene chains of lower molecular weight penetrate an existing brush layer and reach the surface; no penetration was seen with large chains. Segmental adsorption increases grafting by holding chains at the surface. Three-regime kinetics for non-adsorbing; two-regime kinetics for segmental-adsorbing polymers. No significant entrapment of similar free chains by entanglement in brush layer. Solvent change collapses brush layer, affects thickness measured by QCM. [rat]

History-dependent adsorption of proteins at surfaces is “exquisitely” sensitive to slow relaxation of non-equilibrium structure. Multistep adsorption models can help elucidate surface structure and mobility. Determination of apparent adsorption rate constant, desorption rates in different states, and interfacial one-body cavity function from OWLS data. Different kinetics for different packings, multi-layer and unfolded/denatured proteins. [J.McGuire]

Example of use of surface force apparatus to probe adhesion strength of poly(ethylene oxide) and proteins. At low force, the PEO brush is repulsive. Surprisingly, they found that strong attractive forces are produced between PEO and proteins when pressed together with higher forces. This attraction is not due to denaturation, and indicated that PEO does not simply exclude volume, but exhibits competitive interaction with solvent and proteins.

F108 triblock coatings stabilized on silanized surfaces (or often very hydrophobic surfaces) not only show substantially lower levels of protein adsorption compared to untreated and silane-modified substrates, but also retain their protein repelling properties even after vigorous washing with SDS. [M.Ryder]

Adsorption and elution of nisin on F108-coated hydrophobic silica indicates that nisin is adsorbed into the brush in multilayer quantities. History dependent models support the theory of entrapment in the PEO brush layer instead of on the surface of the brush. BSA did not adsorb to the same layers. [M.Ryder]

Adsorption and elution of nisin on F108-coated hydrophobic silica indicates that nisin is adsorbed into the brush in multilayer quantities. History dependent models support the theory of entrapment in the PEO brush layer instead of on the surface of the brush. BSA did not adsorb to the same layers. [M.Ryder]

Protein adsorption on PEO-thiol SAMs was monitored. SAMs formed from ethanol/water mixtures give higher packing density than pure ethanol. For short (EO 2-4) oligos, fibrinogen and lysozyme adsorb more strongly on the denser SAMs, while larger EO’s resist adsorption regardless of solvent. SAMs terminated with -OH were more resistant to protein adsorption than -COOH, -NH₂ or -CH₃-terminated SAMs.

Investigation of mechanisms of protein repulsion, with PEO SAMs of different chain density, chain length and end-group. PEO layers formed near the cloud point gave lowest adsorption of fibrinogen (presumed highest chain density). Salt effects (2-4 M) in the PEO-thiol solution strongly influence the layer density, as evidenced by substantially decreases in adsorbed amount. Above ~0.5 chains/nm², protein adsorption is independent of chain length. Complement activation was observed on all surfaces, and a variety of blood proteins were detected after exposure to plasma. “Vroman peaks” (a peak in adsorption with fibrinogen concentration) were observed.

Chemisorbed thiolated PEO’s with hydroxyl and methoxy termini were adsorbed on gold-coated wafers; chain density (up to 0.5/nm²) is critical for protein repulsion; highest chain density near cloud-point of solution; ellipsometry and refractive index of PEO (ref); estimation of brush length and water/EO ratio; methoxy-PEO (mPEG) exhibits a minimum in protein binding effect while PEO-OH protein repulsion strictly increases with density; solvation of chains approaches crystallinity at high density (very low water/EO ratio; 2-3 minimum for solvation); self-impeding effect of chains on each other; adsorption favored by high methoxy density at surface of mPEG layers due to inter-chain aggregation and/or protein conformation change at methoxy surface (c.f. clumping of CALB at methyl/hydroxyl SAM surfaces, Laszlo and Evans 2007). [J.McGuire]

Molecular dynamics simulation of PEO-peptide conjugates indicates that the PEO spacer has little effect on the conformation of neutral (hydrophobic) peptides, but interacted with and strongly affected the conformation of charged peptides. When immobilized at a solid surface, the peptide portions of the conjugates aggregated and integrated into the PEO layer, displaying only a small amount of the total immobilized peptide to the bulk solution. In solution, PEO is predominantly in a loose helical structure that may be extended or entwining the attached peptide molecule. [Rat]

Describes ability of two selected surfactants to form bilayers on walls of bare silica capillaries, which are stable in absence of surfactant in buffer. Method is useful in separating high mobility proteins. Indicates that proteins are discouraged from adsorbing to hydrophobic surfaces after coating of surfactant. [W.Gray]

An exhaustively comprehensive review of graft polymers on surfaces, with discussion of length/density of grafted chains, surface dynamics and mobility, measurements of surface properties (contact angle, adsorption, etc), uses for surface functionalization and immobilization, and temperature-sensitive chains. Review with 381 references. [rat]

This study used AFM and OWLS to look at charge effects on protein adsorption to PEO/PLL layers. The proteins used [lysozyme, R-lactalbumin, and myoglobin] were all lysozyme-sized, and it was found that charge effects were significant at low PEO coverage and ionic strength. [J.McGuire]

Performed a thermodynamic analysis on the hydrophobic surfactant proteins with pulmonary surfactants to air-water interaces. Conducted surface tension measurements and examined early absorption kinetics. Calculated ΔG, ΔH, and ΔS for the two different surfactant proteins. [A.Alkhatib]

Describes four surface analytical techniques for probing dynamic, in-situ processes at biomaterial interface. Includes total reflection fluorescence spectroscopy, surface Plasmon resonance, ellipsometry, dynamic contact angle. [W.Gray]

A readable and still relevant introduction to various bioconjugation chemistry topics. An excellent source to find out what you want to find out about. Covers the majority of common conjugation reagents (e.g. NHS esters, maleimides, pyridyl disulfides, iodoacetate, etc.) in a concise format. Recommended reading for anyone interested in immobilizing proteins or biomolecules to surfaces. Review with 81 references. [rat]

A cautionary tale. Traut’s reagent (2-iminothiolane) can be used to introduce -SH groups to primary amines (-NH₂). However, in neutral to basic solution, the 2-iminothiolane can rearrange to an unreactive N-substituted ring with release of ammonia. The ‘active’ -SH can be trapped by inclusion of a sulfhydryl-reactive group such as DTNB (Ellman’s reagent) to stabilize the 2-iminothiolane. This is also reported by Mokotoff, et al. (J. Peptide Res. 57, 2001, 383-389). More sterically-hindered iminothiolane analogues (e.g. methyl-2-iminothiolane) have been shown to be much more stable (c.f. Carroll, et al., 1994, ref. 14). [rat]

A nice review of fluorination of alpha, beta, and cyclic amino acids. Includes chemical synthesis figures. A good reference. [J.Auxier]

A comprehensive review with 375 references of silica surface modification strategies. Strong theoretical background and discussion of silane/silica surface chemistry, effects of different silanes and reactive groups, etc. Must-read for surface chemists and workers interested in optimizing silanization reactions. [rat]

Aminosilane surface loading is hindered by hydrogen-bonding of -NH₂ groups with adjacent silanol -OH’s. Amines act as catalysts by H-bonding with surface Si-OH groups, weakening the Si-OH bond. In addition to this, short diamines (e.g. ethylenediamine) increase surface loading by strongly H-bonding with the surface silanols, preventing silane H-bonding. They are displaced during the silane condensation with the surface. See also discussion by McCool and DeSisto, Adv. Funct. Mater. (2005), 15, 1635-1640. [rat]

Silica surface functionalization monitored by AFM and other techniques. Aminosilane layers deposited from hot, concentrated solutions of silane in anhydrous toluene produced thick, smooth layers after long times; deposition from dilute solution at R.T. produced thin, smooth layers at first (1 hr) but quickly become rough. Control of water, silane concentration, reaction time and temperature are key variables in obtaining uniform, smooth APTES layers. [rat]

Lots of goodies here… Immobilization of DNA on H₂N-PEO₂₀₀₀-NH₂ attached to slides by aldehyde generated from periodate oxidation of hydrolyzed epoxides from glycidylpropoxytrimethoxysilane (GPTMS). Higher-density grafting was observed from molten polymer vs. solution methods. Film thickness was measured by XPS attenuation of substrate Si₂p signal. Micropatterning of DNA array by crossed microfluidics networks.

An example of silanization/modification of OWLS waveguides with APTES and GPTMS for attachment of biomolecules. Discusses some practical issues and compares capture of proteins by the two silanes. [J.McGuire]

Adsorption of ¹²⁵I-radiolabeled proteins used to calibrate XPS and TOF-SIMS data. Limit of detection down to 0.1 ng/cm² by TOF-SIMS. Freundlich isotherm “typical for protein adsorption”. Detection to 100 ng/cm² by XPS N_1s atom% (need at least >0.5% N) and N_1s/substrate signals. Amide carbon (288.2eV) peak used to quantify fibrinogen adsorption on polymers. Peak assignments for positive ion fragments of all 20 amino acids, mica and PTFE. Quantitation by PCA of fibrinogen on surfaces. CNO-/CN- negative ion peaks used to detect/quantify low ng/cm² of fibrinogen on polymers. [Rat]

Approachable discussion of methods to detect and quantify protein adsorption within microchannels. Methods described include XPS, silver staining, mass balance (enzymatic), mass spectroscopy (including SIMS), Raman spectroscopy, and others. Focus is on detection of very small amounts (~1% of a monolayer) of protein, and imaging of distribution on the surfaces. [Rat]

Original kinetic silver staining reference. [rat]

A good review of circular dichroism (CD) and its use for investigation of protein structure and interactions. Discussion of complementary information given by far-UV (180-240 nm) and near-UV (260-320 nm) CD. Fine structure in the near-UV spectrum is indicative of protein-protein interactions, especially tryptophan (Trp) and other aromatic residues. [Kain]

An investigation on indium-tin oxide coated glass substrates using the techniques mentioned in the title. The relevant part: the study used chromosulfuric acid for cleaning the coated substrate. They observed cleaner surfaces as compared to N₂ blowing. They also found no pitting from the acid washing.[J.Auxier]

A very comprehensive review of the 2008 literature (1413 articles) on optical biosensor techniques (e.g. OWLS). The authors highlight 10 papers and discuss what makes them “fabulous, middling or abysmal”.

A review of past 15 year contributions in biomaterials and biochemical research. Reviews ellipsometry and other technical progress and provides an overview of protein conformational changes, protein displacement effects, as well as early clotting cascades and complement activation. [Ben S]

A thorough yet readable introduction to the theory of ellipsometry. Discusses Fresnel equations, how Ψ and Δ values are converted to thickness and refractive index, and features and limitations of various methods of ellipsometry. Required reading for anyone who plans to use the ellipsometer. [Rat]

A critical compare-and-contrast experiment with ellipsometry, OWLS and QCM as techniques for measuring protein adsorption kinetics. [J.McGuire]

Detailed description of an algorithm to find the “best” n and d parameters from ellipsometric angles. This is the basis of the old Fortran ellipsometry programs. [rat]

Development of a method for converting thickness and refractive index of a thin film (e.g. adsorbed protein) to immobilized mass (Γ). These equations use calculated optical (molar refractivity) and physical (specific volume) properties to calculate the total adsorbed mass (as µg/cm²) from ellipsometric data. [rat]

Theoretical background and methods for measurement of protein conformation changes by evaluation of “resistance to elution”. As proteins reach the surface and change from native to denatured states, they become more resistant to elution with weak surfactants (e.g. DTAB). The rate of adsorption to a bare and a partially coated surface also provide information about the ability of proteins to rearrange on the surface (e.g. “history dependent” models). [rat]

A review of the problems faced by industry when manufacturing rFVIII. Gives an overview of rFVIII and the history of production, along with major challenges. Explains some of the manufacturing processes in upstream and downstream areas. [A.Alkhatib]

Examines the aggregation behavior of rFVIII at various temperatures. Uses circular dichroism state kinetic model, where the k was found to change at different temperatures. [A.Alkhatib]

A review on the four main types of Factor VIII used in industry. Explains the manufacturing processes on expression systems and cell lines, culture medium, technical culture systems, purification process, and final formulation. [A.Alkhatib]

Describes ability to harvest rFVIII from the milk of transgenic pigs. Justifies use of fibrinogen due to its availability and functional similarity to rFVIII in initial examination and modeling. [W.Gray]

Recent reference for strong dependence of nisin activity on pH. Nisin activity against B. cereus was highest at pH 4, and >90% activity loss was observed at pH 6.0. Activity was abolished completely at neutral or alkaline pH. After contact with nisin, plates were held at 4 ‘C for 4 h to allow nisin to diffuse into the agar before incubation at 37 ‘C to allow substantial growth of bacteria.

Nisin resistance can be transferred between non-nisin producing L. lactis strains by the nsr gene, which codes for a 34 kDa protease that specifically cleaves nisin at the C-terminal Ser₂₉ to produce a 6-residue fragment. Cleavage reduced nisin activity and cell-membrane affinity by at least two orders of magnitude.

Antimicrobial activity of nisin adsorbed on hydrophobic silica was demonstrated by live-dead staining of L. monocytogenes with iodonitrotetrazolium violent. 1-cm^2^ Si wafer discs were adsorbed with nisin and placed on pour plates inoculated with L. mono; antimicrobial activity was evaluated by measurement of inhibition zones formed on the plate. [rat]

Conformation and structure of nisin-lipid II complex. Nice figures of nisin structure. [rat]

Explanation of mechanism of nisin and other lantibiotics. Nice figures of nisin structure and interaction with cell wall. [rat]

During storage, the terminal dipeptide of nisin_1-34 (spontaneously hydrolyzes, leaving the shortened nisin_1-32 chain. Both variants are active, even after autoclaving briefly with diulte acid; however, treatment with hot, strong acid creates a des–ΔAla₅ nisin_1-32 variant that is inactive. [rat]

The sequence/structure of nisin (with unusual residues noted) is presented in a nice figure. This paper describes the aggregation and cell-membrane pore formation of the Lipid II/nisin complex. A classic reference for nisin-related work. [rat]

Nisin and mutants were analyzed by MALDI-TOF and LC-MS. The native nisin molecule has a molecular weight of 3362.0/3353.6 Da (by MALDI-TOF/LC-MS). A degradation product caused by cleavage of the COOH-terminal dipeptide results in a 32-residue peptide with MW 3184.5/3185.6. These values are consistent with the results of MALDI-TOF on the Prime Pharma nisin, which is essentially pure but contains substantial amounts of the 1-32 degradation product (3152Da).

PEGylation of nisin [primarily] at the α-NH₂ with PEO₁₅₀₀-NHS resulted in a complete loss of nisin antibacterial activity. Attempts to PEGylate the -COOH terminus with PEO₁₅₀₀-hydrazine/EDC failed to produce a conjugate. The loss of activity is attributed to the crucial role of the N-terminal domain in nisin-Lipid II complex formation required for activity. The authors suggest that the loss of charge upon PEGylation and/or the increased hydrophilicity and mass of the N-terminal domain upset some critical balance. [rat]

Background information on nisin and investigation of effects of natural postranslational processing of the molecule from its gene encoded peptide. The article elucidates some of the fundamental chemical and physical properties of nisin. [Ben S]

The density of small (less than ~20 kDa) proteins is a strong exponential function of their molecular weight (large proteins have a nearly constant ρ ≈ 1.37). The authors provide the following function for the density of small proteins, where M is the molecular weight (kDa): [Rat]

Amino acid compositions of human fibrinogen and three derivatives were experimentally determined through column chromatography. Contains breakdown of each amino acid in residues/10^5 g protein moiety. This was useful in FITC labeling adventures. [J.Auxier]

One of the original science humor papers. Learn new and exciting ways to inactivate laboratory wares!

An amusing and motivational essay on the importance of being “stupid” in research. If you don’t know what’s going on, it may be a good thing. [rat]