Immobilization Of Ligands Research Articles

Thiomers: Promising Platform For Macromolecular Drug Delivery

The application of macromolecules as therapeutic agents holds great promise for several major disorders such as cancer and cardiovascular disease. However, their use is limited by the lack of efficient, safe and specific delivery strategies. A promising strategy to overcome these challenges might be the use of thiolated polymers or designated thiomers. Thiomers are synthesized by immobilization of sulfhydryl bearing ligands on a polymeric backbone of well-established polymers. These multifunctional polymeric excipients show advantages in mucoadhesion, enzyme and efflux pump inhibition in comparison to unmodified polymers. One obstacle in the use of thiomers is that they are prone to oxidation at lower pH but this could be solved by introducing a completely new generation of thiomers, namely, the preactivated thiomer generation. Preactivated thiomers are mixed disulfides, which exhibit oxidation resistance and, beyond that, improved thiomer features. This review summarizes recent findings of polymeric excipients for macromolecular drug delivery as well as their synthesis and distinctive features.

Molecular modeling of polymeric adsorbent media: The effects of counter‐ions on ligand immobilization and pore structure

Molecular dynamics modeling and simulations are employed to study the immobilization of ligands on the surface of the pores of a base porous polymeric matrix. The results show the significant effects that the counter-ions have on the spatial distribution of the density of immobilized ligands as well as on the pore size and pore connectivity distributions of the porous adsorbent medium being constructed. The results for the systems studied in this work indicate that by using doubly charged counter-ions whose numbers during ligand immobilization are half to those of singly charged counter-ions, the ligand immobilization process proceeds faster and the magnitude of local nonelectroneutrality becomes smaller. More importantly, the pore structures of the adsorbent media resulting from the system using doubly charged counter-ions have porous structures that are characterized by more mid-sized pores and higher pore connectivity than the porous adsorbent structures generated by the system employing singly charged counter-ions and, furthermore, the density distribution of the immobilized ligands in the porous structures where doubly charged counter-ions are employed tends to be more uniform laterally and the ligands are surrounded by fewer counter-ions. These characteristics affected by the use of doubly charged counter-ions could provide important advantages with respect to the transport and adsorption of adsorbate biomolecules of interest. Furthermore, the results of this work indicate that the type of counter-ions being used in the ligand immobilization process could represent an additional control variable for affecting the ligand density distribution as well as the pore size and pore connectivity distributions of the porous structure of the adsorbent medium being constructed.

Comparative Binding of Disulfide-Bridged PEG-Fabs

Protein PEGylation is the most clinically validated method to improve the efficacy of protein-based medicines. Antibody fragments such as Fabs display rapid clearance from blood circulation and therefore are good candidates for PEGylation. We have developed PEG-bis-sulfone reagents 1 that can selectively alkylate both sulfurs derived from a native disulfide. Using PEG-bis-sulfone reagents 1, conjugation of PEG specifically targets the disulfide distal to the binding region of the Fab (Scheme 2 ). PEG-bis-sulfone reagents 1 (10-40 kDa) were used to generate the corresponding PEG-mono-sulfones 2 that underwent essentially quantitative conjugation to give the PEG-Fab product 4. Four Fabs were PEGylated: Fab(beva), Fab'(beva), Fab(rani), and Fab(trast). Proteolytic digestion of bevacizumab with papain gave Fab(beva), while digestion of bevacizumab with IdeS gave F(ab')(2-beva), which after reaction with DTT and PEG-mono-sulfone 2 gave PEG(2)-Fab'(beva). Ranibizumab, which is a clinically used Fab, was directly PEGylated to give PEG-Fab(rani). Trastuzumab was proteolytically digested with papain, and its corresponding Fab was PEGylated to give PEG-Fab(trast). Purification of the PEGylated Fabs was accomplished by a single ion exchange chromatography step to give pure PEG-Fab products as determined by silver-stained SDS-PAGE. No loss of PEG was detected post conjugation. A comparative binding study by SPR using Biacore with low ligand immobilization density was conducted using (i) VEGF(165) for the bevacizumab and ranibizumab derived products or (ii) HER2 for the trastuzumab derived products. VEGF(165) is a dimeric ligand with two binding sites for bevacizumab. HER2 has one domain for the binding of trastuzumab. Binding studies with PEG-Fab(beva) indicated that the apparent affinity was 2-fold less compared to the unPEGylated Fab(beva). Binding properties of the PEG-Fab(beva) products appeared to be independent of PEG molecular weight. Site-specific conjugation of two PEG molecules gave PEG(2×20)-Fab'(beva), whose apparent binding affinity was similar to that observed for PEG-Fab(beva) derivatives. The k(d) values were similar to those of the unPEGylated Fab(beva); hence, once bound, PEG-Fab(beva) remained bound to the same degree as Fab(beva). Biacore analysis indicated that both Fab(rani) and PEG(20)-Fab(rani) did not dissociate from the immobilized VEGF at 25 °C, but ELISA using immobilized VEGF showed 2-fold less apparent binding affinity for PEG(20)-Fab(rani) compared to the unPEGylated Fab(rani). Additionally, the apparent binding affinities for trastuzumab and Fab(trast) were comparable by both Biacore and ELISA. Biacore results suggested that trastuzumab had a slower association rate compared to Fab(trast); however, both molecules displayed the same apparent binding affinity. This could have been due to enhanced rebinding effects of trastuzumab, as it is a bivalent molecule. Analogous to PEG-Fab(beva) products, PEG(20)-Fab(trast) displayed 2-fold lower binding compared to Fab(trast) when evaluated by ELISA. The variations in the apparent affinity for the PEGylated Fab variants were all related to the differences in the association rates (k(a)) rather than the dissociation rates (k(d)). We have shown that (i) Fabs are well-matched for site-specific PEGylation with our bis-alkylation PEG reagents, (ii) PEGylated Fabs display only a 2-fold reduction in apparent affinity without any change in the dissociation rate, and (iii) the apparent binding rates and affinities remain constant as the PEG molecular weight is varied.

A Spatial Model for Integrin Clustering as a Result of Feedback between Integrin Activation and Integrin Binding

Integrins are transmembrane adhesion receptors that bind extracellular matrix (ECM) proteins and signal bidirectionally to regulate cell adhesion and migration. In many cell types, integrins cluster at cell-ECM contacts to create the foundation for adhesion complexes that transfer force between the cell and the ECM. Even though the temporal and spatial regulation of these integrin clusters is essential for cell migration, how cells regulate their formation is currently unknown. It has been shown that integrin cluster formation is independent of actin stress fiber formation, but requires active (high-affinity) integrins, phosphoinositol-4,5-bisphosphate (PIP2), talin, and immobile ECM ligand. Based on these observations, we propose a minimal model for initial formation of integrin clusters, facilitated by localized activation and binding of integrins to ECM ligands as a result of biochemical feedback between integrin binding and integrin activation. By employing a diffusion-reaction framework for modeling these reactions, we show how spatial organization of bound integrins into clusters may be achieved by a local source of active integrins, namely protein complexes formed on the cytoplasmic tails of bound integrins. Further, we show how such a mechanism can turn small local increases in the concentration of active talin or active integrin into integrin clusters via positive feedback. Our results suggest that the formation of integrin clusters by the proposed mechanism depends on the relationships between production and diffusion of integrin-activating species, and that changes to the relative rates of these processes may affect the resulting properties of integrin clusters.

Fabrication of dense, uniform aminosilane monolayers: A platform for protein or ligand immobilization

Designing biointerfaces with enhanced biomolecule binding abilities, along with their distribution and presentation on the surface, largely requires control over underlying surface chemistries. In an effort to correlate experimental parameters to surface properties, ligand distribution and biomolecule binding, the influence of (3-aminopropyl)triethoxysilane (APTES) concentration (1, 2 and 4vol%) and duration of the silanization reaction (5–60min) on the number of available NH2 groups were determined and correlated with the amount of surface bound human immunoglobulin G (HIgG). Surfaces silanized with 2vol% APTES for 30min yielded a densely populated silane monolayer (1.0–1.2nm) where the average molecular orientation was 38±2° with respect to the surface normal. A surface density of NH2 moieties of ∼1015/cm2 was obtained and was significantly higher compared to other conditions evaluated. The combined data from ellipsometry and atomic force microscopy (AFM) analyses supports our findings. Coupling of HIgG to surfaces silanized with 2vol% APTES for 30min yielded ∼1013IgG/cm2, which was significantly higher than values obtained at 1vol% APTES for 30min. The distribution of immobilized HIgG was noted to be dense and uniform on surfaces silanized with 2vol% APTES when visualized using Nanogold® antibody conjugates. This study highlights the critical role of the experimental parameters that impact the biomolecule immobilization process on functionalized surfaces.

Immobilization of ethylene bis-indenyl ligands on functionalized silica gel

Four ethylene bis-indenyl ligands containing tethers of various lengths were successfully immobilized on the surface of functionalized silica gel. The strategy of immobilization was based on catalytic thiol–ene coupling of terminal alkene groups in the tethers with surface thiol groups. Obtained materials have high BET surface area and pore volume. The method developed can be used for immobilization of catalytically active bis-indenyl metallocene complexes, thus preventing their dimerization and deactivation.

Nonlinear Analyte Concentration Gradients for One-Step Kinetic Analysis Employing Optical Microring Resonators

Conventional methods to probe the binding kinetics of macromolecules at biosensor surfaces employ a stepwise titration of analyte concentrations and measure the association and dissociation to the immobilized ligand at each concentration level. It has previously been shown that kinetic rates can be measured in a single step by monitoring binding as the analyte concentration increases over time in a linear gradient. We report here the application of nonlinear analyte concentration gradients for determining kinetic rates and equilibrium binding affinities in a single experiment. A versatile nonlinear gradient maker is presented, which is easily applied to microfluidic systems. Simulations validate that accurate kinetic rates can be extracted for a wide range of association and dissociation rates, gradient slopes, and curvatures, and with models for mass transport. The nonlinear analyte gradient method is demonstrated with a silicon photonic microring resonator platform to measure prostate specific antigen-antibody binding kinetics.

Role of C-C chemokine receptor type 7 and its ligands during neuroinflammation

For decades, chemokines and their receptors have received a great deal of attention for their multiple roles in controlling leukocyte functions during inflammation and immunity. The ability of chemokines to convey remarkably versatile but context-specific signals identifies them as powerful modulators of immune responses generated in response to diverse pathogenic or non-infectious insults. A number of recent studies have speculated that the C-C chemokine receptor type 7 (CCR7), plays important roles in immune-cell trafficking in various tissue compartments during inflammation and in immune surveillance. Using computational modeling and microfluidics-based approaches, recent studies have explored leukocyte migration behavior in response to CCR7 ligands in a complex chemokine environment existing with other coexisting chemokine fields. In this review, we summarize the current understanding of the effects of soluble versus immobilized ligands and of the downstream signaling pathways of CCR7 that control leukocyte motility, directionality, and speed. This review also integrates the current knowledge about the role of CCR7 in coordinating immune responses between secondary lymphoid organs and peripheral tissue microenvironments during primary or secondary antigen encounters. CCR7 seems to influence distinct immunological events during inflammatory responses in the central nervous system (CNS) including immune-cell entry and migration, and neuroglial interactions. The clinical and pathological outcome may vary depending on its contribution in the inflamed CNS microenvironment. Understanding these mechanisms has direct implications for therapeutic developments favoring more protective and efficient immune responses.

Nanohole-Based Surface Plasmon Resonance Instruments with Improved Spectral Resolution Quantify a Broad Range of Antibody-Ligand Binding Kinetics

We demonstrate an affordable low-noise surface plasmon resonance (SPR) instrument based on extraordinary optical transmission (EOT) in metallic nanohole arrays and quantify a broad range of antibody-ligand binding kinetics with equilibrium dissociation constants ranging from 200 pM to 40 nM. This nanohole-based SPR instrument is straightforward to construct, align, and operate, since it is built around a standard microscope and a portable fiber-optic spectrometer. The measured refractive index resolution of this platform is 3.1 × 10(-6) without on-chip cooling, which is among the lowest reported for SPR sensors based on EOT. This is accomplished via rapid full-spectrum acquisition in 10 ms followed by frame averaging of the EOT spectra, which is made possible by the production of template-stripped gold nanohole arrays with homogeneous optical properties over centimeter-sized areas. Sequential SPR measurements are performed using a 12-channel microfluidic flow cell after optimizing surface modification protocols and antibody injection conditions to minimize mass-transport artifacts. The immobilization of a model ligand, the protective antigen of anthrax on the gold surface, is monitored in real-time with a signal-to-noise ratio of ~860. Subsequently, real-time binding kinetic curves were measured quantitatively between the antigen and a panel of small, 25 kDa single-chain antibodies at concentrations down to 1 nM. These results indicate that nanohole-based SPR instruments have potential for quantitative antibody screening and as a general-purpose platform for integrating SPR sensors with other bioanalytical tools.

Immobilization of hydrophobic peptidic ligands to hydrophilic chromatographic matrix: A preconcentration approach

This study presents a methodology for covalent attachment of hydrophobic peptidic ligands to hydrophilic chromatographic matrices with improved coupling efficiency. Preconcentration was introduced through the use of polyethylene glycol (PEG)-based crosslinkers. Immobilization of model hydrophobic peptide pep12 (ITLISSEGYVSS) to hydrophilic silica–amine matrix was investigated in the absence/presence of PEG-based linker. The effect of linker densities 14.2, 27.6, and 56.4μmol/g beads on coupling efficiency was investigated. Whereas a ligand coupling efficiency of 67% was obtained in the absence of the linker, incorporating PEG-based linker at low densities allowed a 30% increase in the coupling efficiency. Although the heterobifunctional crosslinker, maleimide–PEG–NHS (N-hydroxysuccinimide) ester, can be used to couple thiol-bearing ligands to amine-functionalized matrices, no method is available for quenching free amine moieties on the matrix after ligand immobilization. The efficacy of acylating agents, acetyl chloride and oxalyl chloride, in blocking free amine groups when immobilizing the model peptide pep14 (CITLISSEGYVSSK) to silica–amine matrix using maleimide–PEG–NHS ester crosslinker was investigated. Because oxalyl chloride was nonreactive to maleimides, it allowed successful coupling of pep14 to the maleimide termini of the linkers. Adsorption studies between pep14-immobilized microspheres and human immunoglobulin M (hIgM) suggested retention of ligand activity and a 95% decrease in nonspecific binding of proteins to the matrix.

Linear Triphosphines as Ligands for Metal Complexes Immobilization in Ionic Liquids: Palladium-Catalyzed Methoxylation of Alkynes

Several novel palladium triphosphine complexes have been synthesized and tested as recyclable catalysts for the methoxylation of alkynes into acetals in ionic liquids. A complete conversion of phenylacetylene was achieved with only 0.2% of (Pd(Triphos)NCMe)((PF6)2) in a methanol/(BMIM)(BF4) mixture. We discovered that the addition of an ionic liquid to methanol allowed not only to increase the activity of the palladium catalyst but also to provide a recyclable catalyst which can be reused several times with a weaker drop of activity. To complete these catalytic studies, we describe the synthesis of the first poor -electron-donating/strong -electron-acceptor linear Triphosphine which, after palladium coordination, led to a better selectivity compared to its Triphos analogue. The performances of recovered ionic liquid re- action mixtures show for the first time that P-tridentate ligands efficiently immobilize palladium catalysts and lead to se- lective catalytic systems benign for environment.

Boronic acid modified magnetic nanoparticles for enrichment of glycoproteins via azide and alkyne click chemistry

In this study, a novel approach was developed to synthesize aminophenylboronic acid functionalized magnetic nanoparticles (NPs) via Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) ‘click’ chemistry. Firstly, azide-functional Fe3O4 NPs were obtained by a two-step chemical modification process. Then, an alkyne-phenylboronic acid molecule was connected onto the surface of magnetite by the CuAAC reaction. The morphology, structure and composition of the synthesized nanocomposites were characterized by transmission electron microscopy (TEM), X-ray powder diffraction (XRD), vibrating sample magnetometer (VSM), Fourier transform infrared (FTIR) spectroscopy and X-ray photoelectron spectrometry (XPS). Five proteins, including ovalbumin (OB), transferrin (Trf), as glycoprotein templates and lysozyme (Lyz), bovine serum albumin (BSA), horse heart cytochrome c (Cyt C) as nonglycoprotein templates are chosen as target proteins. The as-prepared click-Fe3O4@APBA NPs with a mean diameter of 23.2 nm showed a strong magnetic response to an externally applied magnetic field and exhibited a high adsorption capacity and excellent specificity towards glycoproteins in comparison with nonglycoproteins. The click-Fe3O4@APBA NPs showed the higher adsorption capacity towards glycoproteins than the nonclick-Fe3O4@APBA NPs which were synthesized through a common nucleophilic substitution reaction. The greatly enhanced adsorption capacity towards glycoproteins demonstrated that the ‘click’ method presented great superiority in ligand immobilization. Finally, the click-Fe3O4@APBA NPs could efficiently enrich glycoproteins from real egg white samples as well.

Dip-pen nanolithography of optically transparent cationic polymers to manipulate spatial organization of proteolipid membranes

Herein we report on an approach to use dip-pen nanolithography (DPN) to rapidly prototype nano- and microscale cationic polymer structures that guide the transport of lipid molecules in a fluid membrane. An atomic force microscope (AFM) tip is used to transfer poly(diallyldimethylammonium chloride) (PDAC) to a silica surface that templates the self-assembly of a phospholipid bilayer. Based on AFM and high-resolution optical microscopy data, DPN-generated PDAC structures are optically transparent and composed of up to three molecular layers of polyelectrolyte. We demonstrate that patterns can juxtapose mobile and immobile ligands in a supported lipid bilayer that simultaneously engages the epidermal growth factor receptor (EGFR) and adhesion receptors on the cell surface. In principle, DPN-generated polyelectrolyte structures have important applications in investigating supramolecular protein assemblies in living cells.

Optical mesosensors for monitoring and removal of ultra-trace concentration of Zn(ii) and Cu(ii) ions from water

Optical captor design is necessary for the controlled development of a technique for detecting and removing heavy and toxic metals from drinking water with high flexibility and low capital cost. We designed chemical mesocaptors for optical separation/extraction and monitoring/detection of Cu(II) and Zn(II) ions from water even at trace concentration levels without a preconcentration process. The mesoporous aluminosilica carriers with three-dimensional (3D) structures, high aluminum content, natural surfaces, and active acid sites strongly induced H-bonding and dispersive interactions with organic moieties, thereby leading to the formation of stable captors without chromophore leaching during the removal assays of Cu(II) and Zn(II) ions. Using such a tailored mesocaptor design, the direct immobilization of these hydrophobic ligands (4,5-diamino-6-hydroxy-2-mercaptopyrimidine and diphenylthiocarbazone) into ordered pore-based aluminasilica monoliths enabled the easy generation and transduction of optical colour signals as a response to metal-to-ligand binding events, even at ultra-trace concentrations (~10(-9) mol dm(-3)) of Cu(II) and Zn(II) ions in drinking water, without the need for sophisticated instruments. Theoretical models have been developed to provide insights into the effect of active site surfaces on the enhancement of the optical removal process in terms of long-term stability, reversibility, and selectivity, hence allowing us to understand the role of mesoscopic geometry and nanoscale pore orientation of mesocaptors better. Generally, this ion-capture model enables the development of a simple and effective technique for effective wastewater treatment and management.

One-step synthesis of a thioester chiral PMO and its use as a catalyst in asymmetric oxidation reactions

The study of key aspects for the heterogenization of chiral homogeneous catalysts is addressed in this work. Two different heterogenization routes were evaluated. On the one hand, the immobilization of a tartaric acid derivative (Sharpless catalyst) was accomplished by following a two-step synthesis methodology. In the first step, the mesoporous support SBA-15 was functionalized with different percentages of mercaptopropyl-(trimethoxy)-silane (MPTMS) through a co-condensation method, obtaining very high degrees of MPTMS incorporation. In the second step, the chiral moiety is anchored onto the previously organically modified silica by means of a thiotransesterification reaction. On the other hand, the second route consisted of the formation of mesoporous structure and chiral ligand immobilization in the walls, simultaneously, in only one step. In this case, the chiral organic functionality was incorporated into the wall silica framework, since the built-organic complex is a bis-silane, obtaining a chiral PMO. Finally, once characterized, the synthesized materials were proved in an asymmetric catalytic test in order to study their enantioselective properties. The chosen reaction was the thioanisole enantioselective sulfoxidation, using cumene hydroperoxide as an oxidant. The materials synthesized by the multi-step method reached 40% enantiomeric excess (ee), while the synthesized one-pot chiral PMO materials reached up to 50% ee. Thereby, materials with the chiral Sharpless ligand incorporated into the three dimensional silica framework were successfully accomplished, as well as its ability to induce higher chirality in the enantiomeric tested reaction than the conventional methods of chiral ligand heterogenization onto a silica framework.

Selective recognition of arsenic by tailoring ion-imprinted polymer for ICP-MS quantification

A novel arsenic-ion imprinted polymer (As-IIP) was firstly synthesized for the separation and recovery of trace elemental As from environmental water samples. Polymers prepared from bifunctional monomers with intrinsic metal-binding capability are a platform for tailoring ion-selectivity via imprinting moiety–template interaction, without complex formation and ligand immobilization. In the present study, As-IIPs based on 1-vinylimidazole, 4-vinylpyridine and styrene were designed to investigate the imprinting mechanism in relation to their structural and functional properties. In terms of selectivity as well as imprinting effects compared with the non-imprinted polymer (NIP), 1-vinylimidazole-based As-IIP exhibited superior analyte recognition for As ion among 23 competing elements, with a 25-fold enhancement in the practical dynamic and static adsorption capacity range (0.048–4.925μmolg−1). The robust As-IIP sorbent features good reusability up to 20 cycles and a wide working pH 5–7 for a firstly reported solid-phase extraction (SPE) application. As a result of selective sample clean-up, As-IIP-SPE offered limits of detection (LOD) and quantification (LOQ) down to 0.025 and 0.083μmolL−1, respectively, for environmental sample analysis using inductively coupled plasma-mass spectrometry.

Comparison of anion removal capacities of Octolig® and Cuprilig

Mixtures of sodium salts of phosphate, arsenate, and fluoride were used in chromatography and batch experiments to compare the capacity of two immobilized ligands (IMLIGs) to remove these anions: Octolig® and its copper (II)-based metallolig, Cuprilig. The focus of this study was twofold. First, to find out which material, Octolig® or Cuprilig, would remove these anions most effectively; and secondly to determine the optimal capacity for removal of representative anions. Removal capacity was evaluated by two methods: by chromatography and by a batch method. The methods gave identical results, but the batch method was more convenient. Cuprilig had a lower capacity for removal of phosphate than Octolig®. The capability of Octolig® for removing anions was: phosphate > arsenate > fluoride >>>chloride >>>>bromide. Replication of phosphate removal was good for a given batch of Octolig®. Of three different samples of Octolig®, two seemed to be alike based on the phosphate- removal capacity; the third was different on the basis of a Student's t-test. This distinction indicates the batch procedure could serve as a quality control/assurance technique.

Schiff Base Ligands Immobilized on a Nanosized SiO2–Al2O3 Mixed Oxide as Adsorbents for Heavy Metals

Three adsorbents have been synthesized by immobilization of Schiff base ligands on the surface of SiO2–Al2O3 mixed oxides and evaluated for the removal of Cd(II) and Pb(II) from aqueous solutions. The contact time to attain equilibrium for maximum adsorption was 120 min. These heterogeneous Schiff base ligands were found to be effective adsorbents for the removal of heavy metal ions from solution, with Si/Al-pr-N=salicylaldehyde having a high adsorption capacity for Cd(II) ions and Si/Al-pr-N=pyridine-2-carbaldehyde having a high adsorption capacity for Pb(II) ions. The adsorption of heavy metal ions has been studied in terms of pseudo-first-order and pseudo-second-order kinetics, and the Freundlich, Langmuir, and Langmuir–Freundlich isotherm models have also been applied to the equilibrium adsorption data.

Development of surface chemistry for surface plasmon resonance based sensors for the detection of proteins and DNA molecules

The immobilisation of biological recognition elements onto a sensor chip surface is a crucial step for the construction of biosensors. While some of the optical biosensors utilise silicon dioxide as the sensor surface, most of the biosensor surfaces are coated with metals for transduction of the signal. Biological recognition elements such as proteins can be adsorbed spontaneously on metal or silicon dioxide substrates but this may denature the molecule and can result in either activity reduction or loss. Self assembled monolayers (SAMs) provide an effective method to protect the biological recognition elements from the sensor surface, thereby providing ligand immobilisation that enables the repeated binding and regeneration cycles to be performed without losing the immobilised ligand, as well as additionally helping to minimise non-specific adsorption. Therefore, in this study different surface chemistries were constructed on SPR sensor chips to investigate protein and DNA immobilisation on Au surfaces. A cysteamine surface and 1%, 10% and 100% mercaptoundecanoic acid (MUDA) coatings with or without dendrimer modification were utilised to construct the various sensor surfaces used in this investigation. A higher response was obtained for NeutrAvidin immobilisation on dendrimer modified surfaces compared to MUDA and cysteamine layers, however, protein or DNA capture responses on the immobilised NeutrAvidin did not show a similar higher response when dendrimer modified surfaces were used.

Metal Ion Affinity-based Biomolecular Recognition and Conjugation inside Synthetic Polymer Nanopores Modified with Iron–Terpyridine Complexes

Here we demonstrate a novel biosensing platform for the detection of lactoferrin (LFN) via metal-organic frameworks, in which the metal ions have accessible free coordination sites for binding, inside the single conical nanopores fabricated in polymeric membrane. First, monolayer of amine-terminated terpyridine (metal-chelating ligand) is covalently immobilized on the inner walls of the nanopore via carbodiimide coupling chemistry. Second, iron-terpyridine (iron-terPy) complexes are obtained by treating the terpyridine modified-nanopores with ferrous sulfate solution. The immobilized iron-terPy complexes can be used as recognition elements to fabricate biosensing nanodevice. The working principle of the proposed biosensor is based on specific noncovalent interactions between LFN and chelated metal ions in the immobilized terpyridine monolayer, leading to the selective detection of analyte protein. In addition, control experiments proved that the designed biosensor exhibits excellent biospecificity and nonfouling properties. Furthermore, complementary experiments are conducted with multipore membranes containing an array of cylindrical nanopores. We demonstrate that in the presence of LFN in the feed solution, permeation of methyl viologen (MV(2+)) and 1,5-naphthalenedisulphate (NDS(2-)) is drastically suppressed across the iron-terPy modified membranes. On the basis of these findings, we envision that apart from conventional ligand-receptor interactions, the designing and immobilization of alternative functional ligands inside the synthetic nanopores would extend this method for the construction of new metal ion affinity-based biomimetic systems for the specific binding and recognition of other biomolecules.