Specific Immobilization Research Articles

TMV-Based Adapter Templates for Enhanced Enzyme Loading in Biosensor Applications.

Nanotubular tobacco mosaic virus (TMV) particles and RNA-free lower-order coat protein (CP) aggregates have been employed as enzyme carriers in different diagnostic layouts and compared for their influence on biosensor performance. In the following, we describe a label-free electrochemical biosensor for improved glucose detection by use of TMV adapters and the enzyme glucose oxidase (GOD). A specific and efficient immobilization of streptavidin-conjugated GOD ([SA]-GOD) complexes on biotinylated TMV nanotubes or CP aggregates was achieved via bioaffinity binding. Glucose sensors with adsorptively immobilized [SA]-GOD, and with [SA]-GOD cross-linked with glutardialdehyde, respectively, were tested in parallel on the same sensor chip. Comparison of these sensors revealed that TMV adapters enhanced the amperometric glucose detection remarkably, conveying highest sensitivity, an extended linear detection range and fastest response times. These results underline a great potential of an integration of virus/biomolecule hybrids with electronic transducers for applications in biosensorics and biochips. Here, we describe the fabrication and use of amperometric sensor chips combining an array of circular Pt electrodes, their loading with GOD-modified TMV nanotubes (and other GOD immobilization methods), and the subsequent investigations of the sensor performance.

One-step methodology for the direct covalent capture of GPCRs from complex matrices onto solid surfaces based on the bioorthogonal reaction between haloalkane dehalogenase and chloroalkanes.

Protein immobilization techniques play an important role in the development of assays for disease diagnosis and drug discovery. However, many of these approaches are not applicable to transmembrane proteins. G protein-coupled receptors (GPCRs) are the largest protein superfamily encoded by the human genome and are targeted by a quarter of all prescription drugs. GPCRs are highly dynamic and sensitive to changes in the ambient environment, and current immobilization methodologies are not suitable for GPCRs. We used haloalkane dehalogenase (Halo) as an immobilization tag fused to the β2-adrenoceptor (β2-AR), angiotensin II type 1 (AT1) and angiotensin II type 2 (AT2) receptors. The engineered Halo-tag covalently binds to a specific substrate chloroalkane through Asp 106 in the catalytic pocket. The Halo-tagged GPCRs were expressed in Escherichia coli at a suitable yield. Accordingly, we loaded cell lysate containing Halo-tagged GPCRs onto a macroporous silica gel coated with chloroalkane. Morphological characterization indicated a homogeneous monolayer of immobilized Halo-tagged GPCRs on the silica gel surface. The immobilized receptors proved to be surrounded by specific bound phospholipids including PG C18:1/C18:1. We observed a radio-ligand binding ability and ligand-induced conformational changes in the immobilized GPCRs, suggesting the preservation of bioactivity. This method is a one-step approach for the specific immobilization of GPCRs from cell lysates and validates that immobilized receptors retain canonical ligand binding capacity. Our immobilization strategy circumvents labor-intensive purification procedures and minimizes loss of activity. The immobilized receptors can be applied to high-throughput drug and interaction partner screening for GPCRs.

Label-free electrochemical aptasensor based on gold and polypyrrole nanoparticles for interleukin 6 detection

A highly sensitive, selective, fast and stable electrochemical aptasensor, based on a screen-printed carbon electrode modified with a nanocomposite architecture consisting of polypyrrole and gold nanoparticles was designed for interleukin6 detection in human serum. The simultaneous presence of the conductive polymer and gold at the electrode surface provided a suitable environment for the interleukin6 specific aptamer immobilization of through sulfur-gold bonding. Cyclic voltammetry and electrochemical impedance spectroscopy were used for the monitoring, characterization and optimization of the biosensor during all the steps involved in the fabrication process. After complete optimization, the aptasensor exhibited a good impedimetric response towards the target analyte. The results showed that interleukin6 could be detected in a wide linear range from 1 pg mL−1 to 15 μg mL−1 with a detection limit of 0.33 pg mL−1. The biosensor was tested in the presence of other proteins in human serum samples with good recoveries.

Use of Biochar as an Amendment for Remediation of Heavy Metal-Contaminated Soils: Prospects and Challenges

Biochar (BC), known as the new black gold, is a stable, novel carbonaceous by-product that is synthesized through pyrolysis of biological materials in the absence of O2. Recently, an emerging interest in the application of BC as a robust soil amendment has given rise to a broad research area in science and technology. It is considered a promising remediation option for heavy metal (HM)-contaminated soils to reduce HM bioavailability to plants. Remediation efficacy of BC depends on the porosity, composition, pyrolysis temperature, feedstock, and residence time of pyrolysis. This review article aimed to present an overview of BC use in the immobilization of HMs, i.e., Cd, As, Pb, Zn, Ni, Cu, Mn, Cr, and Sb, in contaminated soils. The remaining uncertain factors, including the specific soil HM immobilization mechanisms, long-term beneficial effects, and potential environmental risks associated with BC application are analyzed. Future research must be conducted to ensure that the management of environmental pollution is in accord with ecological sustainability and adaptation of the black gold biotechnology on a commercial basis for immobilization of HMs in contaminated soils.

Molecular Recognition and Immobilization of Ligand-Conjugated Redox-Responsive Polymer Nanocontainers.

We present the preparation of ligand-conjugated redox-responsive polymer nanocontainers by the supramolecular decoration of cyclodextrin vesicles with a thin redox-cleavable polymer shell that displays molecular recognition units on its surface. Two widely different recognition motifs (mannose-Concanavalin A and biotin-streptavidin) are compared and the impact of ligand density on the nanocontainer surface as well as an additional functionalization with nonadhesive poly(ethylene glycol) is studied. Aggregation assays, dynamic light scattering, and a fluorometric quantification reveal that the molecular recognition of ligand-conjugated polymer nanocontainers by receptor proteins is strongly affected by the multivalency of interactions and the association strength of the recognition motif. Finally, microcontact printing is used to prepare streptavidin-patterned surfaces, and the specific immobilization of biotin-conjugated nanocontainers is demonstrated. As a prototype of a nanosensor, these tethered nanocontainers can sense a reductive environment and react by releasing a payload.

Mussel-Inspired Universal Bioconjugation of Polydiacetylene Liposome for Droplet-Array Biosensors.

Most solid-state biosensor platforms require a specific immobilization chemistry and a bioconjugation strategy separately to tether sensory molecules to a substrate and attach specific receptors to the sensory unit, respectively. We developed a mussel-inspired universal conjugation method that enables both surface immobilization and bioconjugation at the same time. By incorporating dopamine or catechol moiety into self-signaling polydiacetylene (PDA) liposomes, we demonstrated efficient immobilization of the PDA liposomes to a wide range of substrates, without any substrate modification. Moreover, receptor molecules having a specificity toward a target molecule can also be attached to the immobilized PDA liposome layer without any chemical modification. We applied our mussel-inspired conjugation method to a droplet-array biosensor by exploiting the hydrophilic nature of PDA liposomes coated on a hydrophobic polytetrafluoroethylene surface and demonstrated selective and sensitive detection of vascular endothelial growth factor down to 10 nM.

Severe neonatal thrombocytopenia due to fetomaternal anti-A alloimmunization: A case report

Severe neonatal thrombocytopenia due to fetomaternal anti-A alloimmunization: A case report

Self-Immobilizing Fusion Enzymes for Compartmentalized Biocatalysis

The establishment of microfluidic enzyme cascades is a topical field of research and development, which is currently hampered by the lack of methodologies for mild and efficient immobilization of isolated enzymes. We here describe the use of self-immobilizing fusion enzymes for the modular configuration of microfluidic packed-bed reactors. Specifically, three different enzymes, the (R)-selective alcohol dehydrogenase LbADH, the (S)-selective methylglyoxal reductase Gre2p and the NADP(H) regeneration enzyme glucose 1-dehydrogenase GDH, were genetically fused with streptavidin binding peptide, Spy and Halo-based tags, to enable their specific and directional immobilization on magnetic microbeads coated with complementary receptors. The enzyme-modified beads were loaded in four-channel microfluidic chips to create compartments that have the capability for either (R)- or (S)-selective reduction of the prochiral CS-symmetrical substrate 5-nitrononane-2,8-dione (NDK). Analysis of the isomeric hydroxyketone and ...

Bioinstructive Coatings for Hematopoietic Stem Cell Expansion Based on Chemical Vapor Deposition Copolymerization.

We report the chemical vapor deposition (CVD) of dual-functional polymer films for the specific and orthogonal immobilization of two biomolecules (notch ligand delta-like 1 (DLL1) and an RGD-peptide) that govern the fate of hematopoietic stem and progenitor cells. The composition of the CVD polymer and thus the biomolecule ratio can be tailored to investigate and optimize the influence of the relative surface concentrations of biomolecules on stem cell behavior. Prior to cell experiments, all surfaces were characterized by infrared reflection adsorption spectroscopy, time-of-flight secondary ion mass spectrometry, and X-ray photoelectron spectroscopy to confirm the presence of both biomolecules. In a proof-of-principle stem cell culture study, we show that all polymer surfaces are cytocompatible and that the proliferation of the hematopoietic stem and progenitor cells is predominantly influenced by the surface concentration of immobilized DLL1.

Efficient, ultra-high-affinity chromatography in a one-step purification of complex proteins

Protein purification is an essential primary step in numerous biological studies. It is particularly significant for the rapidly emerging high-throughput fields, such as proteomics, interactomics, and drug discovery. Moreover, purifications for structural and industrial applications should meet the requirement of high yield, high purity, and high activity (HHH). It is, therefore, highly desirable to have an efficient purification system with a potential to meet the HHH benchmark in a single step. Here, we report a chromatographic technology based on the ultra-high-affinity (Kd ∼ 10-14-10-17 M) complex between the Colicin E7 DNase (CE7) and its inhibitor, Immunity protein 7 (Im7). For this application, we mutated CE7 to create a CL7 tag, which retained the full binding affinity to Im7 but was inactivated as a DNase. To achieve high capacity, we developed a protocol for a large-scale production and highly specific immobilization of Im7 to a solid support. We demonstrated its utility with one-step HHH purification of a wide range of traditionally challenging biological molecules, including eukaryotic, membrane, toxic, and multisubunit DNA/RNA-binding proteins. The system is simple, reusable, and also applicable to pulldown and kinetic activity/binding assays.

Perspective: A toolbox for protein structure determination in physiological environment through oriented, 2D ordered, site specific immobilization.

Revealing the structure of complex biological macromolecules, such as proteins, is an essential step for understanding the chemical mechanisms that determine the diversity of their functions. Synchrotron based X-ray crystallography and cryo-electron microscopy have made major contributions in determining thousands of protein structures even from micro-sized crystals. They suffer from some limitations that have not been overcome, such as radiation damage, the natural inability to crystallize a number of proteins, and experimental conditions for structure determination that are incompatible with the physiological environment. Today, the ultra-short and ultra-bright pulses of X-ray free-electron lasers have made attainable the dream to determine protein structures before radiation damage starts to destroy the samples. However, the signal-to-noise ratio remains a great challenge to obtain usable diffraction patterns from a single protein molecule. With the perspective to overcome these challenges, we describe here a new methodology that has the potential to overcome the signal-to-noise-ratio and protein crystallization limits. Using a multidisciplinary approach, we propose to create ordered, two dimensional protein arrays with defined orientation attached on a self-assembled-monolayer. We develop a literature-based flexible toolbox capable of assembling different kinds of proteins on a functionalized surface and consider using a graphene cover layer that will allow performing experiments with proteins in physiological conditions.

Clickable Antifouling Polymer Brushes for Polymer Pen Lithography.

Protein-repellent reactive surfaces that promote localized specific binding are highly desirable for applications in the biomedical field. Nonspecific adhesion will compromise the function of bioactive surfaces, leading to ambiguous results of binding assays and negating the binding specificity of patterned cell-adhesive motives. Localized specific binding is often achieved by attaching a linker to the surface, and the other side of the linker is used to bind specifically to a desired functional agent, as e.g. proteins, antibodies, and fluorophores, depending on the function required by the application. We present a protein-repellent polymer brush enabling highly specific covalent surface immobilization of biorecognition elements by strain-promoted alkyne-azide cycloaddition click chemistry for selective protein adhesion. The protein-repellent polymer brush is functionalized by highly localized molecular binding sites in the low micrometer range using polymer pen lithography (PPL). Because of the massive parallelization of writing pens, the tunable PPL printed patterns can span over square centimeter areas. The selective binding of the protein streptavidin to these surface sites is demonstrated while the remaining polymer brush surface is resisting nonspecific adsorption without any prior blocking by bovine serum albumin (BSA). In contrast to the widely used BSA blocking, the reactive polymer brushes are able to significantly reduce nonspecific protein adsorption, which is the cause of biofouling. This was achieved for solutions of single proteins as well as complex biological fluids. The remarkable fouling resistance of the polymer brushes has the potential to improve the multiplexing capabilities of protein probes and therefore impact biomedical research and applications.

TGF-β1 activation in human hamstring cells through growth factor binding peptides on polycaprolactone surfaces.

Our study describes the possibility to deliver hTGF-β1 to human derived hamstring cells using a non-covalent bioactive strategy. The significance of our results in vivo with our functionalized biomaterial with TGF-β1-binding peptides lies in the fact that these materials can now be employed to capture endogenous TGF-β1 in a spatially-controlled manner, overcoming the need for exogenous administration of supra-physiological TGF-β1 doses. Our method is different from current solutions that rely on global TGF-β1 administration, soaking the devices with TGF-β1, etc. Therefore we believe that our method is a significant change from current state-of-the-art in the types of devices that are used for ligament/tendon repair and that following our method can endow current and future medical devices with TGF-β1 binding properties.

Orthogonal Surface Tags for Whole-Cell Biocatalysis.

We herein describe the engineering of E. coli strains that display orthogonal tags for immobilization on their surface and overexpress a functional heterologous "protein content" in their cytosol at the same time. Using the outer membrane protein Lpp-ompA, cell-surface display of the streptavidin-binding peptide, the SpyTag/SpyCatcher system, or a HaloTag variant allowed us to generate bacterial strains that can selectively bind to solid substrates, as demonstrated with magnetic microbeads. The simultaneous cytosolic expression of functional content was demonstrated for fluorescent proteins or stereoselective ketoreductase enzymes. The latter strains gave high selectivities for specific immobilization onto complementary surfaces and also in the whole-cell stereospecific transformation of a prochiral CS -symmetric nitrodiketone.

All-Electrical Graphene DNA Sensor Array.

Electrical sensing of biomolecules has been an important pursuit due to the label-free operation and chip-scale construct such sensing modality can enable. In particular, electrical biomolecular sensors based on nanomaterials such as semiconductor nanowires, carbon nanotubes, and graphene have demonstrated high sensitivity, which in the case of nanowires and carbon nanotubes can surpass typical optical detection sensitivity. Among these nanomaterials, graphene is well suited for a practical candidate for implementing a large-scale array of biomolecular sensors, as its two-dimensional morphology is readily compatible with industry standard top-down fabrication techniques. In our recent work published in 2014 Nature Communications, we demonstrated these benefits by creating DNA sensor arrays from chemical vapor deposition (CVD) graphene. The present chapter, which is a review of this recent work, outlines procedures demonstrating the use of individual graphene sites of the array in dual roles--electrophoretic electrodes for site specific probe DNA immobilization and field effect transistor (FET) sensors for detection of target DNA hybridization. The 100fM detection sensitivity achieved in 7 out of 8 graphene FET sensors in the array combined with the alternative use of the graphene channels as electrophoretic electrodes for probe deposition represent steps toward creating an all-electrical multiplexed DNA array.

High performance dendrimer functionalized single-walled carbon nanotubes field effect transistor biosensor for protein detection

We report a single-walled carbon nanotube (SWNT) field-effect transistor (FET) functionalized with Polyamidoamine (PAMAM) dendrimer with 128 carboxyl groups as anchors for site specific biomolecular immobilization of protein antibody for C-reactive protein (CRP) detection. The FET device was characterized by scanning electron microscopy and current-gate voltage (I-Vg) characteristic studies. A concentration-dependent decrease in the source-drain current was observed in the regime of clinical significance, with a detection limit of ∼85 pM and a high sensitivity of 20% change in current (ΔI/I) per decade CRP concentration, showing SWNT being locally gated by the binding of CRP to antibody (anti-CRP) on the FET device. The low value of the dissociation constant (Kd = 0.31 ± 0.13 μg ml−1) indicated a high affinity of the device towards CRP analyte arising due to high anti-CRP loading with a better probe orientation on the 3-dimensional PAMAM structure.

Mixture of PLA-PEG and biotinylated albumin enables immobilization of avidins on electrospun fibers.

The application of nanotechnology in biomedical field has enormous potential in basic and applied research. Micro or nanofibers produced by electrospinning technique offer excellent properties because of large specific surface area, high porosity, and ability to incorporate functional additives. Here we embedded biotinylated bovine serum albumin into polylactic acid (PLA)-polyethylene glycol (PEG) fibers, which enabled specific immobilization of fluorescently labelled avidin. An alkaline phosphatase enzyme was immobilized via biotin-streptavidin interaction on the hybrid nanofibers, demonstrating the suitability of the material for biosensing applications. These functional nanofibers provide a promising platform for development of biosensors and other biofunctional materials utilizing avidin-biotin as a generic and robust immobilization method. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 356-362, 2017.

Multifunctional biomaterial coatings: synthetic challenges and biological activity.

A controlled interaction of materials with their surrounding biological environment is of great interest in many fields. Multifunctional coatings aim to provide simultaneous modulation of several biological signals. They can consist of various combinations of bioactive, and bioinert components as well as of reporter molecules to improve cell-material contacts, prevent infections or to analyze biochemical events on the surface. However, specific immobilization and particular assembly of various active molecules are challenging. Herein, an overview of multifunctional coatings for biomaterials is given, focusing on synthetic strategies and the biological benefits by displaying several motifs.

(Keynote) Enzymatic Biofuel Cell Based on Porous Carbon Materials

Enzymatic biofuel cells (EBFCs) are a type of fuel cells, which employ enzymes instead of conventional noble metal catalysts. The working principle is the same as in conventional polymer electrolyte membrane fuel cells, namely fuel is oxidized at the anode side and the electrons reach the cathode, where they combine with an oxygen to water. EBFCs are promising for sustainable green energy applications; however, they are still at an early stage of development, with many yet-to-be-resolved fundamental scientific and engineering problems. Two critical problems are short lifetime and poor power density, both of which are related to enzyme stability, electron transfer rate, and enzyme loading. To achieve the practical application of EBFCs, a promising approach is to use porous carbon materials. Strategies for the designing of hierarchically structured supports composed of mesoporous and macroporous are considered: the large surface area of mesoporous materials can increase the enzyme loading and electron transfer efficiency, and the macropores enable the efficient fuel transport. Our recent paper demonstrated that Magnesium oxide (MgO)-templated porous carbon forms are promising candidates as electrode materials for the elaboration of efficient bioelectrochemical devices. MgO-templated mesoporous carbon (MgOC, mean pore diameter 10, 20, 40, 80 nm) were used to increase the effective specific surface area for specific enzyme immobilization. MgOC particles were deposited on a current collector by an electrophoretic deposition method to generate micro meter-scale macropores to improve the mass transfer of glucose and electrolyte (buffer) ions. To create a glucose bioanode, the porous-carbon-modified electrode was further coated with a biocatalytic hydrogel composed of a conductive redox polymer, flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH), and a cross-linker. The electrode coated with FAD-GDH and a redox polymer showed a 33-fold increase in glucose oxidation current density (ca. 100 mA cm−2) compared to that of the flat electrode. MgOC-modified electrode can be applied for the biocathode using bilirubin oxidase (BOD) as an oxygen reduction reaction catalysts. Our recent wearable EBFC based on MgOC will be presented. FAD-GDH was used as an anodic catalyst. 1,4-naphtoquinone was modified on the MgOC mesopore surface. Bilirubin oxidase and ABTS mediator were modified on the MgOC surface. Aiming at the application of EBFCs to the wearable power devices, we modified the MgOC on the surface of flexible carbon cloth. The cell operation was initiated after addition of 0.4 mL of 1 M phosphate buffer solution containing various concentrations of glucose, ranging from 0.1 – 3 M. The maximum output power density per geometric surface area was 2 mW cm-2, which was limited by the cathode performance, especially O2 supply from the air. The operation time, i.e. cell capacity, was quantitatively determined by the mole number of applied glucose evaluated by galvanostatic experiments at 0.25 – 4 mA cm-2 of constant current density, indicating that the capacity was determined by the consumption of β-d-glucose. After consumption of the fuel, the cells were able to restart by adding the glucose solution. The operation time linearly depended on the mole number of glucose added. In this presentation, recent developments in EBFCs technology using porous carbon materials are highlighted. The essential properties of the resulting materials with respect to the EBFC application are also discussed. A combination of electron transfer technology and porous carbon material would be helpful in achieving a much higher and stable current output, thus contributing to a practical advance in the sustainable energy field.

Positive Dendritic Effect on Maleimide Surface Modification of Core-Shell (γ-Fe2O3/Polymer) Nanoparticles for Bio-Immobilization

Maleimide functionalization of γ-Fe2O3/Polymer MNPs (Magnetic Nanoparticles) was investigated via covalent grafting of a linear and two dendritic functional coupling agents. Chemical surface modification was monitored by ATR FTIR spectroscopy and we emphasized a positive dendritic effect on the surface functionalization. Indeed, the amount of functional group on MNPs’ surface is directly proportional to the dendron's generation of the coupling agent. The immobilization of biomolecules (via thiol/maleimide reaction) was achieved using a two-steps strategy based on (i) biotinylation of maleimide surfaces and (ii) subsequent specific immobilization of streptavidin coated gold 15 nm NPs (SA−Au). The efficiency of maleimide modified MNPs to react with thiol groups of biomolecules was assessed by UV-Vis quantification of SA−Au NPs. The selectivity and the specificity of this immobilization was undoubtedly established, and a positive dendritic effect was demonstrated by the immobilization of Streptavidin coated gold nanoparticles on the surface of MNPs.