Click Chemistry Mediated Functionalization of Vertical Nanowires for Biological Applications

Semiconductor nanowires (NWs) are gaining significant importance in various biological applications, such as biosensing and drug delivery. Efficient and controlled immobilization of biomolecules on the NW surface is crucial for many of these applications. Here, we present for the first time the use of the Cu^I‐catalyzed alkyne–azide cycloaddition and its strain‐promoted variant for the covalent functionalization of vertical NWs with peptides and proteins. The potential of the approach was demonstrated in two complementary applications of measuring enzyme activity and protein binding, which is of general interest for biological studies. The attachment of a peptide substrate provided NW arrays for the detection of protease activity. In addition, green fluorescent protein was immobilized in a site‐specific manner and recognized by antibody binding to demonstrate the proof‐of‐concept for the use of covalently modified NWs for diagnostic purposes using minute amounts of material.     

A General Strategy for Site‐Directed Enzyme Immobilization by Using NiO Nanoparticle Decorated Mesoporous Silica

Mesoporous materials have recently gained much attention owing to their large surface area, narrow pore size distribution, and superior pore structure. These materials have been demonstrated as excellent solid supports for immobilization of a variety of proteins and enzymes for their potential applications as biocatalysts in the chemical and pharmaceutical industries. However, the lack of efficient and reproducible methods for immobilization has limited the activity and recyclability of these biocatalysts. Furthermore, the biocatalysts are usually not robust owing to their rapid denaturation in bulk solvents. To solve these problems, we designed a novel hybrid material system, mesoporous silica immobilized with NiO nanoparticles (SBA‐NiO), wherein enzyme immobilization is directed to specific sites on the pore surface of the material. This yielded the biocatalytic species with higher activity than free enzyme in solution. These biocatalytic species are recyclable with minimal loss of activity after several cycles, demonstrating an advantage over free enzymes.     

Immobilization of histidine-tagged proteins on electrodes

The development of new enzyme immobilization techniques that do not affect catalytic activity or conformation of a protein is an important research task in biotechnology including biosensor applications and heterogeneous reaction systems. One of the most promising approaches for controlled protein immobilization is based on the immobilized metal ion affinity chromatography (IMAC) principle originally developed for protein purification. Here we describe the current status and future perspectives of immobilization of His-tagged proteins on electrode surfaces. Recombinant proteins comprising histidine-tags or histidine rich native proteins have a strong affinity to transition metal ions. For metal ion immobilization at the electrode surface different matrices can be used such as self-assembled monolayers or conductive polymers. This specific technique allows a reversible immobilization of histidine-tagged proteins at electrodes in a defined orientation which is an important prerequisite for efficient electron transfer between the electrode and the biomolecule. Any application requiring immobilized biocatalysts on electrodes can make use of this immobilization approach, making future biosensors and biocatalytic technologies more sensitive, simpler, reusable and less expensive while only requiring mild enzyme modifications.     

Electron paramagnetic resonance spin label titration: a novel method to investigate random and site-specific immobilization of enzymes onto polymeric membranes with different properties

The immobilization of biological molecules onto polymeric membranes to produce biofunctional membranes is used for selective catalysis, separation, analysis, and artificial organs. Normally, random immobilization of enzymes onto polymeric membranes leads to dramatic reduction in activity due to chemical reactions involved in enzyme immobilization, multiple-point binding, etc., and the extent of activity reduction is a function of membrane hydrophilicity (e.g. activity in cellulosic membrane?polysulfone membrane). We have used molecular biology to effect site-specific immobilization of enzymes in a manner that orients the active site away from the polymeric membrane surface, thus resulting in higher enzyme activity that approaches that in solution and in increased stability of the enzyme relative to the enzyme in solution. A prediction of this site-specific method of enzyme immobilization, which in this study with subtilisin and organophosphorus hydrolase consists of a fusion tag genetically added to these enzymes and subsequent immobilization via the anti-tag antibody and membrane-bound protein A, is that the active site conformation will more closely resemble that of the enzyme in solution than is the case for random immobilization. This hypothesis was confirmed using a new electron paramagnetic resonance (EPR) spin label active site titration method that determines the amount of spin label bound to the active site of the immobilized enzyme. This value nearly perfectly matched the enzyme activity, and the results suggested: (a) a spectroscopic method for measuring activity and thus the extent of active enzyme immobilization in membrane, which may have advantages in cases where optical methods can not be used due to light scattering interference; (b) higher spin label incorporation (and hence activity) in enzymes that had been site-specifically immobilized versus random immobilization; (c) higher spin label incorporation in enzymes immobilized onto hydrophilic bacterial cellulose membranes versus hydrophobic modified poly(ether)sulfone membranes. These results are discussed with reference to analysis and utilization of biofunctional membranes.     

Reversible immobilization of proteins with streptavidin affinity tags on a surface plasmon resonance biosensor chip

Dissociation of biotin from streptavidin is very difficult due to their high binding affinity. The re-use of streptavidin-modified surfaces is therefore almost impossible, making devices containing them (e.g. surface plasmon resonance (SPR) sensor chips) expensive. This paper describes a new protocol for reversible and site-directed immobilization of proteins with streptavidin affinity tags on the streptavidin-coated SPR biosensor chip (SA chip). Two streptavidin affinity tags, nano-tag and streptavidin-binding peptide (SBP tag), were applied. They both can specifically interact with streptavidin but have weaker binding force compared to the biotin–streptavidin system, thus allowing association and dissociation under controlled conditions. The SA chip surface could be regenerated repeatedly without loss of activity by injection of 50 mM NaOH solution. The fusion construct of a SBP tag and a single-chain antibody to mature bovine prion protein (scFv-Z186-SBP) interacts with the SA chip, resulting in a single-chain-antibody-modified surface. The chip showed kinetic response to the prion antigen with equilibrium dissociation constant K _D≈4.01×10⁻⁷. All results indicated that the capture activity of the SA chip has no irreversible loss after repeated immobilization and regeneration cycles. The method should be of great benefit to various biosensors, biochips and immunoassay applications based on the streptavidin capture surface.     

毛细管柱固定蛋白质方法的研究进展

在熔融石英毛细管中,固定化的蛋白质(酶)在手性拆分、蛋白质肽谱、药物筛选、样品预浓缩等方面具有重要应用.本文对用于毛细管中蛋白质固定的溶胶-凝胶法、物理吸附法、离子鳌合吸附法、基于脂质的蛋白固定和共价键合法进行了综述.     

Light-Controlled Chemoenzymatic Immobilization of Proteins towards Engineering of Bioactive Papers

Efficient and reliable methods for the generation of bioactive papers are of growing interest in relation to point-of-care testing devices that do not require extensive analytical equipment. Herein, we report the immobilization of functional proteins on paper fibers using a modular chemoenzymatic approach. The synthetic strategy relies on a combination of highly efficient spatially controllable photo-triggered cycloaddition followed by site-specific sortase A-catalyzed transamidation. This site-directed and regiospecific method has allowed unidirectional and covalent immobilization of several proteins displaying different functional properties, with ramifications for application in paper-based diagnostics.     

Enzyme‐Mediated,Site‐Specific Protein Coupling Strategies for Surface‐Based Binding Assays

Covalent surface immobilization of proteins for binding assays is typically performed non‐specifically via lysine residues. However, receptors that either have lysines near their binding pockets, or whose presence at the sensor surface is electrostatically disfavoured, can be hard to probe. To overcome these limitations and to improve the homogeneity of surface functionalization, we adapted and optimized three different enzymatic coupling strategies (4′‐phosphopantetheinyl transferase, sortase A, and asparaginyl endopeptidase) for biolayer interferometry surface modification. All of these enzymes can be used to site‐specifically and covalently ligate proteins of interest via short recognition sequences. The enzymes function under mild conditions and thus immobilization does not affect the receptors’ functionality. We successfully employed this enzymatic surface functionalization approach to study the binding kinetics of two different receptor–ligand pairs.     

Protein‐Structure‐Directed Metal–Organic Zeolite‐like Networks as Biomacromolecule Carriers

Fabrication of zeolite‐like metal–organic frameworks (ZMOFs) for advanced applications, such as enzyme immobilization, is of great interest but is a great synthetic challenge. Herein, we have developed a new strategy using proteins as structure‐directed agents to direct the formation of new ZMOFs that can act as versatile platforms for the in situ encapsulation of proteins under ambient conditions. Notably, protein incorporation directs the formation of a ZMOF with a sodalite ( sod ) topology instead of a non‐porous diamondoid ( dia ) topology under analogous synthetic conditions. Histidines in proteins play a crucial role in the observed templating effect. Modulating histidine content thereby influenced the resultant MOF product (from dia to dia + sod mixture and, ultimately, to sod MOF). Moreover, the resulting ZMOF‐incorporated proteins preserved their activity even after exposure to high temperatures and organic solvents, demonstrating their potential for biocatalysis and biopharmaceutical applications.     

Supramolecular Protein Immobilization on Lipid Bilayers

Protein immobilization on surfaces, and on lipid bilayers specifically, has great potential in biomolecular and biotechnological research. Of current special interest is the immobilization of proteins using supramolecular noncovalent interactions. This allows for a reversible immobilization and obviates the use of harsh ligation conditions that could denature fragile proteins. In the work presented here, reversible supramolecular immobilization of proteins on lipid bilayer surfaces was achieved by using the host–guest interaction of the macrocyclic molecule cucurbit[8]uril. A fluorescent protein was successfully immobilized on the lipid bilayer by making use of the property of cucurbit[8]uril to host together a methylviologen and the indole of a tryptophan positioned on the N‐terminal of the protein. The supramolecular complex was anchored to the bilayer through a cholesterol moiety that was attached to the methylviologen tethered with a small polyethylene glycol spacer. Protein immobilization studies using a quartz crystal microbalance (QCM) showed the assembly of the supramolecular complexes on the bilayer. Specific immobilization through the protein N‐terminus is more efficient than through protein side‐chain events. Reversible surface release of the proteins could be achieved by washing with cucurbit[8]uril or buffer alone. The described system shows the potential of supramolecular assembly of proteins and provides a method for site‐specific protein immobilization under mild conditions in a reversible manner.     

Functionalization of thioctic acid-capped gold nanoparticles for specific immobilization of histidine-tagged proteins

This paper presents an efficient strategy for the specific immobilization of fully functional proteins onto the surface of nanoparticles. Thioctic acid-derivatized gold clusters are used as a scaffold for further stepwise modification, leading to a cobalt(II)-terminated ligand shell. A histidine tag introduced by genetic engineering into a protein is coordinated to this transition metal ion. The specific immobilization has been demonstrated for the cases of a genetically engineered horseradish peroxidase and ferredoxin-NADP(+) reductase, confirming the attachment of the fully functional proteins to the Co(II)-terminated nanointerface. The absence of nonspecific protein adsorption and the specificity of the binding site have been verified using several analogues of the enzymes without the histidine tag.     

Interaction of proteins with polymeric latexes

Syntheses, modifications and properties of latexes containing polyacrolein, suitable for the covalent immobilization of proteins, are described. Results of the studies of physical adsorption and covalent attachment of human serum albumin and gamma globulins are discussed. Procedures allowing immobilization of the enzyme, horseradish peroxidase, are described. Changes in protein molecules, owing to their contact with latex particles, are analyzed. Applicability of polyacrolein containing latexes for diagnostic purposes is discussed.     

Fabrication and Characterization of Cysteine-Functionalized Zinc Oxide Nanoparticles for Enzyme Immobilization

A green approach is reported for the synthesis of cysteine-functionalized zinc oxide nanoparticles using potato extract as a nontoxic and economical reducing agent. The cysteine-functionalized nanoparticles were used as a support for enzyme immobilization. The structural morphology, crystallinity, and surface functionalization were characterized by scanning electron microscopy, X-ray diffraction, and infrared spectroscopy, respectively. Spherical nanoparticles from 150 to 200?nm were used to evaluate the immobilization efficiency for urease through covalent attachment on the glutaraldehyde-activated amino group of cysteine. In comparison to the unmodified nanoparticles, 62.9% enzyme loading with 72.45% of enzyme specific activity was recovered which was 56% higher than on bare zinc oxide nanoparticles. The point of addition of cysteine during the nanoparticle synthesis had a direct effect on the immobilization efficiency. The immobilized enzyme-specific activity was reduced to 34.32% when cysteine was added following the nanoparticle synthesis. With a facile synthesis procedure and significant immobilization efficiency, cysteine-functionalized zinc oxide nanoparticles were shown to be suitable for various clinical and industrial applications.     

The Preparation and Enzyme Immobilization of Hydrophobic Polysiloxane Supports

Enzymes are versatile biocatalysts and find increasing applications in many areas. The major advantages of using enzymes in biocatalytic transformations are their chemo‐, regio‐, and stereospecificity, as well as the mild reaction conditions that can be used. However, even when an enzyme is identified as being useful for a given reaction, its application is often hampered by its lack of long‐term stability under process conditions, and also by difficulties in recovery and recycling. For ease of application and stabilization purposes, enzymes are often immobilized on solid supports. Among support matrices, hydrophobic biomaterials have been extensively used as supports for enzyme immobilization because the hydrophobic interactions not only can effectively increase the amount of enzyme immobilization, but also exhibit higher activity and retention of activity compared with hydrophilic supports. On the other hand, polysiloxane can evidently increase the amount of enzyme immobilization because of its hydrophobicity and strong affinity with enzyme. Therefore, this research details the first preparation and use of a hydrophobic polysiloxane support for enzyme immobilization in which the structural and functional characteristics of new supports have been investigated by using glucose oxidase (GOD) and a simple Fenton's assay method, and extremely interesting features were revealed. The results showed that the amount of GOD immobilization and the stability of GOD loaded, which are fundamental properties for enzyme separation and purification, can be significantly improved by adsorption. Moreover, the results indicated that hydrophobic polysiloxane supports can effectively increase the enzymatic affinity and durability of GOD, and decrease the rate of GOD desorbed.

     

Enzymes immobilized in mesoporous silica: A physical–chemical perspective

Mesoporous materials as support for immobilized enzymes have been explored extensively during the last two decades, primarily not only for biocatalysis applications, but also for biosensing, biofuels and enzyme-controlled drug delivery. The activity of the immobilized enzymes inside the pores is often different compared to that of the free enzymes, and an important challenge is to understand how the immobilization affects the enzymes in order to design immobilization conditions that lead to optimal enzyme activity. This review summarizes methods that can be used to understand how material properties can be linked to changes in enzyme activity. Real-time monitoring of the immobilization process and techniques that demonstrate that the enzymes are located inside the pores is discussed by contrasting them to the common practice of indirectly measuring the depletion of the protein concentration or enzyme activity in the surrounding bulk phase. We propose that pore filling (pore volume fraction occupied by proteins) is the best standard for comparing the amount of immobilized enzymes at the molecular level, and present equations to calculate pore filling from the more commonly reported immobilized mass. Methods to detect changes in enzyme structure upon immobilization and to study the microenvironment inside the pores are discussed in detail. Combining the knowledge generated from these methodologies should aid in rationally designing biocatalyst based on enzymes immobilized in mesoporous materials.     

Effect of Motif‐Programmed Artificial Proteins on the Calcium Uptake in a Synthetic Hydrogel

Motif‐programmed artificial proteins with mineralization‐related activity were covalently immobilized onto the surface of a hydrogel, poly(2‐hydroxyethyl methacrylate) (PHEMA). We investigated the influence of assaying conditions upon the ability of three selected proteins (PS64, PS382 and PS458) to modulate calcification in vitro. A long‐term assay measuring the real amount of calcium phosphate phase in the protein‐modified PHEMA showed that all proteins enhanced the uptake of calcium by the hydrogel. For PS382 and PS458, this is a behaviour opposite to that displayed when the same proteins were tested in a free state by a rapid solution assay. Such difference may be attributed to a restricted mobility of the proteins due to immobilization.

     

Covalent attachment of trypsin on plasma polymerized allylamine

This paper focuses on the immobilization of a proteolytic enzyme, trypsin, on plasma polymerized allylamine (ppAA) films. The later have been deposited onto silicon substrate by means of radiofrequency glow discharge. The covalent attachment of the enzyme was achieved in three steps: (i) activation of the polymer surface with glutaraldehyde (GA) as a linker, (ii) immobilization of trypsin and (iii) imino groups reduction treatment. The effects and efficiency of each step were investigated by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). Fluorescent spectroscopy was used to evaluate the change of the biological activity following the immobilization steps. The results showed that enzyme immobilization on GA-modified substrate increases the enzyme activity by 50% comparing to adsorbed enzymes, while the imino reduction treatment improves the enzyme retention by about 30% comparing to untreated samples. In agreement with XPS and AFM data, UV–vis absorption spectroscopy, used to quantify the amount of immobilized enzyme, showed that allylamine plasma polymer presents a high adsorption yield of trypsin. Although the adsorbed enzymes exhibit a lower activity than that measured for enzymes grafted through GA linkers, the highest catalytic activity obtained was for the enzymes that underwent the three steps of the immobilization process.     

Lysozyme Immobilized on Micro-Sized Magnetic Particles: Kinetic Parameters at Wine pH

In order to use lysozyme as an anti-microbial agent during the winemaking process, hen egg-white lysozyme (LYZ) was covalently immobilized on two different micro-size magnetic particles (tosyl-activated and carboxylated, TSA and CA, respectively). A cell suspension of Oenococcus oeni, an oenological strain involved in the winemaking process, was utilized as LYZ substrate. Both a kinetic study and a study of the stability of free and immobilized LYZ were performed in McIlvane buffer at pH 3.2, that is the average minimum pH value in wine. The activity and kinetic parameters measured for the free LYZ at pH 3.2 are lower than those reported at the optimum pH (4.5); however the residual activity at pH 3.2 is sufficient to be of interest for further immobilization and applications in winemaking. All kinetic parameters of both biocatalysts (LYZ-CA and LYZ-TSA) are altered after immobilization, probably due to the structural modifications in the active site caused by covalent attachment to the supports. The half-life calculated at 25 °C was 39 h for free LYZ, while it increased to 280 and 134 h for LYZ-TSA and LYZ-CA, respectively. This result indicates that immobilization improves the enzyme stability and that LYZ can be utilized in wine applications in its immobilized forms. In addition, LYZ-TSA seems to be the best biocatalyst for further applications in winemaking.     

An Amperometric Glucose Sensor Based on Isoporous Crystalline Protein Membranes as Immobilization Matrix

Abstract

S-layer ultrafiltration membranes (SUMs) with an active filtration layer composed of coherent two-dimensional, isoporous protein crystals (S-layers) have been used as matrix for immobilizing monolayers of enzymes. Since S-layers are formed by periodic repetition of identical protein subunits, functional groups are present on the crystalline array in an identical position and orientation. As a consequence monolayers of enzymes can bind in a geometrically well defined way. For the covalent immobilization of enzymes carboxyl groups from the S-layer protein were activated with carbodiimide and allowed to react with amino groups of the enzyme. SUMs were employed as a new type of immobilization matrix for the developement of an amperometric glucose sensor using glucose oxidase (GOD) as the biologically active component. Glucose oxidase covalently bound to the surface of the S-layer protein retained approximately 40% of its activity. The enzyme loaded SUMs were covered with a layer of gold or platinum to function as working electrodes. These sensors yielded high signals (150nA/mm²/mmol glucose), fast response times (10–30s) and a linearity range up to 12 mM glucose. The stability under working conditions was more than 48 hours. There was no loss in activity after a storage period of 6 month.     

Chemoselective and Site‐Selective Lysine‐Directed Lysine Modification Enables Single‐Site Labeling of Native Proteins

The necessity for precision labeling of proteins emerged during the efforts to understand and regulate their structure and function. It demands selective attachment of tags such as affinity probes, fluorophores, and potent cytotoxins. Here, we report a method that enables single‐site labeling of a high‐frequency Lys residue in the native proteins. At first, the enabling reagent forms stabilized imines with multiple solvent‐accessible Lys residues chemoselectively. These linchpins create the opportunity to regulate the position of a second Lys‐selective electrophile connected by a spacer. Consequently, it enables the irreversible single‐site labeling of a Lys residue independent of its place in the reactivity order. The user‐friendly protocol involves a series of steps to deconvolute and address chemoselectivity, site‐selectivity, and modularity. Also, it delivers ordered immobilization and analytically pure probe‐tagged proteins. Besides, the methodology provides access to antibody‐drug conjugate (ADC), which exhibits highly selective anti‐proliferative activity towards HER‐2 expressing SKBR‐3 breast cancer cells.